Combination studies of trifluorothymidine with antifolates (AG337, ZD1694, GW1843) and the DNA synthesis inhibitors gemcitabine, irinotecan and oxaliplatin

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Trifluorothymidine (TFT) is a thymidine analog which is activated by thymidine kinase to TFT-MP which may inhibit thymidylate synthase (TS). TS converts dUMP to dTMP and its inhibition leads to dTTP depletion in cells causing dUTP to be misincorporated into DNA. TFT-TP can be incorporated into DNA, possibly resulting in DNA damage and cell death. The antifolates AG337, ZD1694 and GW1843 are direct TS inhibitors. We studied the effect of these antifolates and gemcitabine (dFdC), irinotecan (as SN38) and oxaliplatin (OHP) on TFT sensitivity by using cytotoxicity assays (SRB and MTT assay). A panel of 5 colon cancer cell lines (Colo320, H630, SNU-C4, SW1116, WiDr) were used for the experiments. WiDr/F is a WiDr variant adapted to grow on low folate conditions (2.5 nM leucovorin). Multiple drug effect analysis was performed using Calcusyn software (Biosoft, 1996) and expressed as mutually nonexclusive combination index (CI) in which a CI≤0.9 indicates synergism and CI≥1.1 antagonism. Three experimental procedures were used to test the interaction of the drugs: either one of the drugs was kept at a constant concentration (∼IC25) or two drugs were added in a 1:1 IC50 equimolar ratio. For the antifolates we also determined the effect of the combinations on TS inhibition and DNA damage induction. The combination of TFT-antifolate in which one of the drugs was kept at a constant concentration was not more than additive and was cell line dependent (TFT-AG337: 0.9<CI<2.3; TFT-ZD1694: 0.9<CI<1.3; TFT-GW1843: 0.8<CI<1.7), except for WiDr/F (all CI≤0.8). The procedure in which the two drugs were added in a 1:1 IC50 equimolar ratio showed antagonism for all three combinations in all cell lines (CI≥2.8). When TFT was combined with GW1843 more TS inhibition (15%) and DNA damage (8%) was seen in WiDr/F cells than expected (p<0.05), in contrast to ZD1694 and AG337, which showed inhibiting effects as expected (additive). The TFT-GW1843 combination is being evaluated in a preincubation schedule. The combination of TFT-dFdC showed synergistic to slightly antagonistic effects, depending on the cell line and experimental procedure used (0.5<CI<1.3). The combination of TFT-SN38 showed additive to antagonistic effects in most cell lines (0.8<CI<4.3), except Colo320 (CI∼0.7), also depending on the experimental procedure used. The combination of TFT-OHP, which showed synergism in 3 cell lines (0.4<CI<0.8) and additivity in SNU-C4 and Colo320 (CI around 0.8) for each procedure, is currently being evaluated for OHP-DNA adduct formation. It can be concluded that the combination of TFT with these antifolates is mainly additive when the drugs are given simultaneously and that TS is the major target for TFT. Results showed that WiDr/F is more sensitive to two-sided inhibition of TS than WiDr indicating that folate concentrations can influence induced synergism in colon cancer cells.