Combination studies with 3'-azido-3'-deoxythymidine (AZT) plus ICI D1694. Cytotoxic and biochemical effects

The cytotoxicity of 3'-azido-3'-deoxythymidine (AZT), a thymidine analogue, and ICI D1694, a folate-based thymidylate synthase (TS) inhibitor, was examined individually and in combination in two human tumor cell lines, MGH-U1 bladder cancer and HCT-8 colon cancer cells, grown as a monolayer culture with and without thymidine (TdR). In addition, TS inhibition, [3H]AZT incorporation into DNA, [3H]AZT-MP (monophosphate) production, and DNA double-strand breaks were measured. Twenty-four hour exposure of AZT at 0.5, 5, 50 and 500 microM was not cytotoxic to MGH-U1 or HCT-8 cells in a colony-forming assay. ICI D1694 cytotoxicity increased with drug concentration, and the IC50 and IC90, respectively, were 0.0064 and 0.01 microM in MGH-U1 cells and 0.009 and 0.018 microM in HCT-8 cells. TdR in concentrations of 0.1 to 1.0 microM did not affect ICI D1694 cytotoxicity in either cell line. AZT at 5, 50 or 500 microM increased ICI D1694 cell kill. The IC50 and IC90 for MGH-U1 were 0.0037 and 0.0075 microM for 50 microM AZT combined with ICI D1694. The IC50 and IC90 for HCT-8 were 0.0075 and 0.015 microM for 50 microM AZT plus ICI D1694. The incorporation of [3H]AZT into DNA increased with increasing concentrations of ICI D1694. Concentrations producing an IC50 and IC90 of ICI D1694, respectively, increased incorporation of [3H]AZT into DNA by 319 and 569% in MHG-U1, and 243 and 400% in HCT-8 cells. The formation of [3H]AZT-MP paralleled the increase in [3H]AZT incorporated into DNA. AZT, ICI D1694 and the combination of AZT and ICI D1694 caused DNA double-strand breaks, with the combination of these agents being additive. CFU-GM survival, exposed to drug concentrations, as those used in the tumor cell lines, revealed that the therapeutic index was greater for AZT plus ICI D1694 than for ICI D1694 alone. These findings suggest that AZT plus ICI D1694 may increase antitumor effect with minimal myelosuppression. We conclude that AZT increases the cytotoxicity of ICI D1694 with increasing AZT incorporation into DNA.