In this application we propose to continue our studies of the pharmacology of cyclophosphamide and other clinically utilized alkylating agents. During the period of funding requested we shall focus on three principal goals. The first is to continue and bring toward a state of completion and clinical relevance our studies of the mechanism through which elevated gluthathione (GSH) and glutathione S-transferases produce cellular resistance to bischlorethylamine antiumor agents. These studies initially defined and subsequently have supported the hypothesis that GSH, an avid nucleophile, competes with DNA for the reactive aziridinyl moiety of these agents. This reaction is catalysed by human GSTs alpha and mu. We initially identified phosphoramide mustard (PM) as a product of the microsomal metabolism of cyclophosphamide, and have provided evidence it is principal alkylating agent produced by the metabolism of cyclophosphamide. Recent NMR studies on this grant have demonstrated that the formation of the aziridinium of PM at physiologic pH leads to P-N bond scission of PM, releasing chlorethylaziridine (CEZ). This raises the possibility that CEZ plays a significant role in DNA alkylation and crosslinking. We will study the roles of PM and CEZ derived from PM utilizing doubly labeled PM (3H and 32P) to ascertain the relative contributions to DNA alkylation and crosslinking from PM and CEZ respectively. The microsomal activation of cyclophosphamide and ifosfamide to the active species is well established, and recently the P450's associated with the activation of each compound have been described. However, little is know about the mechanism of this activation. With methodology previously developed under this grant, it is possible for us to facilely and quantitatively study the activation and dechlorethylation of the oxazaphosphorines, and through kinetic isotope studies (deuterium) to examine the mechanism of these reactions. In one set of experiments we propose to examine the relative properties of cyclophosphamide and ifofamide by altering he metabolism of ifosfamide by kinetic switching of the dechlorethylation to activation to 4-hydroxyifosfamide.
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