In our work we have contributed to the definition of the nature and properties of cyclophosphamide metabolites and have devised quantitative techniques to measure these compounds. We have examined the cellular pharmacology of the active metabolites in murine L1210 cells, defined two mechanisms of cellular resistance in L1210 cells and helped establish the probable basis for bone marrow stem cell resistance to cyclophosphamide. We propose to continue the study of cyclophosphamide metabolites, to characterize their cellular pharmacology and to make comparative studies in human and murine tumor cells. The value of these studies lies in the definition of optimum dose scheduling of cyclophosphamide and the examination of potential modulation of cyclophosphamide therapy by other agents. We also plan to extend our work on resistance to finding and characterizing other mechanisms of resistance in human tumor. This work is essential to devising approaches to circumvent resistance and has the potential for establishing rapid biochemical techniques for detecting resistance in human tissue samples. We plan to explore the nature of DNA alterations produced by alkylating metabolites of cyclophosphamide and the biological consequence of these alterations. For these studies we will use DNA sequencing and nuclease digestion. Finally, we plan to develop new analytical techniques to study the clinical pharmacology of activated metabolites using methods which can be readily adapted to other laboratories so that pharmacologic measurements will be readily available for use in clinical studies. While the primary focus of this proposal is the study of cyclophosphamide, many of the techniques will be applicable to the study of other alkylating agents, and comparative studies will be performed as they seem appropriate.
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