Azathioprine in the therapy of paediatric inflammatory bowel disease – part II: pharmacodynamics, pharmacokinetics, and the possibilities of measuring its metabolites in clinical practice

Summary Background: Thiopurines (predominantly azathioprine and mercaptopurine) are widely used in paediatrics to maintain remission in the treatment of inflammatory bowel disease. After its absorption from the gastrointestinal tract, azathioprine is converted into 6 mercaptopurine in approximately 90%. Several enzymes (such as thiopurine methyltransferase, xanthine oxidase and hypoxanthine-guanine phosphoribosyl transferase) participate in its further metabolism, producing non-active methylated metabolites (6 methylmercaptopurine) and thiouric acid and active 6-thioguanine nucleotide. The concentration of these metabolites can be measured in red blood cells. Aim: To map the benefits and possibilities of thiopurine metabolites measurements in patients suffering from inflammatory bowel disease. Conclusion: The measurement of active and non-active metabolites can help evaluate the bioavailability of those drugs, identify some causes of adverse effects and reveal non-adherence. Keywords Crohn’s disease, merkaptopurin, pediatrie, thiopuriny, ulcerative colitis

Therapeutic drug monitoring of thiopurines: Effect of reduced 6-thioguanine nucleotide target levels in IBD patients

Aim: The effect of the Dutch nationwide adjustment of reduced 6-TGN target values (from 600-1200 pmol/8x108 RBC to 320-630 pmol/8x108 RBC) on toxicity and clinical outcome of thiopurine treatment in patients with inflammatory bowel disease (IBD) has not yet been established. Therefore the authors determined the incidence of toxicity-induced discontinuations and efficacy at both target concentrations. Methods: This retrospective study was performed in IBD patients treated with azathioprine or mercaptopurine. Two groups were defined: the former target (FT) group with target concentrations of 600-1200 pmol/8x10^8 RBC and the adjusted target (AT) group with target concentrations of 320-630 pmol/8x10^8 RBC. Patients were followed for maximum 52 weeks or until discontinuation of thiopurine therapy. Data were collected from the local hospital electronic health software of Rijnstate Hospital. Results: 151 patients were included, 76 in the FT group and 75 in the AT group. At week 52, 100 out of 150 patients (66%) of the total population discontinued thiopurine therapy. Forty-eight of this discontinuations were due toxicity (48%). The estimated cumulative incidence of toxicity was higher in the FT group compared to the AT group (47% and 35% respectively, p=0.25). No loss of efficacy was seen in the AT group. Conclusion: Reduction of the target range may lead to less toxicity induced discontinuations. In addition, this study did not find any indication that the reduction of the target range diminished efficacy.

Usefulness of Measuring Thiopurine Metabolites in Children with Inflammatory Bowel Disease and Autoimmunological Hepatitis, Treated with Azathioprine

Introduction. Thiopurines, such as azathioprine (AZA) and 6-mercaptopurine (6-MP), are immunomodulatory agents, used for the maintenance of remission in children with inflammatory bowel disease (IBD)—Crohn’s disease (CD) and ulcerative colitis (UC), as well as with autoimmunological hepatitis (AIH). Measurements of thiopurine metabolites may allow identifying patients at risk for toxicity and nonadherence. It can also provide an explanation for the ineffectiveness of the treatment, observed in some patients. Patients and Methods. A retrospective analysis was carried out of sixty-eight patients (thirty-six patients with CD, eighteen with UC, and fourteen with AIH), treated with AZA. Thiopurine metabolites, 6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine (6-MMP), were assayed by high-performance liquid chromatography (HPLC), and the AZA dose was adjusted when 6-TGN concentration was known. Result. Only twenty-five (41%) children had therapeutic 6-TGN concentrations, ten (16%) subjects had suboptimal 6-TGN concentrations, and twenty-six subjects (43%) had 6-TGN concentrations above the recommended therapeutic range. 6-MMP was not above the therapeutic range in any case. Seven subjects revealed undetectable 6-TGN and 6-MMP levels, indicating nonadherence. The mean AZA dose after the 6-TGN concentration-related adjustment did not differ, in comparison to the initial dose, either in IBD or AIH groups. The mean AZA dose was lower in AIH than in IBD. The subjects with an optimal 6-TGN level presented with a higher ratio of remission (88%) than the under- or overdosed patients (60% and 69%), respectively ( Chi − square test = 3.87 , p < 0.05 ). Conclusion. Timely measurements of thiopurine metabolites can be a useful tool to identify nonadherent patients before a decision is taken to switch to another drug. We may also spot the patients who receive either too low or too high doses, compensating dose deviations in an appropriate way. The patients with optimal 6-TGN levels presented a higher percentage of remission than the under- or overdosed patients. In most patients, both initial and adjusted AZA doses, lower than suggested in guidelines, appeared to be sufficient to maintain remission.

DNA-thioguanine nucleotide as a treatment marker in acute lymphoblastic leukemia patients with NUDT15 variant genotypes

Background Large inter-individual variations in drug metabolism pose a challenge in determining 6-mercaptopurine (6MP) doses. As the last product of 6MP metabolism, DNA-thioguanine nucleotide (DNA-TGN) could reflect the efficacy of 6MP, especially in patients harboring variants in the 6MP metabolism pathway. The aim of this study was to investigate the clinical significance of DNA-TGN monitoring in Korean pediatric acute lymphoblastic leukemia (ALL) patients, focusing on the NUDT15 genotype. Methods The subjects of this study were patients who underwent ALL treatment with 6MP. Tests for the NUDT15 and TPMT genotypes were performed, and prospective DNA-TGN and erythrocyte TGN samples were collected after two weeks or more of 6MP treatment. DNA-TGN was quantified using the liquid chromatography-tandem mass spectrometry method. Results A total of 471 DNA-TGN measurements in 71 patients were analyzed, which ranged from 1.0 to 903.1 fmol thioguanine/μg DNA. The 6MP intensity demonstrated a significant relationship with DNA-TGN concentration (P<0.001). Patients harboring NUDT15 variants were treated with a lower dose of 6MP (P<0.001); however, there was no significant difference in DNA-TGN concentration when compared to patients carrying wild-type NUDT15 (P = 0.261). These patients also presented higher variation in DNA-TGN levels (P = 0.002) and DNA-TGN/6MP intensity (P = 0.019) compared to patients carrying wild-type NUDT15. DNA-TGN concentration did not show a significant correlation with WBC count (P = 0.093). Conclusions Patients harboring NUDT15 variants demonstrated similar DNA-TGN concentrations even at low doses of 6MP and showed high variability in DNA-TGN. Particularly in patients with NUDT15 variants who need a reduced 6MP dose, DNA-TGN could be applied as a useful marker to monitor the therapeutic effect of 6MP.

Normal Ranges of Thiopurine Methyltransferase Activity Do Not Affect Thioguanine Nucleotide Concentrations With Azathioprine Therapy in Inflammatory Bowel Disease

Background Thiopurine methyltransferase (TPMT) activity influences azathioprine conversion into active metabolite 6-thioguanine nucleotide (6-TGN). Low TPMT activity correlates with high 6-TGN and risk for myelosuppression. Conversely, normal-to-high TPMT activity may be associated with low 6-TGN and drug resistance, the so-called hypermetabolizers. Our aim was to identify the effect of normal-to-high TPMT activity on 6-TGN concentrations in an inflammatory bowel disease population. Methods A retrospective chart review of patients aged ≥18 with inflammatory bowel disease, on azathioprine, with documented TPMT activity and 6-TGN concentration was performed. Correlations were evaluated via the Spearman rho correlation coefficient. Linear regression was used to determine the effect of TPMT activity on 6-TGN accounting for confounders. Relationships between TPMT activity, drug dose, and 6-TGN levels were defined via average causal mediation effects. Results One hundred patients were included. No correlation was observed between TPMT activity, azathioprine dosing, and metabolite concentrations. Overall, 39% of the cohort had a therapeutic 6-TGN level of &gt;230 pmol/8 × 108 red blood cells (RBCs). No patient under 1 mg/kg achieved a therapeutic 6-TGN level, whereas 42% of patients taking 2.5 mg/kg did. The median 6-TGN concentration was higher for those in remission (254 pmol/8 × 108 RBCs, interquartile range: 174, 309) versus those not in remission (177 pmol/8 × 108 RBCs, interquartile range: 94.3, 287.8), though not significantly (P = 0.08). Smoking was the only clinical factor associated with 6-TGN level. On multivariate linear regression, only age, azathioprine dose, and obese body mass index were predictive of metabolite concentration. Conclusions Variations within the normal range of TPMT activity do not affect 6-TGN concentration.

P339 Assessment of DNA-thioguanine nucleotide identifies patients at risk of thiopurine-induced leukopoenia in inflammatory bowel disease

Background Thiopurine drugs plays an important role for immunosuppressive therapy in inflammatory bowel disease. The molecular mechanism of the thiopurine toxicity is primarily mediated by DNA-incorporated thioguanine nucleotides (DNA-TGN). The aim of this study was to assess the impact of DNA-TGN levels on thiopurine-induced toxicity. Methods Patients over 18 years who were diagnosed with IBD and prescribed on thiopurines from February 2019 to August 2019 were recruited. All of them were genotyped with NUDT15 R139C before the thiopurine therapy. A novel assay was established to measure levels of DNA-TGN in blood leucocytes. The DNA-TGN and 6TGN levels were correlated with clinical toxicity. Results 185 patients with Crohn’s disease and 5 patients with ulcerative colitis were included in this study. DNA-TGN and 6TGN levels could be quantified in 229 patients’ blood samples. Thiopurine-induced leukopoenia (TIL) arose in 19 individuals with the median 6TGN level of 308.0 pmol/8×108 RBC which was not significantly different from the patients without TIL (p = 0.050). However, there was a significant association between DNA-TGN levels and TIL. Patients who developed TIL were identified with a higher DNA-TGN levels compared with those who did not (p = 0.00030, 456.9 (63.3–879.2) vs. 268.9 (45.6–916.8) fmol/μg DNA). The area under the ROC curves of the continuous DNA-TGN concentration to predict TIL was 0.75 (95% CI: 0.63~0.87) and almost 84% (16/19) of the TIL could be explained based on the DNA-TGN cut-off value of 343.9 fmol/μg DNA Conclusion This study shows that quantification of DNA-TGN levels can be applied to gauge thioprine therapy and avoid TIL after pre-genotyping NUDT15 R139C in inflammatory bowel disease patients.

Allopurinol reverses mercaptopurine-induced hypoglycemia in patients with acute lymphoblastic leukemia

Fasting hypoglycemia is a known complication of mercaptopurine (6MP) maintenance therapy for acute lymphoblastic leukemia (ALL). It is associated with high levels of the methylated metabolite 6-methyl-mercaptopurine (6MMP). Symptoms of hypoglycemia include morning tremulousness, nausea and vomiting. We have previously shown that switching 6MP dosing from evening to morning resolved hypoglycemia by reducing 6MMP; however, the reduction of 6MMP was only transient, potentially resulting in return of hypoglycemia. In children and adults with Crohn’s disease, co-prescribing allopurinol with 6MP blocks the activity of thiopurine methytransferase (TPMT), reducing 6MMP and improving its tolerance. As a consequence of inhibiting TPMT, 6MP is shunted toward the production of 6-thioguanine nucleotide (6TGN), which will result in pancytopenia if the dose of 6MP is not reduced. We demonstrate that allopurinol with a reduced dose of 6MP in two patients with ALL and 6MMP-associated hypoglycemia resulted in a complete and sustained suppression of 6MMP and rapid reversal of hypoglycemia and its symptoms.