Recent experiments suggest that tumor cell levels of 5,10-methylene tetrahydrofolate are insufficient for complete inhibition of thymidylate synthase (TS) by 5-fluorouracil (5-FU) and that the accumulation of deoxyuridylate (dUMP) protects TS in some tumors. The objectives of this proposal are to investigate the biochemical factors that limit the inhibition of DNA synthesis by 5-FU and to evaluate whether enhanced host toxicity or improved therapeutic selectivity would result from combination chemotherapy that removed these obstacles. The toxicity of combination of 5-FU and reduced folates will be compared with that of 5-FU alone on various schedules in laboratory animals. Subsequently, the antitumor activity of the combination will be compared with that of 5-FU using equitoxic dosage regimens in mice bearing a series of colon adonocarcinomas that are sensitive and resistant to 5-FU. During these studies, the pharmacokinetics and distribution of both isomers of leucovorin will be studied after the administration of large doses (50mg./kg.) of 3H-d and 3H-1-leucovorin to mice. The intracellular tetrahydrofolate content of these tumors and of normal mouse tissues will be determined after administration of leucovorin and these levels will be correlated with the stability of the fluorodeoxyuridylate: tetrahydrofolate: thymidylate synthase ternary complex in vivo. The accumulation of dUMP in animal tumors will be studied. The proposed biochemical studies would seek (1) to limit this accumulation with inhibitors of robonucleotide reductase, (2) to define the effects of deoxycytidylate deaminase inhibition on dUMP pools (3) to explain the large difference in basal dUMP levels and extent of accumulation of this metabolite that have been reported and (4) to determine the effect of dUMP pool limitation on inhibition of TS by 5-FU. Subsequently, we would examine the therapeutic advantage afforded by combinations of 5-FU and effective inhibitors of dUMP accumulation in murine models of human colonic adenocarcinoma.
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