Release of a votile factor from solutions of oxazaphosphorines which damage normal and malignant cells

Oxazaphosphorines such as cyclophosphamide and ifosfamide must undergo hydroxylation at the carbon-4-atom of the oxazaphosphorine ring by liver enzymes before they spontaneously degrade to form the alkylating moiety. Some new oxazaphosphorines with different substitutions at the carbon-4-atom, however, undergo spontaneous hydrolysis when dissolved in water. Biological properties of these "activated" oxazaphosphophorines, which thus do not require biotransformation to liberate the alkylating moiety, have been studied extensively. We now demonstrate that the "activated" oxazaphosphorines, 4-(2-sulfonatoethylthio)-cyclophosphamide, 4-hydroperoxy-cyclophosphamide and 4-hydroperoxy-ifosfamide, present as aqueous solutions in wells of conventional microtest tissue culture plates release a votile factor which may penetrate into the media of neighboring cultures and strongly reduce their growth and viability. This phenomenon, which was temperature dependent, could be inhibited by cells or serum present in the drug solutions. Cyclophosphamide, ifosfamide or nitrogen mustard dissolved in water did not release detectable amounts of toxic factor. Acrolein, which is the only known votile metabolite of oxazaphosphorines, was also found to reduce cell growth in neighboring cultures, and its evaporation could be inhibited by cells and serum. It is concluded that the toxic votile factor, most likely acrolein, which is spontaneously released from certain oxazaphosphorines, may strongly affect cells in in vitro culture systems.