thioguanine-anhydrous and Purine-Pyrimidine-Metabolism--Inborn-Errors

Azathioprine and mercaptopurine are widely used in the treatment of inflammatory bowel disease. However, its use is limited by adverse drug event related to the relatively narrow therapeutic index of the active metabolites. Several patients discontinue treatment because of intolerable adverse events or toxicity such as leucopenia and hepatotoxicity. High 6-thioguanine nucleotides and 6-methylmercaptopurine ribonucleotides levels are associated with toxicity. Variations in the thiopurine S-methyltransferase (TPMT) gene can lead to diminished TPMT enzyme activity and to an increased incidence of myelotoxicity due to high 6-methylmercaptopurine ribonucleotides levels after treatment with azathioprine and mercaptopurine. Unlike azathioprine and mercaptopurine, thioguanine is more directly metabolized to the active metabolites without formation of the toxic 6-methylmercaptopurine ribonucleotides. Taking this into account, it seems likely that thioguanine is less associated with myelotoxicity due to TPMT deficiency. However, we report the case of a Crohn's disease patient with life-threatening complications on 6TG treatment due to TPMT deficiency. Our patient developed a severe pancytopenia on thioguanine therapy, with 6-thioguanine nucleotides levels more than 10 times higher than the upper limit of the therapeutic window and was found to be a TPMT poor metabolizer (TPMT *3A/*3A). This case strongly illustrates that knowledge of TPMT enzyme activity is very important in the use of all thiopurines, including thioguanine. In conclusion, clinicians should be aware of the impact of TPMT deficiency on the metabolism of thioguanine and should consider performing preemptive TPMT genotyping in combination with frequent blood test monitoring when using thiopurines in general.

Topics: Aspergillosis; Drug Hypersensitivity; Female; Humans; Inflammatory Bowel Diseases; Middle Aged; Pancytopenia; Purine-Pyrimidine Metabolism, Inborn Errors; Severity of Illness Index; Thioguanine

The activity of the enzyme thiopurine methyltransferase (TPMT) is regulated by a common genetic polymorphism. One in 300 individuals lack enzyme activity and 11% are heterozygous for a variant low activity allele and have an intermediate activity. The thiopurine drugs azathioprine, mercaptopurine and thioguanine are substrates for TPMT; these drugs exhibit well documented myelosuppressive effects on haematopoietic cells and have a track record of idiosyncratic drug reactions. The development of severe bone marrow toxicity, in patients taking standard doses of thiopurine drugs, is associated with TPMT deficiency whilst the TPMT heterozygote is at an increased risk of developing myelosuppression. Factors influencing TPMT enzyme activity, as measured in the surrogate red blood cell, are discussed in this review to enable an appreciation of why concordance between TPMT genotype and phenotype is not 100%. This is particularly important for lower/intermediate TPMT activities to avoid misclassification of TPMT status. TPMT testing is now widely available in routine service laboratories. The British National Formulary suggests TPMT testing before starting thiopurine drugs. Dermatologists were quick to adopt routine TPMT testing whilst gastroenterologists do not specifically recommend TPMT screening. TPMT testing is mandatory prior to the use of mercaptopurine in childhood leukaemia. Thiopurine drug dose and other treatment related influences on cell counts explain some of the differing recommendations between clinical specialities. TPMT testing is cost-effective and the major role is in the identification of the TPMT deficient individual prior to the start of thiopurine drugs.

Topics: Antimetabolites, Antineoplastic; Azathioprine; Cost-Benefit Analysis; Drug Hypersensitivity; Drug Labeling; Genetic Testing; Genotype; Humans; Mercaptopurine; Methyltransferases; Phenotype; Purine-Pyrimidine Metabolism, Inborn Errors; Thioguanine

Thiopurine compounds are commonly used in the treatment of childhood acute lymphoblastic leukemia, and as immunosuppressants following organ transplantation or for treatment of various autoimmune disorders. Thiopurine S-methyltransferase (TPMT) is required for detoxification, through S-methylation, of 6-thioguanine nucleotides (TGNs), a byproduct of thiopurine metabolism. Single nucleotide polymorphisms (SNPs) in the TPMT gene have been shown to affect its function, with some variants associated with serious clinical manifestations including severe to fatal myelosuppression and organ transplant rejection following treatment with standard thiopurine doses. In this study, we describe a TaqMan real time PCR allelic discrimination assay requiring minimal DNA input for TPMT genotyping.. We designed controls for the homozygous wild type and allelic variants of TPMT*2, *3B, and *3C. Genomic DNA was extracted from an additional 412 human blood samples. The samples were tested for the TPMT*2, *3B, *3C, and *3A polymorphisms by TaqMan genotyping assays using the AB 7500 FAST Real-Time PCR instrument. Allelic discrimination plots were used to identify each mutation.. The TaqMan assay correctly genotyped all custom control DNA samples. Of the 412 tested samples, our assay identified 375 samples as wild-type *1/*1 (91.02%), 3 as *1/*2 (0.73%), 1 as *1/*3B (0.24%), 3 as *1/*3C (0.73%), 27 presumed to be *1/*3A (6.55%), and 3 as *3B/*3A (0.73%).. The clinical implications of TPMT genotyping, along with the simplicity and specificity of the TaqMan genotyping assays make this test highly suitable for use in a clinical laboratory.

Topics: Drug Hypersensitivity; Genotype; Genotyping Techniques; Humans; Methyltransferases; Mutation; Polymorphism, Single Nucleotide; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Purine-Pyrimidine Metabolism, Inborn Errors; Thioguanine

Thiopurine S-methyltransferase (TPMT) is an excellent example of an enzyme whose pharmacogenetic polymorphisms affect efficacy and toxicity of a drug. The association between TPMT activity and thiopurine-related myelosuppression is well recognized. To study the significance of TPMT deficiency in thiopurine metabolism and immunosuppressive activity in vitro, we established RNA interference-based TPMT knockdown (kd) in a Jurkat cell line.. In Jurkat TPMT kd cells, TPMT expression was reduced to 73% at the RNA level and 83% at the protein level. TPMT kd cells were more sensitive to 6-mercaptopurine (6-MP) (10 μmol/L) and 6-thioguanine (6-TG) (8 μmol/L) than wild-type (wt) cells, (32% versus 20%) and (18% versus 9%), respectively. Both Jurkat wt and kd cells were more sensitive to 6-TG-induced apoptosis than to 6-MP. 6-TG activity was also more affected by TPMT levels than was 6-MP as reflected by IC60, concentrations that is, 6-MP [4.6 μmol/L (wt) and 4.7 μmol/L (kd)], 6-TG [2.7 μmol/L (wt) and 0.8 μmol/L (kd)]. IC60 concentrations induced significant apoptosis in both Jurkat wt and kd cells (257%, versus 314%) with 6-MP and (323% versus 306%) with 6-TG, respectively. At IC60 (6-MP) 6-thioguanine nucleotides (6-TGN) accumulation in cells was 518 versus 447 pmol/million cells in wt and kd cells, respectively. On the other hand 6-TGN accumulation at IC60 (6-TG) was 477 versus 570 pmol/million cells in wt and kd cells, respectively. 6-Methylated mercaptopurine (6-MeMP) concentrations were more affected than 6-TGN by TPMT kd (194 versus 10 pmol/million cells) in wt and kd cells, respectively.. We conclude that TPMT kd cells are an appropriate in vitro model to investigate the significance of TPMT deficiency with thiopurine therapy and could be helpful in understanding possible clinical consequences of TPMT polymorphism.

Topics: Apoptosis; Cell Line, Tumor; Drug Hypersensitivity; Gene Knockdown Techniques; Guanine Nucleotides; Humans; Immune Tolerance; Jurkat Cells; Mercaptopurine; Methyltransferases; Polymorphism, Genetic; Purine-Pyrimidine Metabolism, Inborn Errors; T-Lymphocytes; Thioguanine; Thionucleotides