Glutathione Conjugation of the Cytostatic Drug Ifosfamide and the Role of Human Glutathione S-Transferases

Development of drug resistance against alkylating cytostatic drugs has been associated with higher intracellular concentrations of glutathione (GSH) and increased expression of glutathione S-transferase (GST) enzymes. Therefore, enhanced detoxification by the glutathione/glutathione S-transferase pathway has been proposed as a major factor in the development of drug resistance toward alkylating agents. In this paper we describe 31P NMR and HPLC studies on the spontaneous and glutathione S-transferase catalyzed formation of glutathionyl conjugates of two metabolites of ifosfamide, i.e., 4-hydroxyifosfamide and ifosfamide mustard. At 25 degrees C activated ifosfamide (= 4-hydroxyifosfamide + aldoifosfamide) disappeared faster in the presence of a 10-fold excess of GSH (t1/2 = 107 min) compared to incubations without GSH (t1/2 = 266 min). No evidence for the formation of 4-glutathionyl ifosfamide was found. The ultimate alkylating species of ifosfamide is ifosfamide mustard (IM). In the absence of glutathione, the rate constant for the disappearance of the ifosfamide mustard signal at 25 degrees C (pH 7) was 1.98 x 10(-3) min-1 (t1/2 = 350 min). In the presence of a 10-fold molar excess of glutathione, this rate constant was 1.95 x 10(-3) min-1 (t1/2 = 355 min), indicating that the spontaneous formation of an aziridinium ion is the rate-limiting event in the reaction with glutathione. The aziridinium ion formed from IM can deprotonate upon formation, leading to the formation of a (noncharged) aziridine species. This intermediate (N-(2-chloroethyl)-N'-phosphoric acid diamide) was characterized by 31P, 1H, and 13C NMR spectra. When 2 mM ifosfamide mustard was incubated with 1 mM GSH in the presence of 40 microM GST P1-1, the formation of monoglutathionyl ifosfamide mustard was 2.3-fold increased above the spontaneous level. The other major human isoenzymes tested (A1-1, A2-2, and M1a-1a) did not influence the formation of monoglutathionyl ifosfamide mustard. The results of these studies demonstrate that increased levels of GST P1-1 can contribute to an enhanced detoxification of ifosfamide.