Analytic aspects of monitoring therapy with thiopurine medications

The thiopurine medications 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), and azathioprine are used in treatment of childhood acute lymphoblastic leukemia, autoimmune diseases, and, in the case of azathioprine, in solid organ transplantation. They are converted in vivo to the active 6-thioguanine nucleotides (6-TGN). One person in 300 in white populations has low or undetectable TPMT activity and is at risk for accumulating 6-TGN with the consequence of severe, life-threatening myelosuppression. A rational therapeutic strategy for thiopurine drug use is to first determine TPMT phenotype/genotype and then to adjust the dosage on an individual basis. Determination of erythrocyte 6-TGN levels can further help to optimize therapy. TPMT activity (phenotype) is determined in erythrocytes using radiochemical or HPLC procedures. Recent HPLC procedures show good agreement with the original radiochemical method, while offering simplified sample pretreatment and improved precision. To date, 12 mutant alleles responsible for TPMT deficiency have been published. Restriction fragment length polymorphism PCR and allele-specific PCR have been used for detection of TPMT mutations. Genotyping methods that allow a higher throughput include real-time PCR (LightCycler) and denaturing HPLC. Numerous HPLC methods have been reported for quantification of 6-TGN. The majority involve acid hydrolysis to 6-TG at high temperature. There are substantial differences in the hydrolysis step, extraction procedure, chromatographic conditions and method of detection. Erythrocyte 6-TGN concentrations can vary up to 2.6-fold depending on the HPLC method. The method that has found the greatest application in clinical studies is that of Lennard. This has served as the basis for the establishment of treatment-related therapeutic ranges for thiopurine therapy. These ranges will not necessarily be applicable when other methodology is used. There is an urgent need to harmonize the analytic procedures for 6-TGN.