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Eighteen patients with hepatic metastases primarily from colorectal carcinoma were treated on a phase I protocol employing hepatic artery infusion (HAI) of 5-fluorouracil (FUra) and 5-iodo-2'-deoxyuridine (IdUrd) via implantable infusion pump. Patients received a 14-day continuous HAI of 300 mg/day FUra. During days 8-14 of therapy, patients received IdUrd as a separate 3-h HAI daily x 7. Treatment cycles were repeated every 28 days. IdUrd was escalated from 0.1 to 2.86 mg/kg/day x 7. Myelosuppression and stomatitis were mild and not dose limiting. Hepatotoxicity was dose limiting and similar to that reported for 5-fluoro-2'deoxyuridine alone administered as a 14-day infusion every month. One patient developed a clinical picture consistent with sclerosing cholangitis and another had biopsy-proven cholestasis and triaditis. Catheter complications occurred in 7 of 18 patients. Plasma concentrations of FUra during the 7-day continuous HAI of FUra alone were consistently either undetectable or very low (less than or equal to 0.1 microM). At level 3 (1.0 mg/kg/day IdUrd) and beyond, measurable plasma concentrations of FUra, iodouracil, and IdUrd were found at the end of the daily 3-h infusion of IdUrd. The maximum tolerated dose of IdUrd as administered in this trial is 2.2 mg/kg/day x 7 and the recommended starting dose for further clinical investigation is 1.7 mg/kg/day x 7.

The phosphorylation of thymidine and iododeoxyuridine by thymidine kinase was stimulated by 5-iodo-5'-amino-2',5'-dideoxyuridine ( AIdUrd ). Antagonism of the feedback inhibition that is normally exerted by the 5'-triphosphates of thymidine and iododeoxyuridine appears to account for the stimulation. The effect of AIdUrd on thymidine kinase purified from HeLa cells by affinity column chromatography was critically dependent on the presence of these feedback inhibitors. In the presence of thymidine triphosphate or iododeoxyuridine triphosphate, AIdUrd could markedly stimulate ( deinhibit ) enzyme activity, whereas, in their absence, AIdUrd inhibited thymidine kinase with an apparent Ki of 0.7 microM. Stimulation was evident over a wide range of concentrations of both iododeoxyuridine and adenosine triphosphate. In intact HeLa and Vero cells, phosphorylation of thymidine and iodoeoxyuridine was strongly enhanced by AIdUrd . Large increases in the intracellular levels of nucleotides derived from exogenous thymidine and iododeoxyuridine were apparent. As a consequence, the cytotoxicity of both nucleosides was exacerbated by AIdUrd . The reductions in cellular replication rates and colony formation produced by iododeoxyuridine were enhanced by AIdUrd . Although the replication of HeLa cells was not inhibited by either thymidine (30 microM) or AIdUrd (300 microM), in combination they were strongly synergistic and produced a 60% inhibition of cellular growth. Under these conditions, the uptake of thymidine was increased over 300% by AIdUrd . AIdUrd represents a new regulatory antagonist of thymidine kinase which may be useful in novel chemotherapeutic strategies.

Iododeoxyuridine (IdUrd) potentiated the lethal but not the growth inhibitory properties of fluorouracil (FUra) and fluorodeoxyuridine (FdUrd) in human bladder cancer cells (T24). The rate of incorporation of IdUrd into DNA was enhanced by both fluoropyrimidines, but to a significantly greater extent by FdUrd. Both inhibition of iododeoxyridylate dehalogenation and the depletion of thymidine triphosphate pools contributed to the increased incorporation rate. Inhibition of dehalogenation accounted for 67% of the observed stimulation in the case of FUra, but only 37% of the increase produced by FdUrd. The depletion of dTTP pools, both in the presence and absence of IdUrd, was greater after FdUrd than FUra exposure. The observed increase in the rate of incorporation of IdUrd appears to account for the enhanced toxicity seen with FdUrd, but other factors may be involved in the case of FUra. Since FUra and IdUrd appear to be mutually potentiating and do not share a dependence on thymidine kinase activity, this drug combination warrants further investigation.

5'-Aminothymidine represents a novel class of compounds capable of antagonizing the feedback inhibition which normally regulates thymidine kinase. As a consequence, the uptake of iododeoxyuridine, a substrate of thymidine kinase, can be substantially increased by 5'-aminothymidine. in this study the phosphorylation, uptake, and cytotoxicity of iododeoxyuridine was markedly enhanced by 5'-aminothymidine in three human bladder cancer cell lines, T24, HT1197 and 647V. In contrast, neither the uptake nor the toxicity of iododeoxyuridine was increased by 5'-aminothymidine in normal human urothelial cells propagated in vitro. Although 30 microM 5'-aminothymidine increased the uptake of 3 microM iododeoxyuridine 4- to 5-fold in the HT1197 and 647V cells and 2.5-fold in the T24 cells, no stimulation was produced in the normal urothelial cells. The modulation of iododeoxyuridine uptake was associated with parallel changes in the inhibition of cellular replication. The cytotoxicity of iododeoxyuridine was strongly augmented by 5'-aminothymidine in the HT1197 and 647V cells, modestly increased in the T24 cells, and unchanged in the normal urothelial cells. The degree to which iododeoxyuridine phosphorylation was stimulated did not correlate with cellular replication rates or with intracellular thymidine triphosphate concentrations. Iododeoxyuridine uptake was markedly increased in the HT1197 (doubling time = 52 h) and 647V (doubling time = 24 h) cells, moderately in the rapidly growing cells T24 (doubling time = 20 h), and minimally in the normal urothelial cells which doubled every 32 h. In exponentially growing cells the thymidine triphosphate pools were approximately 18 microM in the normal cells and about 21, 24, and 35 microM in the HT1197, T24, and 647V cells, respectively. The use of 5'-aminothymidine and other compounds capable of antagonizing feedback inhibition may provide a new means of increasing the efficacy of cytotoxic nucleosides.

We have previously reported that 5'-aminothymidine (5'-AdThd), an antagonist of the feedback inhibition exerted by dTTP that regulates thymidine kinase, enhances the uptake and cytotoxicity of 5-iododeoxyuridine in various human bladder cancer cell lines but not in normal human urothelial cells (HU) propagated in vitro. In this work we have analyzed the factors that could potentially account for the differential effect of 5'-AdThd among various cell types: 647V (a human bladder cancer cell line); HU; SV-HU (a SV40-transformed human urothelial cell line), and C3H/10T1/2 mouse embryo fibroblasts (10T1/2) cells. 5'-AdThd enhanced the uptake of IdUrd in SV-HU cells (greater than 400%), similar to what we have observed before for 647V cells. However, in 10T1/2 and HU cells, 5'-AdThd only minimally increased the uptake of 5-iododeoxyuridine (about 160%). Thymidine kinases purified from the different sources were similarly sensitive to inhibition by dTTP or 5'-AdThd and to deinhibition of the dTTP-induced regulation of enzyme activity by 5'-AdThd. Furthermore, [3H]-5'-AdThd permeated and accumulated intracellularly in all cell types. In none of these cultures was nucleoside phosphorylase activity detected, as indicated by the inability of the cells to produce thymine or iodouracil after exposure to the appropriate nucleosides. Also, 5'-AdThd did not affect the breakdown of dTMP by crude preparations of cytosolic 5'-nucleotidase from the different cells. We found that intracellular dTTP pools in the various cell types were substantially high (15-26 microM) compared to the sensitivity of thymidine kinase to inhibition by dTTP (IC50 2-4 microM). This suggests that thymidine kinase is in a strongly inhibited state in situ. To test the sensitivity of thymidine kinase (in situ) to regulation by dTTP we investigated: (a) the effect of depleting intracellular dTTP pools with methotrexate on the uptake of thymidine (dThd); and (b) the effect of pH on the uptake of dThd and its perturbation by 5'-AdThd, since the inhibition of thymidine kinase activity by dTTP is known to be pH dependent. We found that a 47% reduction of dTTP pools by methotrexate in 10T1/2 and HU cells did not result in an increase in thymidine kinase activity, as indicated by the lack of an effect on the uptake of dThd. However, we have previously shown that, under similar conditions, 647V cells show a substantial increase in dThd uptake.(ABSTRACT TRUNCATED AT 400 WORDS)

Isopycnic centrifugation in CsCl gradients was used to quantify the incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus type 1 DNA. A parallelism between the degree of incorporation into viral DNA and the inhibition of herpes simplex virus type I replication was found for both thymidine analogs. A concentration of 5-iodo-5'-amino-2',5'-dideoxyuridine approximately 100 times greater than 5-iodo-2'-deoxyuridine was required to achieve similar levels of antiviral activity. However, the inhibitory effects of these compounds are similar when compared with respect to the percent of substitution for thymidine in herpes simplex virus type I DNA. Damage to the viral DNA, as indicated by the presence of single or double-stranded breaks, was assessed by centrifugation in alkaline and neutral sucrose gradients. The incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine into herpes simplex virus type I DNA produced single and, to a lesser extent, double-stranded breaks in a dose-dependent manner. 5-Iodo-2'-deoxyuridine did not, however, induced DNA breakage. These data indicate that the additional presence of a phosphoramidate bond in the DNA produced the extensive damage detected under these conditions, but that such damage is not required for antiviral activity.

In HeLa and Vero cells the antiproliferative effects of iododeoxyuridine (IdUrd) were modulated in a biphasic manner by 5'-amino-5'-deoxythymidine (5'-AdThd). Low concentrations of 5'-AdThd increased the cytotoxicity of IdUrd whereas high concentrations of 5'-AdThd were antagonistic. Opposing effects on two enzymes, thymidine kinase (EC 2.7.1.21) and thymidylate kinase (EC 2.7.4.9), account for this unusual biphasic interaction. In the case of thymidine kinase, 5'-AdThd was found to antagonize the feedback inhibition which is normally exerted by the 5'-triphosphates of thymidine and IdUrd. Consequently, 5'-AdThd increased the rate of IdUrd phosphorylation. This stimulation (deinhibition) of enzyme activity was demonstrable in cell-free extracts and with a purified preparation of thymidine kinase provided that the 5'-triphosphates of IdUrd or thymidine were present. In their absence only enzyme inhibition was detected. In intact cells this stimulatory effect of 5'-AdThd was seen as a rapidly apparent, sustained increase in the steady-state levels of the phosphorylated IdUrd metabolites. As a result, IdUrd cytotoxicity was increased. Under these conditions, 5'-AdThd did not alter the relative abundance of the mono-, di-, and triphosphates of IdUrd. However, as the concentration of 5'-AdThd was raised, the percentage of IdUrd nucleotides present as iododeoxyuridylate increased dramatically. Corresponding reductions in the incorporation of IdUrd into cellular DNA and the associated cytotoxic effects were seen. These data suggested a second site of interaction, thymidylate kinase, the enzyme responsible for the conversion of iododeoxyuridylate to the diphosphate. In experiments measuring thymidylate kinase activity in cell-free extracts, 5'-AdThd effectively inhibited the phosphorylation of iododeoxyuridylate but not that of thymidylate. Additionally, 5'-AdThd did not produce an accumulation of thymidylate in intact cells. Thus, the ability of high concentrations of 5'-AdThd to antagonize the cytotoxicity produced by IdUrd without concomitantly inhibiting the phosphorylation of thymidylate and, thereby, reducing DNA synthesis was explained. Although the modulation of IdUrd metabolism produced by 5'-AdThd was qualitatively similar in Vero and HeLa cells, key quantitative differences were evident. Thus, 100 microM 5'-AdThd stimulated the uptake of 3 microM IdUrd in Vero cells but it was inhibitory in HeLa cells. Perturbation of nucleoside metabolism by agents such as 5'-AdThd may provide an important new way to achieve selective toxicity in cancer chemotherapy.