Effects of long-term administration of UFT plus leucovorin on colorectal tumors induced with 1,2-dimethylhydrazine in rats.

Thymidylate synthase and thymidine kinase are key enzymes involved in the de novo and salvage pathways for pyrimidine nucleotide synthesis, respectively. Thymidylate synthase is inhibited by 5-fluorodeoxyuridine monophosphate, forming an inactive ternary complex with intracellular folate. We investigated the effects of 1-(2-tetrahydrofuryl)-5-FU plus uracil (UFT) with or without leucovorin on 1,2-dimethylhydrazine-induced rat colorectal carcinomas. Thirty-week administration of UFT with or without leucovorin markedly suppressed both colorectal carcinogenesis and tumor growth, resulted in the increase of thymidylate synthase inhibition and the decrease of thymidine kinase activity in the tumor cells. These results indicate that the combination of UFT with leucovorin could be useful in the development of pre- and post-operative adjuvant chemotherapy programs.

Thymidylate synthase inhibition after administration of fluorouracil with or without leucovorin in colon cancer patients: implications for treatment with fluorouracil.

PURPOSE: To determine the time-dependence of fluorouracil (5FU)-induced thymidylate synthase (TS) inhibition in colon cancer patients, the effect of leucovorin (LV), and the relation to response. PATIENTS AND METHODS: A 5FU injection (500 mg/m2) was given to 47 patients with advanced colorectal cancer; tumor biopsy specimens were obtained 1 to 72 hours after laparotomy. Eleven patients received LV (2-hour infusion of 500 mg/m2) with 5FU midinfusion; biopsies were obtained after 45 hours. TS inhibition was evaluated by comparing the number of total and free 5-fluoro-2'-deoxy-uridine-5'- monophosphate (UMP) (FdUMP) binding sites and the total and residual catalytic activity of TS. RESULTS: The total catalytic TS activity varied from 0 to 621 pmol/h/mg protein and the total number of FdUMP binding sites varied from 0 to 976 fmol/mg protein. The residual catalytic TS activity after 2, 23, and 45 hours was 41%, 65%, and 74% of the total catalytic activity; the number of free FdUMP binding sites was 12%, 27%, and 49% of the total number, respectively. LV enhanced TS inhibition after 45 hours; the residual catalytic activity decreased from 74% to 49%, and the number of free FdUMP binding sites from 49% to 24%. Eleven of 19 patients treated with hepatic arterial infusion of 5FU had a partial response (PR). In the nonresponding patients, total TS activity was significantly higher (P < .05) than in responding patients. A high TS activity with a poor inhibition correlated with no response. CONCLUSION: Residual and total TS activity are predictive for response to 5FU. The findings may be applicable for treatment of patients with advanced disease and TS should be evaluated as a prognostic factor in adjuvant chemotherapy studies.

Potentiation of the antitumor activity of 5-fluorouracil in colon carcinoma cells by the combination of interferon and deoxyribonucleosides results from complementary effects on thymidine phosphorylase.

alpha-Interferon (IFN alpha) potentiates the cytotoxicity of 5-fluorouracil (FUra) in vitro, and the combination has clinical efficacy in advanced colorectal cancer. We have reported previously an IFN alpha-mediated elevation in cellular FdUMP levels accompanied by the stimulation of thymidine phosphorylase (TP) activity in extracts from HT-29 human colon carcinoma cells treated with IFN alpha. We have now found that this effect of IFN alpha can be measured in vivo as an increase in thymine incorporation in intact cells. The increase was only 3-fold, however, compared to the 12-fold increase seen in TP activity in cell extracts. This suggested that the cosubstrate for TP, deoxyribose-1-phosphate, was rate limiting in the cells. Since the synthetic pathway of TP can also proceed via a transferase reaction, natural and modified deoxyribonucleosides were tested as deoxyribosyl donors. TP activity was measurable in cell extracts using deoxyinosine as cosubstrate with either thymine or FUra, although activity was only 10% of that measured with deoxyribose-1-phosphate. The pyrimidine analogue 5-propynyloxy-2'-deoxyuridine (PO-dUrd) had 15% of the maximal TP activity in cell extracts and also increased thymine incorporation in intact cells 10-fold. Both 2'-deoxyinosine and PO-dUrd potentiated the cytotoxicity of FUra by 8-11-fold. IFN alpha potentiated the cytotoxicity of FUra by 1.8-fold, and the combination of IFN alpha and PO-dUrd produced a 25-fold increase in the cytotoxicity of FUra. Neither the corresponding analogue riboside, 5-propynyloxyuridine, nor the analogue base, 5-propynyloxyuracil, had any effect on FUra cytotoxicity. There was a significant correlation between the ability of a nucleoside and/or IFN alpha combination to increase thymine incorporation and to reduce the 50% inhibitory concentration for FUra. IFN alpha and PO-dUrd also potentiated the inhibition by FUra of thymidylate synthase activity. These findings suggest that the use of a deoxyribonucleoside to provide the rate limiting cosubstrate would complement the stimulation of TP by IFN alpha, and together they should further enhance the antitumor activity of FUra.

[High-dose leucovorin and 5-FU].

Leucovorin (LV), given intravenously the orally becomes 5, 10-methylene tetrahydrofolate in both cancer and normal cells. FdUMP which is an active metabolite of 5-FU binds tightly to thymidylate synthase in the presence of the cofactor 5, 10-methylene tetrahydrofolate. This interaction leads to potentiate the cytotoxic effect of 5-FU by prolonged inhibition of thymidylate synthase. Clinically, the combination of LV and 5-FU is given parenterally by two schedules; 5 consecutive days schedule and weekly schedule. Five 5 consecutive days-schedule is divided into 2 methods. One is a 200 mg/m2/day of LV by Machover, and the other is 20 mg/m2/day of LV by O'Connell. The weekly schedule is a 2-hour infusion of dl-LV (500 mg/m2) and iv bolus of 5-FU (600 mg/m2), given 1 hour after the beginning of LV infusion by Petrelli. A multicenter cooperative study in Japan was conducted to evaluate the clinical efficacy of LV and 5-FU using the weekly schedule by Petrelli. Response rates were 31.5% and 41.2% against advanced gastric and colorectal cancer respectively. Then, we carried out a randomized early phase II study using 250 mg/m2 of l-LV weekly (similar to the schedule of Petrelli's, armA) and 100 mg/m2 (similar to the schedule of Machover's, arm B) or 10 mg/m2 (similar to the schedule of O'Connell's, arm C) of l-LV for 5 consecutive days against gastric cancer. The response rate was 33.3% in arm A, 24.1% in arm B and no response in arm C. Toxicity was within acceptable limits, Toxic effects included diarrhea, stomatitis, anorexia and myelohypoplasia. Our data suggests that high-dose LV and 5-FU seems to be a very promising combination but, there was no responder using low dose (10 mg/m2) of l-LV schedule against gastric cancer patients.

Detection of differential sensitivity to 5-fluorouracil in Ehrlich ascites tumour cells by 19F NMR spectroscopy.

Quantitative analysis of extracts from two Ehrlich ascites tumour cell lines (Lettre cells) by 19F NMR in vitro demonstrated that one Lettre cell line (designated LLM) metabolized 30-50% less 5-fluorouracil to 5-fluoronucleotides when compared to the other cell line (designated LHM). HPLC analysis of these cellular extracts showed a significant decrease in the concentration of the cytotoxic nucleotide 5-fluorouridine triphosphate in LLM cells compared to LHM cells. No major differences could be observed in the 31P and 1H NMR spectra of the two cell lines. Growth inhibition studies in vitro demonstrated that LLM cells were less sensitive to 5-fluorouracil than LHM cells. These results are consistent with the hypothesis that 19F NMR visible levels of 5-fluoronucleotides can predict the cytotoxicity of the anti-cancer drug 5-fluorouracil.

Crystal structure of Escherichia coli thymidylate synthase containing bound 5-fluoro-2'-deoxyuridylate and 10-propargyl-5,8-dideazafolate.

The crystal structure of an Escherichia coli thymidylate synthase (TS) ternary complex containing 5-fluoro-2'-deoxyuridylate (FdUMP) and 10-propargyl-5,8-dideazafolate (PDDF) has been determined and refined at 2.3 A resolution. Each of the two chemically identical subunits folds into a three-layer domain anchored by a large six-stranded mixed beta-sheet. The backside of one sheet is juxtaposed against the corresponding face of the equivalent sheet in the second protomer creating a beta-sandwich. In contrast to other proteins of known structure in which aligned beta-sheets stack face to face with a counterclockwise rotation, sheets in the TS dimer are related by a clockwise twist. The substrate-binding pocket is a large funnel-shaped cleft extending some 25 A into the interior of each subunit and is surrounded by 30 amino acids, 28 from one subunit and two from the other. FdUMP binds at the bottom of this pocket covalently linked through C-6 to the sulfur of Cys146. Up-pointing faces of the pyrimidine and ribose rings are exposed to provide a complementary docking surface for the quinazoline ring of PDDF. The quinazoline inhibitor binds in a partially folded conformation with its p-aminobenzoyl glutamate tail exposed at the entrance to the active site cleft. Ternary complex formation is associated with a large conformational change involving four residues at the protein's carboxy terminus that close down on the distal side of the inhibitor's quinazoline ring, capping the active site and sequestering the bound ligands from bulk solvent.

Crystal structure of Escherichia coli thymidylate synthase with FdUMP and 10-propargyl-5,8-dideazafolate.

The crystal structure of an E. coli TS ternary complex containing FdUMP and PDDF has been determined and refined at 2.3A resolution. Each of the two chemically identical subunits folds into a three-layer domain anchored by a large six-stranded mixed beta sheet. The backside of one sheet is juxtaposed against the corresponding face of the equivalent sheet in the second protomer creating a beta sandwich. In contrast to other proteins of known structure in which aligned beta sheets stack face to face with a counterclockwise rotation, sheets in the TS dimer are related by a clockwise twist. The substrate binding pocket is a large funnel-shaped cleft extending some 25A into the interior of each subunit and surrounded by 28 amino acids, 26 from one subunit and 2 from the other. FdUMP binds at the bottom of this pocket covalently linked through C6 to the sulfur of Cys-146. Up-pointing faces of the pyrimidine and ribose rings are exposed to provide a complementary docking surface for the quinazoline ring of PDDF. The quinazoline inhibitor binds in a partially folded conformation with its p-aminobenzoylglutamate tail exposed at the entrance to the active site cleft. Ternary complex formation is associated with a large conformational change involving 4 residues at the protein's carboxy-terminus that close down on the distal side of the inhibitor's quinazoline ring, capping the active site and sequestering the bound ligands from bulk solvent.

Selective accumulation of 3',5'-dioctanoyl-5-fluoro-2'-deoxyuridine (FdUrd-C8) and sustained release of its active metabolites in VX-2 rabbit hepatoma following intraarterial administration of FdUrd-C8 solution in Lipiodol.

Selective accumulation/retention of 3',5'-dioctanoyl-5-fluoro-2'-deoxyuridine (FdUrd-C8) and sustained release of its active metabolites, 5-fluoro-2'-deoxyuridine (FdUrd) and 5-fluoro-2'-deoxyuridylate (FdUMP), in the rabbit hepatoma (VX-2) were achieved following intrahepatic arterial administration of FdUrd-C8 solution in Lipiodol. Though no significant difference in the FdUrd-C8 levels among the tumor and nontumorous liver was observed immediately after administration, slower elimination of FdUrd-C8 from the tumor (t 1/2 = 15.8 h) than that from nontumorous sites (t 1/2 = 3.8-4.2 h) resulted in selective retention of FdUrd-C8 (17- to 157-fold) in the tumor. Selectively higher levels of FdUrd and FdUMP in the tumor were also achieved (5- to 35-fold) and kept for 72 h after administration. The selective accumulation was also demonstrated in radioactivity distribution after administration of [6-3H]-FdUrd-C8. The ratio of radioactivity in the tumor divided by that in the blood (T/B ratio) was in a range of 870 to 5400 during a 15- to 1440-min period after administration. A trace of radioactivity was found in the stomach, duodenum, kidneys, and bone marrow. Roles of activation and deactivation enzymes on the selective distribution of FdUrd-C8 were also investigated. Esterase activity, which is responsible for the regeneration of FdUrd from FdUrd-C8, was relatively low in the tumor before administration and gradually increased after administration. Phosphorylase activity, which is related to phosphorolytic cleavage of FdUrd, in the tumor was about 3/5 as much as that in the nontumorous liver. These enzyme activities seem to play limited roles in the selective accumulation/retention and regeneration of the drug.