Thiopurine Metabolites Research Articles

S3979 Dramatic Changes in Thiopurine Metabolite Levels in a Patient with Inflammatory Bowel Disease Treated with Tirzepatide for Weight Loss: A Case Report

S3979 Dramatic Changes in Thiopurine Metabolite Levels in a Patient with Inflammatory Bowel Disease Treated with Tirzepatide for Weight Loss: A Case Report

Early therapeutic drug monitoring helps to identify inflammatory bowel disease patients with a high risk to fail thiopurine treatment.

Conventional thiopurines (azathioprine and mercaptopurine) remain standard therapy to maintain steroid sparing remission in inflammatory bowel disease (IBD), but are regularly discontinued due to adverse drug reactions (ADRs). Measurement of the metabolites 6-thioguanine nucleotides (6-TGN), 6-methylmercaptopurine ribonucleotides (6-MMPR) and the 6-MMPR/6-TGN ratio, may predict the development of these ADRs. Our aim was to evaluate whether early thiopurine metabolite measurements were associated with clinical outcomes. A post-hoc analysis was conducted of a multicentre, prospective, observational study on thiopurine-induced hepatotoxicity. IBD patients who initiated thiopurine therapy were included and thiopurine metabolite concentrations were assessed after 7days (±1) (T1). Patients were monitored for 12 weeks to document the occurrence of ADRs, early treatment discontinuation and effectiveness. In total, 181 patients were evaluated. At T1, 6-MMPR concentrations and 6-TGN/6-MMPR ratios were independently related to treatment discontinuation within 12 weeks after correction for sex, age and body mass index (BMI) (P= .034 and .002, respectively). The largest effects were observed for 6-MMPR ≥3000pmol/8 × 108 RBC and 6-TGN/6-MMPR ratio ≥17. Furthermore, 6-MMPR concentrations and 6-TGN/6-MMPR ratios at T1 were independently related to skewed metabolism at steady state (Week 8, 6-MMPR/-6TGN ratio ≥11 and ≥20) (both P< .001). The occurrence of ADRs and effectiveness were not independently related to T1 thiopurine metabolite concentrations. Thiopurine metabolite concentrations at T1 were related to early treatment discontinuation and skewed metabolism at steady state, but not to effectiveness, helping to identify patients with a high risk of thiopurine treatment failure.

Optimizing thiopurine therapy in autoimmune hepatitis: A multicenter study on monitoring metabolite profiles and co-therapy with allopurinol.

In autoimmune hepatitis, achieving complete biochemical remission (CBR) with current weight-based thiopurine dosing is challenging. We investigated whether patients could be stratified regarding CBR according to a target range of thiopurine metabolites. Moreover, we explored the effects of azathioprine dosage increases and co-therapy of allopurinol with low-dose thiopurines on metabolite profiles and treatment response. The relation between metabolites and treatment response was assessed in 337 individuals from 4 European centers. In a global, cross-sectional analysis, active metabolites 6-thioguanine nucleotides (6TGN) were similar in those with and without CBR. However, analyzing patients with sequential measurements over 4 years (N = 146) revealed higher average 6TGN levels in those with stable CBR (260pmol/0.2mL) compared to those failing to maintain CBR (181pmol/0.2mL; p = 0.0014) or never achieving CBR (153pmol/0.2mL; p < 0.0001), with an optimal 6TGN cutoff of ≥223pmol/0.2mL (sensitivity: 76% and specificity: 78%). Only 42% exhibited 6TGN ≥223pmol/0.2mL following weight-based dosing, as doses weakly correlated with 6TGN but with 6-methylmercaptopurine (6MMP), a metabolite associated with toxicity. Azathioprine dose increases led to preferential 6MMP formation (+127% vs. 6TGN +34%; p < 0.0001). Conversely, adding allopurinol to thiopurines in difficult-to-treat patients (N = 36) raised 6TGN (168→321pmol/0.2mL; p < 0.0001) and lowered 6MMP (2125→184pmol/0.2mL; p < 0.0001), resulting in improved transaminases in all patients and long-term CBR in 75%. Maintaining CBR in autoimmune hepatitis was associated with 6TGN ≥223pmol/0.2mL. For patients who fail to achieve CBR and therapeutic 6TGN levels despite thiopurine dose increase due to preferential 6MMP formation, comedication of allopurinol alongside low-dose thiopurines represents an efficient alternative.

Therapeutic drug monitoring of vedolizumab therapy in inflammatory bowel disease.

Therapeutic drug monitoring is effective for optimizing anti-tumor necrosis factor therapies in inflammatory bowel disease, but for vedolizumab, a gut-selective leucocyte migration inhibitor, data are scarce. Observational cohort study including 116 bio-experienced inflammatory bowel disease patients treated with vedolizumab for active luminal disease. Biobanked trough blood samples (n=676) covering 96% of patients were analyzed using a drug-binding immunofluorometric assay. Steroid-free treatment outcomes were classified by clinical disease activity indices and objective findings, primarily endoscopy. Patients with clinical remission to vedolizumab induction therapy (37%) had significantly higher trough levels than those without at weeks 6 (mean 34.1 vs 28.0μg/mL, P=0.03) and 10 (34.8 vs 27.5μg/mL, P=0.01). Optimal thresholds for discrimination were 32.4μg/mL (AUCROC 0.66, P=0.04) and 23.5 (AUCROC 0.67, P=0.01), respectively. This positive association persisted during maintenance phase with 11.9μg/mL (AUCROC 0.69, P<0.01) associated with clinical remission (37%) and 15.3 (AUCROC 0.74, P<0.001) for objective remission (46%). Stratification by temporal evolution of treatment effects revealed higher induction and maintenance vedolizumab levels in persistent and slow responders as compared to secondary or persistent failures. Pharmacokinetics was influenced by rare formation of anti-vedolizumab antibodies (2%), and to a lesser extent gender and albumin during induction, but not disease severity, concomitant steroids, or thiopurine metabolites. Switching to subcutaneous administrations resulted in 2.3-fold increase in steady-state trough levels. Our study supports maintaining adequate drug exposure being essential for sustained positive outcomes of vedolizumab and emphasizes individualized, therapeutic drug monitoring-based treatment regimens. Controlled trials and pharmacokinetic modeling are, however, needed.

Thiopurine Metabolite Shunting in Late Pregnancy Increases the Risk of Intrahepatic Cholestasis of Pregnancy in Women With Inflammatory Bowel Disease, and Can be Managed With Split Dosing.

The risk of intrahepatic cholestasis of pregnancy [ICP] is increased in thiopurine-exposed pregnancies. Thiopurine 'shunting', with a 6-methylmercaptopurine [MMP] to 6-thioguanine [TGN] ratio of >11, progresses over pregnancy, and may promote ICP development. We aimed to explore the association between thiopurine exposure and ICP, including the hypothesised impact of thiopurine shunting, and identify risk minimisation strategies. This prospective multicentre cohort study compared thiopurine and biologic monotherapy-exposed pregnant participants. Disease activity and obstetric outcome data, thiopurine metabolites, bile acids, and transaminases were obtained before conception, in each trimester, at delivery, and postpartum. Thiopurine dose management was at the discretion of the treating physician. Included were 131 thiopurine and 147 biologic monotherapy-exposed pregnancies. MMP/TGN ratio increased from preconception to third trimester [p <0.01], with approximately 25% of participants shunting in pregnancy. Second trimester split dosing led to a decrease in the median MMP/TGN ratio from 18 (interquartile range [IQR] 6-57) to 3 [IQR 2-3.5] at delivery [p = 0.04]. The risk of ICP was increased in thiopurine-exposed pregnancies (6.7% [7/105] vs 0% [0/112], p <0.001), with all ICP cases occurring in the setting of antenatal thiopurine shunting. Thiopurine dose increases (risk ratio [RR] 8.10, 95% confidence interval [CI] 1.88-34.85, p = 0.005) and shunting in third trimester [6.20, 1.21-30.73, p = 0.028] and at delivery [14.18, 1.62-123.9, p = 0.016] were associated with an increased risk of ICP. Thiopurine exposure is associated with an increased risk of ICP, particularly following dose increases antenatally and with shunting in late pregnancy. The latter may be effectively managed with split dosing, although further studies are warranted.

Thrombocytosis and Transaminitis in Infants Born to Women With Inflammatory Bowel Disease Is Associated With Exposure to Maternal Inflammation In Utero

Abstract Background Despite reassuring clinical safety data, thrombocytosis, anemia, lymphopenia, and liver function derangements have been observed in infants born to women with inflammatory bowel disease (IBD) treated with thiopurines and biologics. We aimed to define the prevalence, course, associations, and clinical impact of hematological and biochemical abnormalities in such infants. Methods This multicenter prospective cohort study assessed clinical, hematologic, and biochemical outcomes of infants exposed to thiopurines or biologics in utero for management of maternal IBD. Liver transaminases, full blood examination, and infant thiopurine metabolites (where exposed) were taken at delivery and 6 weeks of age. Abnormal results were repeated until normalization. Infants were followed clinically by a pediatric gastroenterologist up to 2 years of age. Results A total of 130 infants were included. Thrombocytosis and elevated alanine transaminase (ALT) were seen in over half of infants up to 6 months of age with no significant clinical impact. Elevated ALT was associated with increasing maternal C-reactive protein in second trimester, while thrombocytosis was associated with increasing maternal C-reactive protein and fecal calprotectin in third trimester. Preceding infection and vaccination were associated with an increased risk of elevated alkaline phosphatase at 3 months. In those exposed to thiopurines, increasing maternal 6-methylmercaptopurine at delivery was associated with increased ALT to 6 months. Conclusions Infants born to women with IBD commonly developed thrombocytosis, elevated alkaline phosphatase, and elevated ALT. These findings were associated with exposure to maternal inflammation, elevated 6-methylmercaptopurine at delivery, and infant vaccinations and infections, and had minimal clinical consequence.

P012 Intestinal epithelial cells as targets of thiopurines in Inflammatory Bowel Disease paediatric patients-derived organoids

Abstract Background Thiopurines, azathioprine (AZA) and mercaptopurine (MP), are immunomodulators used to maintain remission in patients with inflammatory bowel disease (IBD). Intestinal epithelial cells play a key role in the pathogenesis of IBD, however, their role as target cells for thiopurines has not been elucidated. The aim of the project is to establish an in vitro IBD model using organoids starting from intestinal biopsies of IBD paediatric patients to investigate thiopurine effects on intestinal epithelium. Methods Forty-four IBD patients (mean age 14.4 years, 21 males) were enrolled. Intestinal biopsies were used to isolate crypts and generate adult stem cell-derived organoids. The cytotoxicity of thiopurines (0.2-200 µM for 72 hours) was evaluated by CellTiter-Glo 3D assay. Gene expression was evaluated using TaqMan assays. Thiopurine metabolites were quantified by LC-MS/MS analysis. Proteomic profiles were analyzed by Q-Exactive Plus mass spectrometer, western blot and REACTOME pathway software. Statistical analysis was performed using GraphPad and R software. Results IBD patients-derived organoids were sensitive to thiopurine treatment in a dose-dependent manner with a high interpatient variability (Two-way ANOVA p-value&amp;lt;0.0001; IC50 2.24 µM for AZA (95% CI: 1.28–5.53 µM) and 2.65 µM for MP (95% CI: 1.43–8.27 µM)).A significant negative correlation was observed between the viability after thiopurine exposure and mRNA expression of candidate genes involved in thiopurine pharmacokinetics (ITPA, TPMT, PACSIN2 and NUDT15). The most abundant thiopurine metabolite measured on organoids treated with IC50 of MP was MeTIMP, with a concentration correlated with cell viability after MP exposure (p=0.0056, ρ=-0.99). Proteomic analysis on organoids treated with IC20 of thiopurines (0.2 μM) showed 45 differentially expressed proteins in common between AZA and MP (p&amp;lt;0.05). REACTOME enrichment analysis revealed as the most altered pathways: vesicular traffic, autophagy, protein ubiquitination and interferon signalling, while no proteins related to apoptosis were induced. Among these proteins, TRIM32, a ubiquitin ligase involved in STING activity, a key regulator of both interferon I expression and NF-kB activation, resulted downregulated by the treatment (t-test AZA: p=0.0006; MP: p=0.014) and its basal levels negatively correlated with viability rates after thiopurine exposure (AZA: p=0.006, ρ=-0.85; MP: p=0.002, ρ=-0.88). Organoid exposure to thiopurines also decreased p-STING/STING ratio, particularly after MP treatment (t-test p=0.025), and increased autophagy, assessed as LC3-II levels (t-test AZA: p=0.0006; MP: p=0.014). Conclusion Organoid cultures provide new insights on the mechanism of action of thiopurines in the intestinal epithelium.

Quantification of Thiopurine Metabolites in Human Erythrocytes by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

The thiopurine drugs, azathioprine, mercaptopurine, and thioguanine, are widely used in the treatment of several malignant and nonmalignant diseases. These inactive prodrugs undergo extensive metabolism to form active cytotoxic metabolites, which act mainly by incorporating into DNA and affecting cell replication. Thiopurine methyltransferase is a highly variable cytosolic enzyme that catalyzes the S-methylation of the thiopurine bases-an inactivating pathway. Patients with low-activity variants of TPMT can be affected by pronounced pharmacologic effects when receiving thiopurine medications. Clinical studies have reported significant interpatient variability in intracellular thiopurine metabolite concentrations in patients receiving thiopurine therapy. In this chapter, we present an LC-MS/MS method to monitor the thiopurine metabolites: 6-thioguanine nucleotides and 6-methylmercaptopurine derivatives in human erythrocytes. This method utilizes acid hydrolysis to release the bases and improves upon previously published procedures by utilizing stable isotope internal standards and a more efficient chromatographic separation.

Association between 6-thioguanine nucleotide levels and preventing production of antibodies to infliximab in patients with inflammatory bowel disease

ObjectiveCombination therapy with infliximab and a thiopurine has been shown to be more effective than monotherapy in patients with inflammatory bowel disease (IBD). The therapeutic efficacy of thiopurines is correlated...

Humoral immune response to SARS-CoV-2 vaccination in patients with inflammatory bowel disease on immunosuppressive medication: association to serum drug levels and disease type

Objectives Immune responses following SARS-CoV-2 vaccination in patients with inflammatory bowel disease (IBD) are not well characterized. The aims of this study were to explore the serological response associated with IBD, and immunosuppressive medications including serum concentrations of biologics and thiopurine metabolites. Materials and methods This prospective, observational study included adult patients with ulcerative colitis (UC) and Crohn’s disease (CD), and healthy controls. Antibodies to the receptor-binding domain of SARS-CoV-2 spike proteins, and serum concentrations of ongoing biologic and immunomodulatory medications were assessed prior to, and 2-5 weeks after the second vaccine dose. Serologic response was defined as anti-Spike antibodies ≥70 AU/ml. Results In 958 IBD patients (380 UC, 578 CD) and 323 healthy controls, the median (Q1; Q3) anti-Spike antibody level (AU/ml) was lower in patients (618 (192; 4370)) compared to controls (3355 (896; 7849)) (p < 0.001). The antibody levels were lower in CD (439 (174; 3304)) compared to UC (1088 (251; 5975)) (p < 0.001). No associations were demonstrated between antibody levels and serum drug concentrations for TNF inhibitor (TNFi), vedolizumab and ustekinumab. Patients receiving TNFi + thiopurines with a subtherapeutic 6-thioguanine nucleotide (6-TGN) level had higher response rate (93%) compared to patients with 6-TGN within the therapeutic range (53%) (p = 0.003). A diagnosis of UC, mRNA-1273 vaccine, and other treatments than TNFi + thiopurines were associated with humoral response. Conclusions Patients with CD had an attenuated humoral response to SARS-COV-2 vaccination as compared to patients with UC. The lack of association between serum levels of biologics and serologic response indicates vaccination regardless of proximity to drug administration.

ITPA Polymorphisms and the Incidence of Toxicities in Children with Acute Lymphoblastic Leukemia.

6-Mercaptopurine (6-MP), a thiopurine agent, is a essential medication for treating pediatric acute lymphoblastic leukemia (ALL). However, its side effects of neutropenia and hepatotoxicity might interrupt treatment, resulting in poor outcomes. Inosine triphosphate pyrophosphatase (ITPA), an enzyme in the thiopurine pathway, may prevent the accumulation of toxic thiopurine metabolites. Studies on ITPA and thiopurine-associated toxicities are scarce. This study retrospectively investigated 1- to 15-year-old children with ALL who received 6-MP during the maintenance phase of treatment between 2000 and 2020. Toxicity during the first year of maintenance therapy and the mean dose of 6-MP were analyzed. The 209 patients had a median age of 4.8 (0.3-14.8) years. Of these, 124 patients (59.3%) had wild-type ITPA, 73 patients (34.9%) had heterozygous ITPA 94C>A (hetITPA), and 12 patients (5.7%) had homozygous ITPA 94C>A (homITPA), with an allele frequency of 0.23. The incidence of neutropenia among ITPA polymorphisms did not significantly differ (P = 0.813). In patients harboring homITPA, transaminitis was more frequent than other polymorphisms but without a significant difference (P = 0.063). The mean dose of 6-MP for patients with homITPA was significantly lower than that for patients with hetITPA or wild-type ITPA (P = 0.016). HomITPA had a higher incidence of transaminitis and required a significantly larger dose reduction of 6-MP than wild-type ITPA. Further study is warranted to elucidate the effects of ITPA polymorphisms on toxicity in patients with ALL treated with 6-MP.

Impact of Duodenal Pathology on Oral Drug Bioavailability and Disease Outcomes in Pediatric Crohn’s Disease

Crohn's disease with upper gastrointestinal tract involvement occurs more often in children than adults and has the potential to interfere with oral drug absorption. We aimed to compare disease outcomes in children receiving oral azathioprine for the treatment of Crohn's disease with (DP) and without (NDP) duodenal pathology at diagnosis. Duodenal villous length, body mass index (BMI), and laboratory studies were compared in DP vs. NDP during the first year post-diagnosis, using parametric/nonparametric tests and regression analysis (SAS v9.4); the data are reported as the median (interquartile range) or the mean ± standard deviation. Thiopurine metabolite concentration (pmol/8 × 108 erythrocytes) 230-400 was considered therapeutic for 6-thioguanine nucleotides (6-TGN), and >5700 was considered hepatotoxic for 6-methylmercaptopurine (6-MMPN). Twenty-six of the fifty-eight children enrolled (29 DP, 29 NDP) started azathioprine for standard medical care, including nine DP and ten NDP who had normal thiopurine methyltransferase activity. Duodenal villous length was significantly shorter in DP vs. NDP (342 ± 153 vs. 460 ± 85 μm; p < 0.001) at diagnosis; age, sex, hemoglobin, and BMI were comparable between groups. A trend toward lower 6-TGN was observed in the DP vs. NDP subset receiving azathioprine (164 (117, 271) vs. 272 (187, 331); p = 0.15). Compared to NDP, DP received significantly higher azathioprine doses (2.5 (2.3, 2.6) vs. 2.2 (2.0, 2.2) mg/kg/day; p = 0.01) and had an increased relative risk of sub-therapeutic 6-TGN. At 9 months post-diagnosis, children with DP had significantly lower hemoglobin (12.5 (11.7, 12.6) vs. 13.1 (12.7, 13.3) g/dL; p = 0.01) and BMI z-scores (-0.29 (-0.93, -0.11) vs. 0.88 (0.53, 0.99); p = 0.02) than children with NDP. For children with Crohn's disease, duodenal pathology, marked by villous blunting, increased the risk of sub-therapeutic 6-TGN levels, despite higher azathioprine dosing during the first year post-diagnosis. Lower hemoglobin and BMI z-scores at 9 months post-diagnosis suggest the impaired absorption/bioavailability of nutrients, as well as oral drugs, in children with duodenal disease.

P496 Antenatal thiopurine exposure in women with IBD is associated with intrahepatic cholestasis of pregnancy

Abstract Background Pregnant women with Inflammatory Bowel Disease (IBD) continue thiopurines to maintain remission. Intrahepatic cholestasis of pregnancy (ICP) is the most common liver disorder of pregnancy, often causing distressing maternal symptoms and with potential implications for pregnancy outcome. Case studies have reported treatment-resistant intrahepatic cholestasis of pregnancy in thiopurine-exposed IBD pregnancies. We aimed to investigate whether thiopurines are associated with an increased risk of ICP and the course of ICP in the setting of thiopurine therapy. Methods Single-centre retrospective cohort study comparing incidence and course of ICP in thiopurine exposed vs non-exposed patients with IBD and compared with age-matched pregnant controls. Results The IBD cohort consisted of 386 pregnancies in 243 patients with IBD, with 386 age-matched controls without IBD. In patients with IBD, ICP was more common amongst thiopurine exposed pregnancies (n=10 9.0% vs n=5 1.8%; OR 5.34 [1.78-16.02]; p=0.021). Only thiopurine exposure was associated with the development of ICP. Age, disease activity, IBD phenotype, previous surgery, mesalazine, biologic, and steroid exposure were not associated. All thiopurine exposed ICP cases (of which 4 received ursodeoxycholic acid) improved symptomatically without thiopurine discontinuation. The incidence of ICP was significantly lower in women without IBD compared with women with IBD (n=5 1.3% vs n=15 4.1%; p=0.039). Thiopurine-exposed IBD patients had higher odds of experiencing ICP compared to non-IBD controls (n= 10 9.0% vs n=5 1.3%; OR 7.55 [2.52-22.57]; p&amp;lt;0.001). IBD patients not exposed to thiopurines had a comparable ICP incidence to controls (n-=5 1.8% vs n=5 1.3%; p=0.75). Severe ICP (total serum bile acids ≥40 µmol/l) occurred in 80% of thiopurine exposed patients, vs 40% in non-exposed (p=0.25), vs 20% in controls (p=0.09). We found no evidence of an unfavourable course of ICP in thiopurine exposed IBD cases. Median week of delivery was not significantly different among ICP cases in all three groups (p=0.39). Conclusion Thiopurine exposure was associated with a significantly increased risk of ICP amongst patients with IBD compared to non-exposed IBD patients and age-matched general population controls. The course of ICP was not significantly different in thiopurine exposed cases. Clinicians involved in the management of patients with IBD should be aware of the potential risk for ICP and assess bile acids when symptoms occur. Prospective studies are required to understand the potential relationship between thiopurine metabolites and ICP.

The role of therapeutic drug monitoring in the management of inflammatory bowel disease

Therapeutic drug monitoring (TDM) is performed when a drug has a narrow therapeutic range (e.g., thiopurines) or there is no linear drug response relationship (biologic agents). TDM is the measurement of drug and/or antibody concentration and individualizing the dose to maintain the drug concentration in the target range. Maintaining serum drug concentration in a therapeutic range is associated with a higher rate of favorable outcomes. Reactive drug monitoring is performed when the disease is active, and proactive drug monitoring is performed when the disease is in remission. Thiopurines still form an important therapeutic armamentarium in the treatment of inflammatory bowel disease (IBD) but have a narrow therapeutic range. Preemptive thiopurine methyl transferase and nucleoside diphosphate-linked moiety X-type motif (NUDT) genotype measurement can reduce the toxicity with appropriate dose initiation and thiopurine metabolite measurement can optimize the dose and reduce the toxicity. Antitumor necrosis factor agents brought about a paradigm shift in the management of moderate-to-severe IBD at the turn of the past century. Despite their efficacy, up to half the patients discontinue the biologic agents at the end of 1 year due to primary nonresponse or secondary loss of response. TDM can optimize the drug level and improve clinical response, reduce antibody formation, reduce cost, and can improve corticosteroid- free clinical remission. Despite postulated benefits and guidelines, the evidence for objective benefits from meta-analysis is mixed. This article reviews the current concepts regarding TDM.

Investigation of the Association Between the ITPA Gene 94C>A Gene Sequence Variant and Liver Transplant Rejection in Iranian Liver Transplant Recipients.

Inosine triphosphate pyrophosphatase is an enzyme encoded by the ITPA gene and functions to prevent the incorporation of thiopurine nucleotides into DNA and RNA. Thiopurine drug metabolites such as azathioprine and 6-mercaptopurine have been included in the lists of inosine triphosphate pyrophosphatase substrates. Inosine triphosphatase gene alterations are other pharmacogenetic sequence variants possibly involved in thiopurine metabolism. This study aimed to evaluate the possible association between ITPA 94C>A gene sequence variant (C-to-A substitution at nucleotide 94) in liver transplant recipients. The genotyping of ITPA 94C>A was evaluated by the polymerase chain reaction- restriction fragment length polymorphism method in 200 liver transplant recipients as well as 100 controls. Data were analyzed with SPSS statistical software. This study showed statistically significant associations between the CA genotype of the ITPA 94C>A sequence variant and liver transplant in the rejection and nonrejection groups. Moreover, the results reported in this study showed no significant differences between sex, age, and blood group in patients with liver transplant (with or without transplant rejection). Our results indicated that there were statistically significant associations of the CA genotype of ITPA 94C>A sequence variant with liver transplant in the rejection and nonrejection groups. Further studies are recommended.

A Validated HPLC-Diode Array Detection Method for Therapeutic Drug Monitoring of Thiopurines in Pediatric Patients: From Bench to Bedside.

Thiopurine drugs are part of the therapeutic armamentarium for pediatric patients suffering from inflammatory bowel disease (IBD) and acute lymphoblastic leukemia (ALL). The therapeutic drug monitoring of these drugs, consisting of measurements of the thiopurine metabolites thioguanine nucleotides (TGN) and methylmercaptopurine nucleotides (MMPN) are used to optimize the effectiveness of treatment and prevent adverse effects. In this context, we developed and validated a high-performance liquid chromatography-diode array detection (HPLC-DAD) method for the simultaneous quantification of thiopurine metabolites according to the most recent International Council for Harmonisation (ICH) guidelines. The calibration curves were built in the clinically relevant range of concentrations for TGN of 300-12,000 nM and for MMPN of 3000-60,000 nM. The limit of detection and the lower limit of quantification were 100 and 300 nM for TGN and 900 and 3000 nM for MMPN, respectively. The percentage of inter-day accuracy and precision (CV%) varied between 85 and 104% and 1.6 and 13.8%. Stability was demonstrated for both of the metabolites for at least 50 days at -20 °C. The proposed HPLC-DAD method showed an appropriate selectivity, specificity, linearity, accuracy, precision and good applicability to samples from patients with IBD and ALL undergoing thiopurine treatment.

Inflammatory Bowel Disease and Pregnancy

Inflammatory Bowel Disease and Pregnancy

Clinical utility of thiopurine metabolite monitoring in inflammatory bowel disease and its impact on healthcare utilization in Singapore.

Background and AimThiopurines are recommended for maintenance of steroid‐free remission (SFR) in inflammatory bowel disease (IBD). Thiopurine metabolite monitoring (MM) is increasingly used in the West but remains novel in Singapore, with limited information on its therapeutic and economic benefits. Hence, this study aims to investigate MM's clinical utility and its impact on healthcare resource utilization in Singaporean IBD patients.MethodsA retrospective observational study was conducted at Singapore General Hospital outpatient IBD Centre. Patients with IBD, baseline MM during 2014–2017, and weight‐based thiopurine doses for ≥4 weeks were followed up for 1 year. Actions were taken to optimize therapy, and metabolite levels before and after the first action were documented. Outcomes assessed included SFR, no therapy escalation or surgery, healthcare resource utilization, and direct healthcare costs.ResultsNinety IBD patients (50 Crohn's disease, 40 ulcerative colitis) were included. Among them, 40% had baseline metabolite levels within therapeutic range, 31.1% sub‐therapeutic, 21.1% supra‐therapeutic, and 7.8% shunters. Repeated MM with subsequent dose optimization helped 67.2% of patients achieve therapeutic levels after 1 year. Overall, 87.8% of patients achieved SFR and 90% had no therapy escalation or surgery. Despite greater outpatient visits and laboratory investigations with MM, the median total healthcare costs at 1 year only increased marginally (S$6407.66 [shunters] vs S$5215.20 [supra‐therapeutic] vs S$4970.80 [sub‐therapeutic] vs S$4370.48 [control (within therapeutic range)], P = 0.592).ConclusionMM guided timely therapy escalation for non‐responders, identification of non‐adherence, and reversal of shunting. Therefore, it is a useful clinical tool to optimize thiopurines without significantly increasing healthcare costs.

THU488 - Examining the role of thiopurine metabolite testing in the management of patients with autoimmune hepatitis

THU488 - Examining the role of thiopurine metabolite testing in the management of patients with autoimmune hepatitis

Therapeutic Monitoring of Thiopurine Metabolites: Validation of an HPLC Method and Preliminary Findings from a Small Cohort of Malaysian Patients with Inflammatory Bowel Disease

Thiopurine therapy of inflammatory bowel disease (IBD) is guided by the relative blood concentrations 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). However, their action is altered by in vivo phosphorylation, and this is not normally measured in clinical studies. Hence, we trialled a novel method for profiling phosphorylated thiopurine metabolites and revisited the association between thiopurine metabolites and IBD treatment outcomes. We first optimised and validated a published high-performance liquid chromatography (HPLC) method for measuring the blood levels of thioguanosine monophosphate (TGMP), thioguanosine diphosphate (TGDP), thioguanosine triphosphate (TGTP), and methylthioinosine monophosphate (MeTIMP). Then, we assembled a small cohort of IBD patients (n=20), who had been treated with azathioprine for at least three months, and obtained blood samples for analysis of the metabolites. The patients received treatments at the Universiti Kebangsaan Malaysia Specialist Centre between March 2018 and April 2019. They were classified as responders (n = 12) or non-responders (n = 6) to azathioprine based on their disease activity scores (CDAI or Mayo score). The HPLC method was precise with intraday and interday variation &lt; 15% for all the tested metabolites, and the relative accuracy ranged from 40.2 to 114.0%. We noted that the responders had higher median 6-TGN but lower median TGTP levels than the non-responders. However, the differences were not statistically significant (Wilcoxon rank-sum tests; 6-TGN, p = 0.925; TGTP, p = 0.189). The higher median 6-TGN level detected in the responders is in keeping with the findings of prior studies, suggesting that HPLC analysis of phosphorylated thiopurine metabolites is both technically feasible and clinically useful.