In Vitro Study of the Enzymatic and Nonenzymatic Conjugation of Treosulfan with Glutathione

Background and Objectives: Treosulfan (dihydroxybusulfan), licensed for the treatment of ovarian carcinoma, is investigated in clinical trials as a myeloablative agent for conditioning prior to hematopoietic stem cell transplantation. Clinical experience shows that treosulfan exhibits lower organ toxicity than busulfan, including hepatotoxicity. Elimination of busulfan primarily via enzymatic conjugation with glutathione (GSH) in the liver is considered to be the main cause of the drug’s hepatotoxicity and interpatient clearance variability. It is believed that treosulfan undergoes no hepatic metabolism but empirical evidence is lacking. The aim of this kinetic study was to verify if treosulfan is capable of conjugating with GSH. Methods: Treosulfan (200 µM) was incubated at pH 7.2 and 37 °C with 5 mM GSH in the presence or absence of human liver cytosol, the main store of glutathione S-transferase in the body. Concentrations of treosulfan were determined using liquid chromatography–tandem mass spectrometry and then subjected to kinetic analysis. Results: The decay of treosulfan in the solution followed a one-exponential model in the presence of either GSH or liver cytosol and GSH. The first-order reaction rate constants (0.25 h^-1) did not differ statistically from those found for treosulfan conversion in pH 7.2 buffer only. Conclusion: Treosulfan does not undergo either spontaneous or enzymatic conjugation with GSH at a noticeable rate. The result indicates that the clearance of treosulfan is independent of glutathione S-transferase activity, GSH stores, and co-administration of drugs utilizing the GSH metabolic pathway.