Experimental combination chemotherapy with thymidylate synthetase and ribonucleotide reductase inhibitors

The synergistic cytotoxic effects on exponentially growing 9L rat brain tumor cells of several inhibitors of thymidylate synthetase (TS) and ribonucleotide reductase (RNR) used in combination were investigated using a colony forming efficiency assay as the experimental endpoint. A 24 h treatment with nontoxic (0.1 µg/ml) or low (1.0 µg/ml) doses of 5-fluorouracil (FUra), 5-fluorodeoxyuridine, 5,8-dideazaisofolic acid, or 2'-deoxy-2'-fluoro-ara-uracil markedly enhanced cell kill caused by subsequent administration of 100 µg/ml hydroxyurea (HU) for 6 h. When a similar dose of HU or 1-formylisoquinoline thiosemicarbazone was administered for 6 h immediately after a 24 h treatment with either a 0.1 µg/ml or 1.0 µg/ml of FUra, a cell kill of approximately 1 log in addition to that caused by each drug alone was obtained. Thus a synergistic cell kill was consistently obtained when a low dose of TS inhibitors was administered 24 h before a 6 h treatment with another low dose of agents that act as RNR inhibitors. This synergism was not observed when FUra-treated cells were treated with methotrexate, 6-mercaptopurine, vincristine, or l,3-bis(2-chloroethyl)-1-nitrosourea. Similarly, a 6 h treatment with 1 µg/ml of FUra of cells that had been treated for various periods with 100 µg/ml of HU did not increase cell kill more than that obtained with HU alone (30% cell kill).