The proposed study is directed at a growth inhibitory and mechanistic evaluation of combinations of antifolates which exhibit synergy with each other against hepatoma cells in culture. The preliminary studies have shown the dihydrofolate reductase (DHFR) inhibitors can be combined with a folate based thymidylate synthase (TS) inhibitor (N10-propartyl-5,8- dideazafolate, PDDF) to yield synergistic tumor cell growth inhibition and toxicity. Currently the most effective DHFR inhibitors in combination with PDDF are the lipid soluble antifols such as metoprin or trimetrexate. We propose to make evaluations of other DHFR inhibitors to determine which are the most effective and which drug timing is the most effective. The generality of the synergistic combinations are to be tested in other tumor cell systems. The experimental approach will be to evaluate the inhibition by measuring the effects of synergistic combination with outgrowth and clonal assays. Metabolic inhibition by the synergistic combinations will be evaluated by measuring de novo thymidylate biosynthesis in treated cells. The observation that thymidine can protect against combination based growth inhibition suggests that mildly inhibitory concentrations of DHFR inhibitors are sensitizing the cells to PDDF. This could be accounted by a reduction in 5,10-methylenetetrahydrofolate and increase in dUMP, which favors the formation of an inhibited PDDF-TS-dUMP ternary complex. A detailed mechanistic analysis of the synergistic interaction of the two classes of antifolates will be made. The approach in the current phase of the study will be to examine the effects of the DHFR and TS inhibitors on levels of substrates for TS. In addition the functional amounts of the target enzymes and the amounts of PDDF bound to TS during synergistic inhibition will be examined. Other causes for the unique drug interaction are plausible and these will be considered depending upon the outcome of the proposed mechanistic studies. It is hoped that an understanding of this mechanism will result in more knowledge about the use of the antifolates in treatment of neoplasms and the development of the potential for combination of these agents.
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