Thiopurines are prodrugs that are used in the treatment of numerus autoimmune diseases and acute lymphoblastic leukemia. For maintenance therapy of acute lymphoblastic leukemia, a combination of thiopurine 6-mercaptopurine and methotrexate is used. Those drugs are cytotoxic mostly to rapidly proliferating cells like bone marrow cells. Because of the extensive intracellular thiopurine metabolism and several factors that influence the activity of involved enzymes, patients respond differently to the therapy. To provide appropriate therapeutic effects and minimaze the side effects of thiopurines during the treatment, we monitor different parameters with the purpose of modifying the drug doses. With the use of high performance liquid chromatography, we can measure the concentrations of two main 6 mercaptopurine metabolites. 6 thioguanine nucleotides are active metabolites that have therapeutic effects and are capable of incorporation into DNA during replication. Too low concentrations of active metabolites are associated with the lack of therapeutic effects and a higher relapse hazard. Too high values are associated with a higher risk of developing life threatening myelosuppression. 6-mercaptopurine is an inactive metabolite, the exccessive values of which are associated with a higher risk of hepatotoxicity. We evaluated the method for measuring thiopurine metabolites on the new HPLC system in accordance with the recommendations. We measured calibration curves and reference samples and evaluated linearity, accuracy, and precision. We determined the lowest and the highest limit of quantification for both metabolites. Then, we measured the concentrations of metabolites in the samples of patients receiving maintenance therapy with 6 mercaptopurine and methotrexate. We observed possible connections within different parameters like dose of 6 mercaptopurine and methotrexate, leukocyte and neutrophil count and values of catalytic activity of liver enzymes. The results showed no correlation between 6 thioguanine nucleotides and 6 mercaptopurine doze. However, the doze did correlate with 6 methylmercaptopurine. We also noticed that 6 thioguanine nucleotides were negatively correlated with leukocyte and neutrophil count, while 6 methylmercaptopurine was positively correlated with values of catalytic activity of liver enzymes AST and ALT. Monitoring of thiopurines metabolites gives an additional insight into metabolism and patients' conditions during maintenance therapy, aiding more precise doze adjustments and holistic treatment of the patient.
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