5-ETHOXY-2'-DEOXYURIDINE, A NOVEL SUBSTRATE FOR THYMIDINE PHOSPHORYLASE, POTENTIATES THE ANTITUMOR-ACTIVITY OF 5-FLUOROURACIL WHEN USED IN COMBINATION WITH INTERFERON, AN INDUCER OF THYMIDINE PHOSPHORYLASE EXPRESSION

Clinical studies have demonstrated that the combination of 5-fluoroura cil (FUra) and IFN-alpha has activity in the treatment of advanced col orectal cancer. Treatment of human colon carcinoma cells with IFN caus ed a 5-fold increase in the level of thymidine phosphorylase (TP) mRNA and an 8-fold increase in TP enzyme activity, Since TP catalyzes the first step in the direct conversion of FUra to deoxyribonucleotides, i ts induction by IFN is a potential biochemical mechanism for the modul ation of the antitumor activity of FUra. In contrast to the activity m easured in cell extracts, however, thymine utilization by intact cells was increased less than 2-fold by IFN, suggesting that the metabolic activation of PUra by TP in the IFN-treated cells was similarly subopt imal. This was likely due to a rate-limiting amount of cosubstrate for TP, and in this study, a series of 5-substituted 2'-deoxyuridine anal ogues were synthesized and tested as potential deoxyribose donors for TP. One of the compounds, the novel pyrimidine analogue 5-ethoxy-2'-de oxyuridine (EOdU), was found to be a substrate for the transferase rea ction of TP, to have little or no direct cytotoxicity, to selectively increase the cellular levels of 5-fluoro-dUMP, to enhance the inhibito ry effect of FUra on thymidylate synthase activity, and to potentiate the cytotoxicity of FUra and IFN in human colon carcinoma cells, EOdU was tested in vivo against HT-29 cells grown as xenografts in nude mic e. The combination of EOdU + FUra + IFN-alpha 2a produced tumor regres sions and a significantly greater delay in tumor growth when compared to FUra + IFN-alpha 2a, FUra + EOdU, or FUra or IFN used alone; tumors were 72% smaller in the EOdU + FUra + IFN-alpha 2a-treated animals co mpared to the saline control group. A comparable antitumor effect was also found when a related nucleoside analogue, 5-propynyloxy-2'-deoxyu ridine, was used with FUra + IFN, and it also showed modulating activi ty when used with only FUra. The antitumor activity of the three agent combination (nucleoside + IFN + FUra) was comparable to that of a hig her dose of PUra used alone, but it was substantially less toxic to th e animals than the higher dose of FUra, indicating that the modulating agents improved the therapeutic index of FUra. The substitution of a hybrid recombinant IFN-alpha A/D, active in both human and murine tiss ues, in place of the species-specific human IFN-alpha 2a did not furth er increase the efficacy of the combination, suggesting that a direct effect on the tumor cells rather than host-mediated actions of IM was the predominant mechanism for antitumor effects observed in vivo. Thes e studies demonstrate a potential novel approach to increase the effic acy and selectivity of FUra, which incorporates two complementary bioc hemical actions: the selective induction by TPN of expression of a gen e regulating pyrimidine synthesis, coupled with the rational design of a cosubstrate for the induced enzyme.