Evaluation of Stability of Thiopurine Metabolites Using a Validated LC-MS/MS Method

Measurement of thiopurine metabolites is helpful to monitor adverse effects and assess compliance in patients on thiopurine treatment. The purpose of this study was to develop and validate an analytical method for measurement of thiopurine metabolites, thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine nucleotide (6-MMPN), in RBCs. We developed and validated a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the quantification of 6-TGN and 6-MMPN and evaluated the stability of the thiopurine metabolites in RBC and whole blood states without any preprocessing at various storage conditions. The linear range was 0.1–10 µmol/L and 0.5–100 µmol/L for 6-TGN and 6-MMPN, respectively. The mean extraction recovery at the two concentrations was 71.0% and 75.0% for 6-TGN, and 102.2% and 96.4% for 6-MMPN. Thiopurine metabolites in preprocessed RBC samples were stable at 25℃ and 4℃ after storage for 4 hours and at −70℃ for up to 6 months. However, 6-TGN decreased by 30% compared with the initial concentration when stored at −20℃ for 180 days. In whole blood states, 6-TGN decreased by about 20% at four days after storage at 4℃. We validated a reliable LC-MS/MS method and recommend that the patient's whole blood sample be preprocessed as soon as possible.

Analysis of Related Factors of Leukopenia Induced by Azathioprine in the Treatment of Inflammatory Bowel Disease / 中国药房

OBJECTIVE:To explore the clinical related factors of leucopenia induced by azathioprine in the treatment of inflam-matory bowel disease (IBD). METHODS:Clinical information of 114 IBD patients were collected from our hospital during Jan. 2013-Mar. 2015. Steady concentration of AZA metabolite 6-thioguanine(6-TGNs)in red blood cell was determined by HPLC. The correlation of patient’s gender,age,diseases,AZA daily dose and blood concentration of 6-TGNs with leucopenia induced by AZA were investigated. The optimal critical value of leucopenia could be predicted with ROC curves. RESULTS:Among 114 IBD patients,40 patients suffered from leucopenia(35.1%). There was no statistical significance in the proportion of leucopenia among patients with different age,gender,diseases and AZA daily dose(P>0.05). There was statistical significance in the proportion of leucopenia among patients with different concentrations of 6-TGNs(P291.04 pmol/(8 × 108)RBC. CONCLUSIONS:AZA induced leucopenia may be related to the concentration of 6-TGNs in red blood cell of IBD patients,and high concentration of 6-TGNs is risk factors of leucopenia. Clinicians can provide AZA individual treatment for IBD patient to reduce the occurrence of leucopenia according to routine blood test and the concentra-tion of 6-TGNs.

Monitoring Thiopurine Metabolites in Korean Pediatric Patients with Inflammatory Bowel Disease

PURPOSE: This study aimed to assess the role of thiopurine S-methyltransferase (TPMT) and 6-thioguanine nucleotide (6-TGN) as predictors of clinical response and side effects to azathioprine (AZA), and estimate the optimal AZA dose in Korean pediatric inflammatory bowel disease (IBD) patients. MATERIALS AND METHODS: One hundred and nine pediatric IBD patients in whom AZA treatment was required were enrolled. Thiopurine metabolites were monitored since September 2010. Among them, 83 patients who had prescribed AZA for at least 3 months prior to September 2010 were enrolled and followed until October 2011 to evaluate optimal AZA dose, adverse effects and disease activity before and after thiopurine metabolite monitoring. RESULTS: The result of the TPMT genotype was that 102 patients were *1/*1 (wild type), four were *1/*3C, one was *1/*6, one was *1/*16 (heterozygote) and one was *3C/*3C (homozygote). Adverse effects happened in 31 patients pre-metabolite monitoring and in only nine patients post-metabolite monitoring. AZA dose was 1.4+/-0.31 mg/kg/day before monitoring and 1.1+/-0.46 mg/kg/day after monitoring (p<0.001). However, there were no statistical differences in disease activity during metabolite monitoring period (p=0.34). Adverse effects noticeably decreased although reduction of the AZA dose since monitoring. CONCLUSION: TPMT genotype and thiopurine metabolite monitoring could be helpful to examine TPMT genotypes before administering AZA and to measure 6-TGN concentrations during prescribing AZA in IBD patients.

6-Thioguanine-Induced Hepatic Sinusoidal Obstruction Syndrome in a Leukemic Child with TPMT Heterozygote / 임상소아혈액종양

Hepatic sinusoidal obstruction syndrome (hSOS) can be developed as a common complication after hematopoietic stem cell transplantation (HSCT), and rarely after 6-thioguanine-based chemotherapy without HSCT. A four-year-old boy with heterozygotic polymorphism for thiopurine methyltransferase (TPMT) developed hSOS after he received chemotherapy with cytarabine, cyclophosphamide, intrathecal methotrexate and 6-thioguanine (6-TG) as reconsolidation chemotherapy of acute lymphoblastic leukemia (ALL). He was treated with defibrotide, N-acetylcysteine, urusodeoxycholic acid, glutathione, and supportive care. He recovered completely in nine days without long-term complication, and completed chemotherapy with 6-mercaptopurine without severe complication. We report a case of hSOS developed in an ALL patient with TPMT heterozygote after 6-TG based reconsolidation chemotherapy.

6-Thioguanine-Induced Hepatic Sinusoidal Obstruction Syndrome in a Leukemic Child with TPMT Heterozygote / 임상소아혈액종양

Hepatic sinusoidal obstruction syndrome (hSOS) can be developed as a common complication after hematopoietic stem cell transplantation (HSCT), and rarely after 6-thioguanine-based chemotherapy without HSCT. A four-year-old boy with heterozygotic polymorphism for thiopurine methyltransferase (TPMT) developed hSOS after he received chemotherapy with cytarabine, cyclophosphamide, intrathecal methotrexate and 6-thioguanine (6-TG) as reconsolidation chemotherapy of acute lymphoblastic leukemia (ALL). He was treated with defibrotide, N-acetylcysteine, urusodeoxycholic acid, glutathione, and supportive care. He recovered completely in nine days without long-term complication, and completed chemotherapy with 6-mercaptopurine without severe complication. We report a case of hSOS developed in an ALL patient with TPMT heterozygote after 6-TG based reconsolidation chemotherapy.

Monitoração terapêutica da azatioprina: uma revisão: [revisão] / Therapeutic drug monitoring of azathioprine: a review: [review]

Os nucleotídeos de tioguanina (6-TGN), metabólitos ativos da azatioprina (AZA) e da 6-mercaptopurina (6-MP), atuam como antagonistas das purinas, inibindo as sínteses de DNA, RNA e a protéica, e induzindo à citotoxicidade/imunossupressão. A enzima geneticamente determinada, tiopurina metiltransferase (TPMT), está envolvida no metabolismo desses agentes e, hipoteticamente, determina a resposta clínica às tiopurinas. A baixa atividade dessa enzima diminui a metilação das tiopurinas, resultando em potencial sobredose, enquanto altos níveis de TPMT levam à superprodução do metabólito tóxico 6-metilmercaptopurina (6-MMP) e à não-efetividade terapêutica da AZA e da 6-MP. Várias mutações no gene da TPMT têm sido identificadas e correlacionadas com fenótipos de baixa atividade. Neste artigo, também se discute a monitoração terapêutica desses fármacos por meio da medida dos níveis de 6-TGN intra-eritrocitários, os quais se correlacionam com imunossupressão e mielotoxicidade. Já a 6-MMP está diretamente relacionada com hepatotoxicidade. Esses ensaios estão associados ao uso de doses adequadas dessa droga, resultando num melhor controle da doença e menor uso de corticosteróides.

Thioguanine nucleotides (6-TGN), active metabolites of azathioprine (AZA) and 6-mercaptopurine (6-MP), act as purine antagonists, inhibiting DNA, RNA, and protein synthesis and inducing cytotoxicity and immunosuppression. The genetically determined thiopurine methyltransferase enzyme (TPMT) is involved in the metabolism of these agents and, theoretically, determines the clinical response to thiopurines. Low activity of this enzyme decreases the methylation of thiopurines, what results in potential overdosing, whereas high TPMT status leads to overproduction of toxic metabolite 6-methilmercaptopurine (6-MMP) and ineffectiveness of AZA and 6-MP. Several mutations in the TPMT gene have been identified and correlated with low activity phenotypes. In this study, we also discuss the therapeutic monitoring of these drugs by means of red blood cell 6-TGN levels, which correlate with immunosuppression and mielotoxicity. 6-MMP is directly connected with hepatotoxicity. These metabolites assays are associated with the use of appropriate doses of this drug, what results in a better control of the disease and a decreased use of corticosteroids.

Pancreatitis Induced by 6-mercaptopurine and 6-thioguanine in Childhood Acute Lymphoblastic Leukemia / 대한혈액학회지

Although pancreatitis is known as a common complication during the treatment of acute lymphoblastic leukemia, acute pancreatitis that's induced by 6-mercaptopurine or 6-thioguanine is very uncommon. We experienced the case of an 11-year-old boy with consecutive acute pancreatitis, and this was induced by 6-mercaptopurine and 6-thioguanine during maintenance chemotherapy of childhood acute lymphoblastic leukemia. We report here on this along with a review of the pertinent literature.

Determination of Thioguanosine in Human Red Blood Cells by HPLC / 中国药房

OBJECTIVE: To establish HPLC method for the determination of thioguanosine in human red blood cells. METHODS: The determination was performed on Hypersil ODS colum. The mobile phase consisted of methanol-water (10∶90) with detection wavelength at 342nm. 6-Thioguanine(6-TG)was generated from thioguanosine through heating and hydrolyzing. 6-TG was extracted into 0.1mol/L hydrochloric acid for sample injection assay with external reference method for the quantification. RESULTS: The linear range of 6-TG was 30～1 200pmol/8?108RBCs. Its optimum hydrolyzing time was 1 hour and optimal extraction pH value ranged from 11 to 12. The content of phenyl mercury acetate in the extraction solution was 1.3mmol/L. CONCLUSION: Under optimized conditions, HPLC method for the determination of thioguanosine is fast, accurate and sensitive.