A comparison of the effects of ifosfamide vs. mafosfamide treatment on intracellular glutathione levels and immunological functions of immunocompetent lymphocyte subsets

This study compares the effects of ifosfamide treatment with those of mafosfamide treatment with respect to important immunological functions and intracellular glutathione (GSH) levels of immunocompetent lymphocyte subsets such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The proliferative and cytotoxic capacity of human peripheral blood lymphocyte (PBL) subsets was measured by a standard [³H] thymidine uptake assay and a [⁵¹Cr] release assay; the intracellular glutathione levels were determined by using an established HPLC method described by Reed. Following incubation of human PBLs with the activated forms of ifosfamide (4-OH-IF) and mafosfamide (4-OH-CP), the proliferative capacity of recombinant interleukin 2 (rIL-2)-stimulated PBLs was reduced by both drugs in a dose-dependent manner. However, a threefold higher concentration of ifosfamide compared with mafosfamide is needed to achieve a comparable inhibition rate in the proliferative capacity of these lymphocytes. Separation of PBLs into a CD3⁺ CTL and a CD3^- NK subpopulation revealed that proliferative activity was reduced in both subpopulations in a dose-dependent manner by ifosfamide and mafosfamide. However, growth inhibition was much more pronounced in the CD3⁺ CTL compared with the CD3^- NK cells. The intracellular GSH level in CTL, and to a lower extent in NK cells, was reduced more substantially following an ifosfamide treatment compared with a mafosfamide treatment. With respect to our previous finding that an ifosfamide-induced reduction of intracellular GSH levels correlates with decreased cytotoxic function, in this study we compared the effects of ifosfamide treatment with those of mafosfamide treatment on the cytolytic activity of lymphocyte subpopulations. The cytotoxic activity of CD3⁺ CTL against allogeneic target cells (B-lymphoblastoid cells) was significantly reduced aher preincubation with either activated ifosfamide or mafosfamide. In contrast, the lysis of NK-sensitive tumor target cells (K562), mediated by CD3^- NK cells is only affected if the effector cells are exposed to high concentrations (100 pM) of activated ifosfamide. The cytotoxic activity of NK cells pretreated with high concentrations of activated mafosfamide (33 μM) had no significant inhibitory effect on the cytotoxic function. Taken together, our findings were as follows: 1) A threefold higher concentration of activated ifosfamide compared with mafosfamide results in a comparable inhibition of the proliferative activity, in vitro. 2) The intracellular GSH levels of unseparated rIL-2 activated lymphocytes were reduced by ifosfamide and mafosfamide at concentrations above 16 μM. 3) Separated NK cells compared with CTLs are more resistant to treatment with ifosfamide with respect to their intracellular GSH levels. This phenomenon is even more pronounced after treatment with mafosfamide. 4) The reduction in intracellular GSH levels after treatment with ifosfamide and mafosfamide could be correlated with a reduction in the cytotoxic activity.