1843u89 and Colonic-Neoplasms

Trifluorothymidine (TFT) is a fluoropyrimidine that is part of the novel combination metabolite TAS-102, in which TFT is combined with a potent thymidine phosphorylase inhibitor (TPI). TAS-102 is currently tested as an orally chemotherapeutic agent in different schedules in a phase I study. In its monophosphate form, TFT can inhibit thymidylate synthase (TS) activity after binding to the TS-nucleotide binding site leading to dTTP depletion, and in its triphosphate form TFT is incorporated into DNA, eventually leading to DNA damage. In this in vitro study, we investigated whether TFT could potentiate cytotoxicity of the antifolate-based TS inhibitors AG337 (Nolatrexed), ZD1694 (Raltitrexed) and GW1843; and whether increased TS inhibition or DNA damage would be related to this result.. The drug combinations were studied in colon cancer cell lines either grown at low or high folate conditions. Multiple drug effect analysis was performed after measuring growth inhibition when the drugs were combined (MTT Assay) and expressed as Combination Index (CI), where CI<0.9 indicates synergism, CI=0.9-1.1 indicates additivity and CI>1.1 indicates antagonism. Drug target analysis was performed using the TS in situ inhibition assay and the FADU DNA-damage assay. Cells were exposed to either the drugs alone or in combination to determine the effect on TS activity and DNA damage induction, respectively.. Three experimental procedures were used to test the interaction of the drugs: either one of the drugs was kept at a constant concentration (IC25) or two drugs were added in a 1:1 IC50-based molar ratio. The combinations of TFT with one of the antifolates in which one of the drugs was kept at a constant concentration were synergistic for all antifolates in WiDr/F cells, which grow in low folate medium (CI=0.6-0.8), but only additive to antagonistic for the cell lines growing in high folate medium: TFT-AG337: CI=0.9-2.3; TFT-ZD1694: CI=0.9-1.3; TFT-GW1843: CI=0.8-1.7. The procedure in which the two drugs were added in a 1:1 IC50-based molar ratio showed antagonism for all three combinations in all cell lines (CI>2.7). TS inhibition (14.3%) and DNA damage (8%) were more pronounced than expected (P<0.05) when TFT was combined with GW1843 in WiDr/F cells, in contrast to AG337 and ZD1694, which showed inhibiting effects as expected (additive).. The combination of TFT with the antifolates AG337, ZD1694 and GW1843 is mainly additive when the drugs are given simultaneously and this is mediated by an additive TS inhibition and DNA damage. The drug interaction may partly be dependent on the folate homeostasis since WiDr/F cells growing at low folate conditions show pronounced synergism in growth inhibition, two-sided TS inhibition and DNA damage, especially when TFT is combined with the tight-binding TS inhibitor GW1843.

Topics: Antimetabolites; Colonic Neoplasms; DNA Damage; Drug Interactions; Folic Acid; Folic Acid Antagonists; Homeostasis; Humans; Indoles; Isoindoles; Quinazolines; Thiophenes; Trifluridine; Tumor Cells, Cultured

The cytotoxicity and metabolic effects of two thymidylate synthase (TS) inhibitors, Tomudex (Raltitrexed, ZD1694) and GW1843U89, were studied in WiDr colon cancer cells under four different growth conditions: as standard monolayers and as postconfluent multilayers grown under either high (WiDr, 8.8 microM folic acid) or low (WiDr/F, 1 nM leucovorin) folate conditions. Both GW1843U89 and ZD1694 were 13-15-fold more active against WiDr/F than WiDr cells when cultured as monolayers (IC50s in WiDr/F cells were 0.22 and 0.39 nM, respectively). WiDr cells were markedly less sensitive to the drugs when grown as multilayers (4-15-fold), in contrast to the WiDr/F cells, which were equally sensitive. However, total growth inhibition could not be achieved in WiDr multilayers (concentration causing total growth inhibition > 10,000 nM), whereas in WiDr/F multilayers, it could be achieved at 0.42 nM ZD1694 and 150 nM GW1843U89. Growth conditions markedly affected the TS levels when using different enzyme assays. At nonsaturating substrate concentrations, the catalytic activity of TS was similar in mono- and multilayers grown under high folate conditions but lower in multilayers at saturating concentrations. In cells grown under low folate conditions, TS catalytic activity was 3-6-fold lower in multilayers than in monolayers. This was consistent with a decrease in the number of S-phase cells in multilayers. Western blotting revealed less pronounced (2-3-fold) differences in the TS protein content. Exposure of the cells for 24 h to the drugs increased the TS levels by 4-fold. Because this increase in TS levels might explain the decrease in sensitivity to the TS inhibitors, we measured TS inhibition (TSI) by the drugs in intact cells using the TS in situ assay. GW1843U89 was more active than ZD1694. However, after 4 h of exposure in WiDr/F mono- and multilayers, TSI was in the same range for both drugs [50% TSI (TSI50), 0.5-1.7 nM]. In WiDr cells, the TSI50 for ZD1694, but not GW1843U89, was 10 times higher in the multilayers as compared to the monolayers. Despite the increase in TS protein levels, the extent of TSI was similar or even more pronounced in both cell lines grown as either multi- or monolayers. Because the cells were grown under depleted and folate-rich conditions that may affect folate uptake, we measured folate transport using methotrexate (MTX) as the reference drug for the activity of the reduced folate carrier. MTX uptake was 4-fold lower in multilayers

Topics: Blotting, Western; Cell Cycle; Colonic Neoplasms; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Folic Acid Antagonists; Humans; Immunohistochemistry; Indoles; Inhibitory Concentration 50; Isoindoles; Methotrexate; Quinazolines; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured

The combined cytotoxic effects of the thymidylate synthase (TS) inhibitors 5-fluorouracil (5FU) and different antifolates were studied in seven colon cancer cell lines. Growth inhibition of the antifolates, Nolatrexed, Raltitrexed, GW1843U89, or MTA in combination with 5FU, was determined and multiple drug effect analysis showed that the drugs acted mostly additively. The only synergistic interaction was found for 5FU and Nolatrexed in the LS174T cell line. Also Raltitrexed and 5FU were slightly synergistic in WiDr/F cells grown at low folate levels, but for the other cell lines grown at high folate levels this combination was more antagonistic. GW1843U89 and 5FU were mainly additive, while 5FU and MTA showed antagonism in WiDr and additivity in LS174T. The effect of the drugs at their target was evaluated by in situ TS inhibition. We observed lower TS activity in all cells when two drugs were used instead of one. Statistical analysis revealed that none of the values of the combinations was higher or lower than could be expected from the product of the effect of single drugs. We concluded that the effects on TS inhibition were additive for all 5FU/antifolate combinations in all cell lines. DNA strand break formation, as a result of TS inhibition, was measured by means of a fluorometric analysis of DNA unwinding. Raltitrexed-induced DNA damage was significantly increased by 5FU in WiDr cells [single agent: 67% double stranded (ds) DNA, combination: 39% ds DNA, P<0.0001]. In LS174T a trend for antagonistic effects was observed for combinations of MTA, GW1843U89, or Raltitrexed and 5FU. The combinations showed additive effects in WiDr/F cells. The overall conclusion of the three assays in each of the cell lines indicated that 5FU and antifolate combinations were predominantly additive in colon cancer cells.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cell Division; Colonic Neoplasms; DNA Damage; Fluorouracil; Folic Acid Antagonists; Glutamates; Guanine; Humans; Indoles; Isoindoles; Pemetrexed; Quinazolines; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured

Syntheses of several new inhibitors of thymidylate synthase (TS) structurally related to folic acid are described in which the pterin portion of the folate molecule is replaced by a benzo[f]quinazoline moiety, but which retain the natural methyleneamino link to the benzoylglutamate side chain. The effect on enzyme activity and cytotoxicity of various changes in the structure of the (p-aminobenzoyl)glutamate side chain are reported. Replacement of the benzamide portion of the (p-aminobenzoyl)glutamate moiety with 2-fluorobenzamido, 2-isoindolinyl, 1,2-benzisothiazol-2-yl, and 2-thenamido moieties varied in effect from a 9-fold diminution of TS activity to a 5-fold enhancement, while cytotoxic potency on SW-480 and MCF-7 tumor lines showed increases ranging from 3.6- to 450-fold. The detrimental effect on enzyme activity and cytotoxicity of alkyl substitution on the PABA nitrogen is confirmed for these compounds, in contrast with several series of previously reported quinazoline antifolates (2). Substitution of a C3-methyl substituent for 3-amino had little effect on TS activity but was beneficial in terms of solubility and cytotoxicity. The excellent combination of TS inhibitory activity, FPGS substrate activity, and affinity for the reduced folate transport system in the most potent of these derivatives, 3e, resulted in IC50 values of 0.2-0.8 nM against these tumor lines.

Topics: Adenocarcinoma; Antineoplastic Agents; Breast Neoplasms; Colonic Neoplasms; Glutamates; Humans; Indoles; Isoindoles; Quinazolines; Structure-Activity Relationship; Thymidylate Synthase; Tumor Cells, Cultured

The activity of a novel thymidylate synthase inhibitor, 1843U89, against WiDr human colon carcinoma multicellular tumor spheroids was investigated. Continuous exposure of the spheroids to 3 nM 1843U89 for 10 days resulted in spheroid disruption, whereas 100 nM methotrexate (MTX) was required for similar effects. Short-term treatment experiments demonstrated that a 3-day exposure to 100 nM 1843U89 caused spheroid disruption 9 days after drug removal. A 4-day exposure to 10 nM 1843U89 caused spheroid disruption 8 days after drug removal. In contrast, treatment with 10 or 100 nM 1843U89 for 6-48 h or treatment with 1 nM 1843U89 for up to 5 days caused only growth delay. Continuous exposure of spheroids to 30 nM 1843U89 in the presence of 0.05-0.3 microM thymidine was as effective in causing spheroid disruption as treatment in the absence of thymidine, but treatment in the presence of 0.7-3.0 microM thymidine caused partial reversal of spheroid disruption. The results of these experiments suggest that 1843U89 should have potent solid tumor activity in humans but should be less effective in mice due to differences in circulating thymidine levels (0.1 vs 1 microM, respectively).

Topics: Antineoplastic Agents; Carcinoma; Cell Division; Colonic Neoplasms; Humans; Indoles; Isoindoles; Methotrexate; Quinazolines; Thymidine; Thymidylate Synthase; Time Factors; Tumor Cells, Cultured

Studies on a series of benzoquinazoline folate analogues as inhibitors of human thymidylate synthase led to the selection of 1843U89 for further evaluation. This compound had a Ki of 90 pM versus human thymidylate synthase and was noncompetitive with (6R,S)-5,10-methylenetetrahydrofolate. It was a good substrate for the addition of the second glutamate by hog liver folylpolyglutamate synthetase, having a Vmax/Km value 7.8-fold higher than (6R,S)-tetrahydrofolate. The data indicate that 1843U89 was transported into cells via the reduced folate carrier. The Kt for 1843U89 in MOLT-4 cells was 0.33 microM, which was 3-fold lower than that for methotrexate and 16-fold lower than that for (6S-5-formyltetrahydrofolate. V/K values were 20.3 for 1843U89 versus 1.2 and 1.9 for methotrexate and (6S)-5-formyltetrahydrofolate, respectively. It was a potent inhibitor of the growth of human cells, having 50% inhibitory concentrations below 1 nM for all cell lines tested. Growth inhibition was reversed by thymidine alone, indicating that thymidylate synthase was the only site of action of this compound. Growth inhibition was not affected by (6R,S-5-formyltetrahydrofolate at concentrations below 5 microM. However, the 50% inhibitory concentration increased when the concentration in the medium was increased to 100 microM, presumably due to competition for transport. Relative to the human cell lines used, murine cell lines were 80-1300-fold less sensitive to 1843U89 and the other benzoquinazolines tested. This decreased sensitivity appeared to be due, at least in part, to decreased transport or accumulation in murine cells. Ki values for inhibition of methotrexate transport for the benzoquinazolines were 5-17-fold higher in L1210 cells than in MOLT-4 cells. 1843U89, the benzoquinazoline which was transported most efficiently and which was the most potent inhibitor of the growth of human cells, exhibited the largest difference between binding to the MOLT-4 human and L1210 murine transporter. The V/K for L1210 transport was 80-fold less than that for MOLT-4. Initial antitumor studies, using the human thymidine kinase-deficient line GC3TK- to circumvent problems associated with murine transport as well as the high circulating thymidine levels in mice, indicated that 1843U89 had marked in vivo antitumor activity.

Topics: Animals; Binding, Competitive; Breast Neoplasms; Cell Division; Colonic Neoplasms; Female; Humans; Indoles; Isoindoles; Leucovorin; Leukemia L1210; Leukemia, T-Cell; Methotrexate; Quinazolines; Structure-Activity Relationship; Subrenal Capsule Assay; Tetrahydrofolates; Thymidylate Synthase