Thermodynamic analysis of the reaction of phosphoramide mustard with protector thiols

The systemic use of thiol-containing uroepithelial protecting agents, e.g., N-acetylcysteine (NAC) or mesna, in conjunction with the alkylating agent cyclophosphamide is predicated on the assumption that the toxic metabolic by-products will be consumed by thiol without diminishing the cytotoxicity of the active alkylating intermediate, phosphoramide mustard. Studies in murine tumor systems have been with either a single dose or two equally divided doses of thiol, administered within 30 min of the addition of cyclophosphamide, without an observed adverse effect on antitumor activity; however, the relatively short serum half-life of thiol relative to alkylating agent in humans weakens the clinical relevance of these results. This study presents a thermodynamic model for the chemical reaction of phosphoramide mustard with either NAC or mesna. The gas phase thermodynamic parameters for these reactions, enthalpy (H) and entropy (S), were calculated using the semiempirical quantum mechanical method AM1 and were used to predict the free energy (delta G) for these processes. For the reaction of phosphoramide mustard with NAC or mesna, delta G = +3.82 and 2.29 kcal/mol, respectively. In the absence of enzyme catalysis, these results suggest that such reactions are not favored. In order to assess the validity of this gas phase thermodynamic model, the cellular cytotoxicity of phosphoramide mustard in the presence or absence of either NAC or mesna was studied using CCRF-CEM cells in culture. In these experiments the 50% effective dose of phosphoramide mustard was 1.7 micrograms/ml; this result was unchanged in the presence of 10 micrograms/ml concentration of either thiol. This study supports the conclusion that phosphoramide mustard and protector thiols are compatible.