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

Abstract

Clinical studies have demonstrated that the combination of 5-fluorouracil (FUra) and IFN-α has activity in the treatment of advanced colorectal cancer. Treatment of human colon carcinoma cells with IFN caused 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, its induction by IFN is a potential biochemical mechanism for the modulation of the antitumor activity of FUra. In contrast to the activity measured in cell extracts, however, thymine utilization by intact cells was increased less than 2-fold by IFN, suggesting that the metabolic activation of FUra by TP in the IFN-treated cells was similarly suboptimal. This was likely due to a rate-limiting amount of cosubstrate for TP, and in this study, a series of 5-substituted 2′-deoxyuridine analogues were synthesized and tested as potential deoxyribose donors for TP. One of the compounds, the novel pyrimidine analogue 5-ethoxy-2′-deoxyuridine (EOdU), was found to be a substrate for the transferase reaction of TP, to have little or no direct cytotoxicity, to selectively increase the cellular levels of 5-fluoro-dUMP, to enhance the inhibitory 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 mice. The combination of EOdU + FUra + IFN-α2a produced tumor regressions and a significantly greater delay in tumor growth when compared to FUra + IFN-α2a, FUra + EOdU, or FUra or IFN used alone; tumors were 72% smaller in the EOdU + FUra + IFN-α2a-treated animals compared to the saline control group. A comparable antitumor effect was also found when a related nucleoside analogue, 5-propynyloxy-2′-deoxyuridine, was used with FUra + IFN, and it also showed modulating activity when used with only FUra. The antitumor activity of the three agent combination (nucleoside + IFN + FUra) was comparable to that of a higher dose of FUra used alone, but it was substantially less toxic to the 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-αA/D, active in both human and murine tissues, in place of the species-specific human IFN-α2a did not further increase the efficacy of the combination, suggesting that a direct effect on the tumor cells rather than host-mediated actions of IFN was the predominant mechanism for antitumor effects observed in vivo. These studies demonstrate a potential novel approach to increase the efficacy and selectivity of FUra, which incorporates two complementary biochemical actions: the selective induction by IFN of expression of a gene regulating pyrimidine synthesis, coupled with the rational design of a cosubstrate for the induced enzyme.