In this application we propose to continue our studies of the chemistry and pharmacology of cyclophosphamide and ifosfamide. The studies proposed focus on two critical aspects of the pharmacology of these agents, the definitive characterization of the DNA interstrand crosslinks produced by phosphoramide mustard and isophosphoramide mustard and the elucidation of the chemical and enzymatic determinants of the two P450 oxidations which determine the activation and inactivation of cyclophosphamide and ifosfamide. Our studies have demonstrated significant differences in the chemistry of phosphoramide mustard and isophophoramide mustard and in the interstrand crosslinks which will be produced by these molecules and their decomposition products. We have also recently discovered that acrolein produced from 4-hydroxycyclophosphamide gives O[6]-guanylate alkylation, probable crosslinking of DNA, and selects for resistance to BCNU by increased O[6]-alkyltransferase. We will explore the mechanisms of these reactions and determine if chloroacetaldehyde will produce similar effects. The repair of the demonstrated DNA crosslinks will be studied in our separately funded collaboration with Dr. Henry Friedman. We will also continue our studies of the P450 activation and dechlorethylation of cyclophosphamide and ifosfamide - determining the relative ratio of these reactions for different P450 enzymes, measuring deuterium isotope effects, defining the mechanism of the reactions, and examining the pharmacokinetic and antitumor properties of ifosfamide after altering the metabolism of ifosfamide by isotope and alkyl substitution induced kinetic switching of dechlorethylation to activation. We believe the results of these studies will have important implications for improving the clinical effectiveness of these important and unique antitumor agents.
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