mercaptopurine and alpha-2--deoxythioguanosine

Adverse reactions and non-response are common in patients treated with thiopurine drugs. Current monitoring of drug metabolite levels for guiding treatment are limited to analysis of thioguanine nucleotides (TGNs) in erythrocytes after chemical derivatisation. Erythrocytes are not the target tissue and TGN levels show poor correlations with clinical response. We have developed a sensitive assay to quantify deoxythioguanosine (dTG) without derivatisation in the DNA of nucleated blood cells. Using liquid chromatography and detection by tandem mass spectrometry, an intra- and inter-assay variability below 7.8% and 17.0% respectively were achieved. The assay had a detection limit of 0.0003125ng (1.1 femtomoles) dTG and was quantified in DNA samples relative to endogenous deoxyadenosine (dA) in a small group of 20 patients with inflammatory bowel disease, all of whom had been established on azathioprine (AZA) therapy for more than 25 weeks. These patients had dTG levels of 20-1360mol dTG/10(6)mol dA; three patients who had not started therapy had no detectable dTG. This method, comparable to previous methods in sensitivity, enables the direct detection of a cytotoxic thiopurine metabolite without derivatisation in an easily obtainable, stable sample and will facilitate a better understanding of the mechanisms of action of these inexpensive yet effective drugs.

Topics: Blood Cells; Chromatography, Liquid; Deoxyguanosine; DNA; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Mercaptopurine; Tandem Mass Spectrometry; Thionucleosides

Although the thiopurine drugs 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are well established agents for the treatment of leukemia, controversies remain regarding their main mode of action. Previous evidence has suggested that although 6-TG exerts a cytotoxic effect through incorporation of 6-thioguanine nucleotides into newly synthesized DNA (DNA-TGN), an important component of the mode of action of 6-MP is inhibition of purine de novo synthesis (PDNS) through the production of S-methyl-thioinosine 5'-monophosphate (MeTIMP), not formed in cells exposed to 6-TG. We have shown that thiopurine methyltransferase (TPMT) modulates this effect. By transfection of the human TPMT gene using an inducible system to produce a 3.8-fold increase in TPMT activity in the ecdysone receptor 293 embryonic kidney cell line, we demonstrated a 4.4-fold increase in sensitivity to 6-MP. This was associated with a rise in intracellular levels of MeTIMP but a decrease in levels of DNA-TGN. In contrast, induction of TPMT produced a 1.6-fold decrease in sensitivity to 6-TG, a decrease in levels of DNA-TGN, and an increase in levels of methylated thioguanosine monophosphate. Exposure of cells to equitoxic doses of drug showed similar incorporation of DNA-TGN for 6-TG but for 6-MP significantly reduced DNA-TGN in TPMT-induced compared with uninduced cells. For equitoxic doses of 6-MP, equivalent levels of MeTIMP correlated with equivalent amounts of PDNS. These observations suggest that intracellular TGN levels do not give an accurate reflection of cytotoxic potential in patients treated with 6-MP, because different levels of DNA-TGN may be associated with equitoxic effects.

Topics: Cell Line; Cells, Cultured; Deoxyguanosine; DNA; Gene Expression; Humans; Mercaptopurine; Methyltransferases; Nucleic Acid Synthesis Inhibitors; Thioguanine; Thionucleosides; Transfection