The goal of this application is to establish pharmacologic and biochemical bases for understanding clinical response and for guiding protocol design and evaluation. This application extends ongoing evaluations of hypotheses concerning drug actions and interactions in human leukemia cells during therapy to similar investigations in a solid tumor, ovarian carcinoma. To identify correlations between the metabolism and actions of gemcitabine nucleotides in tumor cells with clinical response in a phase II trial, the metabolism and pharmacodynamics of gemcitabine in human ovarian carcinoma cells during therapy will be investigated, and these parameters will be compared among patients. Although aspects of this project focus on nucleoside analogs with recently established (fludarabine) or emerging (gemcitabine) antitumor activity, the first clinical and pharmacodynamic evaluations of the next generation of this class of drugs (clofarabine and arabinosylguanine) is also proposed. These investigations will determine the pharmacokinetics of the active nucleotides of these drugs in leukemia cells during therapy and the pharmacodynamic actions relative to inhibition of DNA synthesis and ribonucleotide reductase. Correlations will be sought with clinical response during phase I and phase II single-drug trials. New targets for nucleotide analog incorporation into DNA are created when quiescent leukemia populations are induced to undergo DNA repair. This strategy will be pursued in the chronic lymphocytic leukemia (CLL) cells of patients receiving therapy with fludarabine and cyclophosphamide. The kinetics of interstrand DNA-cross link formation and removal in circulating leukemic lymphocytes during therapy with cyclophosphamide will be determined, and the effect of prior fludarabine infusion on these processes will be studied. The inherent ability of extracts from CLL cells to repair 4-hydroperoxycyclophosphamide-induced adducts on plasmid DNA in vitro will be compared among patients to seek correlations between these parameters and clinical response to cyclophosphamide/ fludarabine therapy. Finally, because all of these agents induce the death of CLL lymphocytes by initiating apoptosis, this disease will be used as a model for investigating the molecular mechanisms involved in this death process. Correlations will be sought between induction of high molecular weight DNA fragmentation (a measure of apoptosis) in CLL cells during therapy and clinical response to nucleoside analog treatment. The hypothesis that the inability of fludarabine to induce proteins required to execute apoptosis is a mechanism for drug resistance will be investigated.
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