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B van Triest,
H M Pinedo,
Y van Hensbergen,
K Smid,
F Telleman,
P S Schoenmakers,
C L van der Wilt, J A van Laar,
P Noordhuis,
G Jansen,
G J Peters
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ABSTRACT: Thymidylate synthase (TS), a critical enzyme in the de novo synthesis of thymidylate, is an important target for fluoropyrimidines and folate-based TS inhibitors. In a panel of 13 nonselected human colon cancer cell lines, we evaluated the role of TS levels in sensitivity to 5-fluorouracil (5FU) and four folate-based TS inhibitors that have been introduced recently into the clinic: ZD1694 (Tomudex, Raltitrexed, TDX), GW1843U89 (GW), LY231514 (LY), and AG337 (Thymitaq, AG). Because the latter compounds have different transport and polyglutamylation characteristics, we also related these parameters with drug sensitivity, measured by the sulforhodamine B assay after 72 h of drug exposure. For 5FU, the IC50s varied from 0.8 to 43.0 microM. Leucovorin (LV) potentiated the activity of 5FU in only 4 of 13 cell lines. Sensitivity to folate-based TS inhibitors was variable; IC50s were in the range of: 5.3-59.0 nM TDX; 11.0-1570 nM LY; and 0.5-8.9 nM GW. Eleven of 13 cell lines had an IC50 for AG between 1.3 and 5.3 microM. Two cell lines were resistant to AG, Colo201 and SW1116, with IC50s of 27 and 29 microM, respectively. TS catalytic activity (conversion of dUMP to dTMP) varied from 62 to 777 pmol/h/10(6) cells. The number of FdUMP binding sites varied from 32 to 231 fmol/10(6) cells. Regression analysis showed a significant relation between TS catalytic activity and IC50s for 5FU and 5FU/LV. Kis for FdUMP showed a significant Spearman rank correlation with the IC50s of AG and GW. The role of antifolate transport, accumulation, and polyglutamylation was determined with [3H]methotrexate (MTX) as a reference compound. [3H]MTX influx via the reduced folate carrier varied from 18.6 to 150 fmol/10(6) cells/min. Folylpolyglutamate synthetase (FPGS) activity showed a range from 47 to 429 pmol/10(6) cells/h. A total of 24 h of [3H]MTX accumulation showed a 20-fold variation, from 1.2 to 21.8 pmol/10(6) cells. FPGS levels showed a Spearman rank positive correlation with cytotoxicity to TDX. In conclusion, in a heterogeneous nonselected human colon cancer cell line panel, the best predictor for sensitivity to 5FU and 5FU/LV was TS activity. Multiple sensitivity determinants were of importance for antifolate TS inhibitors, including FPGS activity and TS enzyme kinetics.
Clinical Cancer Research 04/1999; 5(3):643-54. · 7.74 Impact Factor
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Endoscopy 07/1998; 30(5):S68. · 5.21 Impact Factor
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ABSTRACT: Despite more than 30 years of intensive studies on new drugs against advanced colorectal cancer, the fluoropyrimidines remain the drugs of choice for systemic treatment and for hepatic artery infusion (HAI). This overview describes new developments in advanced colorectal cancer chemotherapy, providing a rationale for more effective use of the fluoropyrimidines, with biochemical modulation, scheduling or by revealing biochemical mechanisms of action that correlate with antitumour activity. In human colorectal cancer cell lines and various animal tumour model systems 5-fluoro-2'-deoxyuridine (FdUrd) is more effective than 5-fluorouracil (5-FU). Comparably, FdUrd's modulation by leucovorin (LV) is more potent than 5-FU. In animal studies it is shown that intermittent high-bolus administration of FdUrd generates better antitumour activity, compared with equal toxic doses or any other schedule of 5-FU. These effects are related to prolonged-thymidylate synthase (TS) inhibition and the prevention of TS induction, rather than RNA incorporation. Preclinical studies with modulators such as N-phosphonacetyl-L-aspartate (PALA), WR-2721, mitomycin C and platinum derivatives provide a rationale for clinical use in the future. The first choice systemic chemotherapy of patients with advanced colorectal cancer remains 5-FU combined with LV. Some improvement in therapeutic efficacy has been achieved with locoregional HAI. In randomised studies HAI FdUrd improves the quality of life and survival as compared with optimal systemic therapy. Chronomodulation decreases toxicity, allowing dose intensification, while modulators such as LV or dexamethasone increase survival of patients treated with HAI FdUrd to 86% after 1 year. In conclusion, the clinical use of FdUrd has not been fully explored. Intermittent high-dose FdUrd, chronomodulation together with the use of modulators or drugs focused on prolonged TS inhibition, should be studied in large randomised studies.
European Journal of Cancer 03/1998; 34(3):296-306. · 5.54 Impact Factor
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Advances in experimental medicine and biology 02/1998; 431:699-704. · 1.09 Impact Factor
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ABSTRACT: 5-Fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd) are common chemotherapeutic drugs for the treatment of advanced colorectal cancer. Two recognized mechanisms of action of these agents are inhibition of thymidylate synthase (TS) and incorporation of fluorinated UTP into cellular RNA. In previous studies on drug scheduling of both fluoropyrimidines, we observed the highest therapeutic efficacy by using a weekly i.v. push schedule. Furthermore, weekly 400-mg/kg FdUrd is superior to equitoxic weekly 80-mg/kg FUra in murine Colon 26-B carcinoma. We evaluated the most important pharmacokinetic and pharmacodynamic effects of both fluoropyrimidines to delineate the biochemical mechanisms underlying their differences in therapeutic activity in this tumor model. FUra concentrations and elimination in tumors after FdUrd or FUra administration were comparable, and the level of FUra incorporation into cellular RNA following treatment with FUra or FdUrd was similar. Free tumoral 5-fluoro-dUMP levels were initially 3-fold higher after FdUrd but diminished rapidly thereafter. The number of free [3H]5-fluoro-dUMP-binding sites decreased to about 25 and 15% of control values within 2 h after treatment with equitoxic doses of FUra and FdUrd and remained low for 72 h. The duration of TS inhibition was significantly longer following treatment with FdUrd compared with FUra, 168 and 72 h, respectively. The superiority of the antitumor activity of an i.v. push of FdUrd over FUra in the treatment of Colon 26-B tumors correlates with maintenance of TS inhibition and repeated drug administration when TS remains low, whereas FUra incorporation into RNA does not appear to distinguish the antitumor response of FdUrd from that of FUra in this tumor model.
Clinical Cancer Research 09/1996; 2(8):1327-33. · 7.74 Impact Factor
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ABSTRACT: 5-Fluorouracil (FUra) is one of the few effective agents in the treatment of patients with colorectal cancer. Its effects on the target enzyme thymidylate synthase (TS) can be modulated by leucovorin (LV) or cisplatin (CDDP). Tumor size and differentiation of tumor characteristics can influence therapeutic efficacy. We therefore studied the relationship between tumor size (cutoff point 200 mm3) and the antitumor activity of FUra and its modulation by LV in murine Colon 26 and Colon 38 tumors. The doubling time of tumors measuring > 200 mm3 was about 160% longer. The antitumor effect of FUra in these large tumors was decreased and could not be modulated by LV. In addition, three subtypes of Colon 26 (Colon 26-A, Colon 26-B, and Colon 26-10) were identified and characterized for tumor-induced weight loss, TS activity, response to chemotherapy, and histological features. Mice bearing Colon 38 and Colon 26-10 did not lose weight as a result of tumor growth. Colon 26-A caused a weight loss of up to 19%, whereas mice with Colon 26-B tumors remained within 10% of their initial weight and tolerated at least 2.5 times more tumor load than did mice bearing Colon 26-A, which induces cachexia. Among untreated tumors, TS catalytic activity was highest in Colon 26-B (5536 pmol mg protein(-1) h(-1)) and lowest in Colon 38 (799 pmol mg protein(-1) h(-1)); Colon 26-A and Colon 26-10 had intermediate activities (about 2500 pmol mg protein(-1) h(-1)). 5-Fluoro-2'-deoxyuridine monophosphate (FdUMP) binding was comparable in the three Colon 26 subtypes but was lower in Colon 38. The antitumor activity of FUra could be modulated by LV in Colon 38, Colon 26-10, and Colon 26-A but could not in Colon 26-B, with complete responses (CR) being obtained in Colon 26-10 and Colon 38. The latter two were highly sensitive to CDDP, followed by Colon 26-A and Colon 26-B (CRs: 50%, 40%, 25%, and 0, respectively). Furthermore, necrosis was noted in Colon 26-B and Colon 38 but not in Colon 26-A. In conclusion, (1) the antitumor activity of FUra in large tumors is decreased and cannot be modulated by LV and (2) characteristics and sensitivity to chemotherapeutics can vary substantially in closely related tumors of the same origin.
Cancer Chemotherapy and Pharmacology 02/1996; 39(1-2):79-89. · 2.83 Impact Factor
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ABSTRACT: 5-Fluorouracil (5FU) can exert its cytotoxic activity by either inhibition of thymidylate synthase or incorporation into RNA. The extent and importance of the latter in tumors of patients are not clear, due to the lack of sensitive and reproducible methods. RNA from 5FU-treated human WiDr colon tumor cells was isolated and [(14)CL]5FU incorporation into RNA was measured by traditional scintillation counting while that of nonradiolabeled 5FU was measured with the present, new method. For the latter purpose, isolated RNA was incubated with RNase, alkaline phosphatase, and uridine phosphorylase, resulting in a complete degradation of RNA, nucleotides, and nucleoside to 5FU. 5FU was then measured with gas chromatography coupled to mass spectrometry. For both methods RNA incorporation was 0.4 pmol/h/micrograms RNA at 25 microM 5FU while a similar time (up to 4 h) and concentration dependence (25 to 50 microM) were found. Reproducibility of the assay was more than 95%. In a murine colon tumor 5FU incorporation into RNA reached a peak of 10 pmol/micron RNA at 2 h after administration of the the maximum tolerated dose of 80 mg5FU/kg, which was retained until at least 72 h at 2.5 pmol/micron. In tumors from patients treated with 500 mg5FU/m(2) incorporation into RNA after 24 h amounted to 1.0-1.5 pmol/micrograms RNA. In conclusion, a novel approach, combining different sensitive and reproducible techniques, was established to measure 5FU incorporation into RNA in clinical tumor specimens enabling determination of its clinical relevance.
Analytical Biochemistry 11/1995; 231(1):157-63. · 3.00 Impact Factor
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ABSTRACT: Modulation of the therapeutic efficacy of cisplatin (CDDP) and 5-fluoro-2'-deoxyuridine (FdUrd) alone and in combination with N-phosphonacetyl-L-aspartate (PALA) was evaluated in mice bearing colon carcinoma (C-26) using a weekly intravenous (i.v.) push schedule for 3 weeks. A non-toxic dose of PALA (100 mg/kg) was administered i.v. 24 h prior to the i.v. administration of CDDP +/- FdUrd. The maximum tolerated doses (MTD) of CDDP and FdUrd when used as a single agent were 9 and 400 mg/kg, respectively. In combination, however, the MTD of CDDP and FdUrd were 2.5 and 300 mg/kg, respectively. PALA did not significantly affect the MTD. PALA improved the antitumour activity of CDDP or FdUrd when used alone; however, the highest tumour response, 66% complete tumour regression, was achieved with a PALA modulation of CDDP and FdUrd in combination.
European Journal of Cancer 07/1995; 31A(6):974-6. · 5.54 Impact Factor
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ABSTRACT: Continuous infusions of 5-fluorouracil (5-FU) are increasingly used in the treatment of cancer. Their optimal use, however, has still to be determined since the availability of suitable animal models is limited. We studied continuous infusions in mice using subcutaneously implanted pellets that release 5-FU over a period of 3 weeks. At the maximum tolerated dose (MTD) (based on the systemic toxicity in healthy animals) we assessed the antitumour activity, haematological toxicity, inhibition of thymidylate synthase (TS) in tumours and the concentration of 5-FU in plasma during the 3-week period. We also studied the addition of leucovorin in different schedules. The dose-limiting toxicity was weight loss, and at the MTD of 10 mg of 5-FU released in 21 days per mouse myelosuppression was tolerable (nadir for leucocytes and thrombocytes was approximately 40% of pretreatment levels). In several independent experiments using the 5-FU-resistant Colon 26 tumour, a good antitumour activity was observed during the first part of the infusion, but thereafter the growth of the tumours resumed; the overall effect of continuous infusions was thus comparable to that of bolus injections. Coadministration of leucovorin did not enhance the therapeutic results; depending on the schedule used, it proved ineffective or only increased toxicity. Similar results were obtained with head and neck squamous cell carcinomas and with the 5-FU-sensitive tumour Colon 38. In Colon 26 tumours the TS activity (FdUMP-binding assay) initially decreased to 20-30% of controls and returned to normal after 11 days. In the catalytic TS assay a slight inhibition was observed for the continuous infusion, followed after 11 days by a marked (4-fold) increase in activity. 5-FU plasma levels varied from 0.1 to 1 microM following a circadian rhythm (with a peak at 6 h after light onset), and were maintained during the entire period. Subcutaneously implanted pellets represent a suitable model to study prolonged administration of 5-FU in mice and to evaluate the effect of modulating agents in laboratory animals before transferring data obtained in vitro to the clinic.
European Journal of Cancer 02/1995; 31A(9):1517-25. · 5.54 Impact Factor
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Advances in experimental medicine and biology 02/1994; 370:109-14. · 1.09 Impact Factor
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ABSTRACT: We have investigated the effects of N-(phosphonacetyl)-L-aspartate (PALA) administered i.v. as a single dose (100 mg/kg) on the antitumor activity of 5-fluoro-2'-deoxyuridine (FdUrd) and 5-fluorouracil (FUra), on the pharmacokinetic parameters of FdUrd and FUra, and on the tumor pyrimidine ribonucleotide triphosphate pools in mice bearing advanced colon carcinoma 26 and leukemia 1210. The antitumor activity was evaluated with PALA administered i.v. 24 h prior to the maximum tolerated dose of FUra and FdUrd administered by: (a) 4 days of continuous infusion (schedule 1, c.i. days 1-4); (b) daily for 4 days by i.v. push (schedule 2, i.v. days 1-4); and (c) weekly for 3 weeks (schedule 3, i.v. weekly for 3 weeks). The maximum tolerated doses of FdUrd were 20, 150, and 400 mg/kg/day and for FUra were 25, 50, and 80 mg/kg/day for schedule 1, 2, and 3, respectively. At the maximum tolerated doses, the antitumor activity in mice bearing advanced colon carcinoma can be summarized as follows: (a) FdUrd is significantly more active than FUra; (b) for both drugs the weekly for 3 weeks i.v. push schedule is superior to the c.i. or i.v. push daily for 4 days schedules; (c) pretreatment with PALA enhances the antitumor activity of FdUrd and FUra and resulted in 95 and 13% complete responses, respectively; (d) long-term survivors with FUra could only be achieved in the presence of PALA; in mice bearing leukemia 1210 cells, FdUrd or FUra with or without PALA exhibited no significant antitumor activity when PALA was administered in a single dose 24 h prior to fluoropyrimidine treatment; and (e) in C-26 and L1210, PALA reduced the pools of CTP and UTP equally, to about 10% of controls with significant difference in their rates of recovery.
Cancer Research 05/1993; 53(7):1560-4. · 7.86 Impact Factor
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Advances in experimental medicine and biology 02/1993; 339:9-20; discussion 21. · 1.09 Impact Factor
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ABSTRACT: We evaluated the effects of WR-2721 and its metabolite WR-1065 on in vitro growth inhibition by 5-fluorouracil (5FU) and cisplatin (CDDP) and the effect of WR-2721 on in vivo toxicity and antitumor effect of 5FU and CDDP. In cell culture both WR-2721 and WR-1065 were not able to reverse growth inhibition caused by either 5FU or CDDP. Administration of WR-2721 i.p. at 525 mg/kg to mice resulted in a severe temperature drop to 27 degrees C; at 200 mg/kg hypothermia was less severe. WR-2721 failed to prevent 5FU toxicity, but the maximum tolerated dose of CDDP in the combination with 5FU (at 100 mg/kg) could be increased from 3 to 7 mg/kg. CDDP at 7 mg/kg enhanced leukopenia caused by 5FU at 100 mg/kg to 20% and thrombocytopenia to 40%; WR-2721 reduced leukopenia and prevented thrombocytopenia induced by the combination. Combination of CDDP, 5FU, and WR-2721 resulted in an enhanced antitumor activity against the murine colon tumor Colon 26 compared to 5FU alone and to 5FU combined with CDDP at their maximum tolerated dose.
International Journal of Radiation OncologyBiologyPhysics 02/1992; 22(4):785-9. · 4.11 Impact Factor
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ABSTRACT: We evaluated the effects of WR-2721 on the toxicity and antitumor activity of the combination of 5-fluorouracil (5FU) and carboplatin (CBDCA) in BALB/c and C57B1/6 mice. On a weekly schedule, i.p. injection of 200 mg/kg WR-2721 at 5 min prior to the administration of this combination enabled us to increase the CBDCA dose from a nontoxic level of 45 mg/kg to a normally toxic dose of 60 mg/kg in non-tumor-bearing BALB/c mice while maintaining the 5FU dose at 100 mg/kg. When WR-2721 was given 30 min before this combination, the CBDCA dose could not be increased to 60 mg/kg without producing drug-related deaths. WR-2721 protected against CBDCA- and 5-FU-induced thrombocytopenia but did not prevent leukopenia or anemia in C57B1/6 mice. The antitumor activity of the combination against colon 26 tumors in BALB/c mice was increased by pretreatment with WR-2721, which facilitated elevation of the CBDCA dose to 60 mg/kg in combination with 100 mg/kg 5FU. These results reveal better therapeutic efficacy for the combination of 5FU and CBDCA following pretreatment with WR-2721.
Cancer Chemotherapy and Pharmacology 02/1992; 31(2):97-102. · 2.83 Impact Factor
