The long term objective of this research is to improve chemotherapy of cancer by using novel antifolates or novel reduced-folate analogs for biochemical modulation to yield an increased therapeutic index. The approach to be used may also define the role of antifolyl- and folylpolyglutamate metabolites in cytotoxicity and selectivity. The antifolate methotrexate (MTX) is clinically effective in combination chemotherapy regimens, but its efficacy is often limited by resistance which cannot be overcome at higher doses because toxicity is reached, i.e., it is not selective enough. Polyglutamates of MTX are implicated in its cytotoxicity as a single agent, but their contribution to cytotoxicity and (particularly) selectivity in combinations using this agent are unknown. Exogenous reduced folates (as leucovorin (LV); 5-formyltetra- hydrofolate) are used to """"""""rescue"""""""" from the cytotoxic effects of MTX and to augment the cytotoxicity of other drugs (e.g., 5-fluorouracil [FU]). The role of folylpolyglutamate synthesis in achieving these effects as well as in host toxicity and selectivity are unknown. This proposal will determine whether substitution of nonpolyglutamylatable analogs of MTX and LV in combination chemotherapy yields any therapeutic advantage. The analogs of MTX and LV will contain 4-fluoroglutamate instead of glutamate, a change sufficient to block polyglutamylation of the analog. The proposed studies of drug combinations will compare the biochemical, cytotoxic, and therapeutic effects of LV or MTX in a regimen with the same regimen containing these nonpolyglutamylatable """"""""mimics"""""""". Cytotoxicity will be assessed by out-growth and/or clonogenic assays using the human leukemia cell line CCRF-CEM and an appropriate murine model (for extension to in vivo therapeutic studies); time and dose dependence will be investigated in detail. Where feasible the biochemical determinants of cytotoxicity will be explored. The regimens to be investigated are: sequential MTX/5FU, simultaneous LV/5-FU, and LV rescue of MTX cytotoxicity. MTX and LV and their respective fluorinated """"""""mimics"""""""" will be compared to determine if they are biochemically equivalent, except for polyglutamylation. If the analogs are true """"""""mimics, the results from the cytotoxicity and efficacy studies would yield basic knowledge about the role of polyglutamates in these combinations. It is hoped that the knowledge gained in these studies will provide a basis for more sophisticated therapy protocols with improved efficacy.
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