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Phase I study of phosphonacetyl-l-aspartate, 5-fluorouracil, and leucovorin in patients with advanced cancer


Low-dose phosphonacetyl-l-aspartate (PALA) may potentiate both 5-fluorouracil (5-FU) incorporation into RNA and thymidylate synthase inhibition by 5-fluorodeoxyuridylate (5-FdUMP). The gastrointestinal toxicity of 5-FU is not increased by PALA administration. Exogenous leucovorin, on the other hand, which enhances thymidylate synthase inhibition, appears to increase the clinical toxicity of 5-FU in a dose-dependent manner. As a result, the clinical use of high-dose leucovorin requires a marked dose reduction of 5-FU. Extracellular leucovorin levels of 1 μM suffice to maximize the enhancement of thymidylate synthase inhibition in several models. We conducted a trial to add leucovorin to the PALA/5-FU regimen. We chose a leucovorin dose that was predicted to yield end-infusion total reduced folate concentrations of 1 μM. The major endpoint was to determine the maximum tolerated dose of 5-FU in this combination. The regimen consisted of 250 mg/m2 PALA given on day 1 and, 24 h later, escalating 5-FU doses ranging from 1,850 to 2,600 mg/m2 admixed with 50 mg/m2 leucovorin and given by 24-h infusion. Courses were repeated weekly. A total of 24 patients with a median performance status of 1 were entered at three dose levels. Diarrhea was dose-limiting; 6/13 patients had grade II or worse diarrhea at 2,600 mg/m2. Dose modification resulted in a mean dose intensity of 2,300 mg/m2 at both the 2,600- and 2,300-mg/m2 dose levels. The 2,300-mg/m2 dose is suitable for phase II testing of this regimen. Three patients (two with breast cancer and 1 with sarcoma) had a partial remission. We measured steady-state concentrations (Css) of 5-FU in 23 patients. The mean Css increased with dose from 0.738 to 1.03 μg/ml. Total body clearance did not vary with dose in this range. Patients with grade II or worse diarrhea had a higher mean Css (1.10±0.19) than those with grade O or I toxicity (0.835±0.25,P<0.02). Total bioactive folates (bound and free) were measured using a biological assay. Pretreatment values ranged from 2 to 52 nM and were not predictive of toxicity. End-infusion (23-h) values were somewhat lower than predicted and ranged from 400 to 950 nM. The risk of diarrhea was positively correlated with end-infusion total folate values. In a logistic regression analysis, total folate values obtained at 23 h were a more powerful predictor of diarrhea than were 5-FU Css values. These results confirm the contribution of leucovorin to the toxicity of the 5-FU/leucovorin combination and suggest that interpatient differences in folate pharmacology may contribute to the therapeutic index of the 5-FU/leucovorin combination.

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  1. 1.

    Arbuck SG (1987) 5-FU/leucovorin. Biochemical modulation that works? Oncology 1:61–71

  2. 2.

    Arbuck SG (1988) Overview of clinical trials using 5FU and leucovorin for the treatment of colorectal cancer. Cancer 63: 1036–1044

  3. 3.

    Ardalan B, Singh G, Silberman H (1988) Randomized phase I and II study of short-term infusion of high-dose fluorouracil with or withoutN-(phosphonacetyl)-l-aspartic acid in patients with advanced pancreatic and colorectal cancer. J Clin Oncol 6: 1053–1058

  4. 4.

    Ardalan B, Sridhar KS, Benedette P, Richman S, Waldman S, Morrell L, Feun L, Savaraj N, Fodor M, Livingstone A (1991) A phase I, II study of high-dose 5-fluorouracil and high-dose leucovorin with low-dose phosphonacetyl-l-aspartic acid in patients with advanced malignancies. Cancer 68: 1242–1246

  5. 5.

    Casper ES, Vale K, Williams LJ, Martin DS, Young CW (1983) Phase I and clinical pharmacological evaluation of biochemical modulation of 5-fluorouracil withN-(phosphonacetyl)-l-aspartic acid. Cancer Res 43: 2324–2328

  6. 6.

    Danhauser LL, Freimann JH Jr, Gilchrist TL, Gutterman JU, Hunter CY, Yeomans AC, Markowitz AV (1993) Phase I and plasma pharmacokinetic study of infusional fluorouracil combined with recombinant interferon alfa-2b in patients with advanced cancer. J Clin Oncol 11: 751–761

  7. 7.

    Doroshow JH, Multhaul P, Leong L, Margolin K, Lichfield T, Akman S, Carr B, Bertrand M, Goldberg D, Blayney D, Odujinrin O, DeLap R, Shuster J, Newman E (1990) Prospective randomized comparison of fluorouracil versus fluorouracil and high-dose continuous infusion leucovorin calcium for the treatment of advanced measurable colorectal cancer in patients previously unexposed to chemotherapy. J Clin Oncol 8: 491–501

  8. 8.

    Erlichman C, Fine S, Wong A, Elhadim T (1988) Randomized trial of fluorouracil and folinic acid in patients with metastatic colorectal carcinoma. J Clin Oncol 6: 469–475

  9. 9.

    Fleming RA, Milano GA, Etienne M-C, Renee N, Thyss A, Schneider M, Demard F (1992) No effect of dose, hepatic function, or nutritional status on 5-FU clearance following continuous (5-day) 5-FU infusion. Br J Cancer 66: 668–672

  10. 10.

    Grem JL, King SA, O'Dwyer PJ, Leyland-Jones B (1988) PALA: biochemistry and clinical activity ofN-phosphonacetyl-l-aspartate: a review. Cancer Res 48: 4441–4454

  11. 11.

    Houghton JA, Williams LG, Cheshire PJ, Wainer IW, Jadaud P, Houghton PJ (1990) Influence of dose of [6RS]leucovorin on reduced folate pools and 5-fluoracil-mediated thymidylate synthase inhibition in human colon adenocarcinoma xenografts. Cancer Res 50: 3940–3946

  12. 12.

    Hryniuk WM, Figueredo A, Goodyear M (1987) Application of dose intensity to problems in chemotherapy of breast and colorectal cancer. Semin Oncol 14 [Suppl 4]: 3–11

  13. 13.

    Leichman CG, Fleming TR, Muggia FM, Ardalan B, Doroshow J, Rangen C, MacDonald J (1993) Fluorouracil (5-FU) schedules and modulation in advanced colorectal cancer (CRC): a Southwest Oncology Group (SWOG) screening trial. Southwest Oncology Group, Seattle, Washington. Proc Am Soc Clin Oncol 12: 198

  14. 14.

    Leyland-Jones BR, O'Dwyer PJ (1986) Biochemical modulation: application of laboratory models to the clinic. Cancer Treat Rep 70: 219–229

  15. 15.

    Martin DS, Stolfi RL, Sawyer RC, Nayak R, Spiegelman S, Young CW, Woodcock T (1980) An overview of thymidine. Cancer 45: 1117–1128

  16. 16.

    Martin DS, Stolfi RL, Sawyer RC, et al (1981) Biochemical modulation of 5-fluorouracil and cytosine arabinoside with emphasis on thymidine, PALA, and 6-methylmercaptopurine riboside. In: Tattersall MHN, Fox RM (eds) Nucleosides and cancer treatment. Academic Press, Sydney, pp 339–382

  17. 17.

    Martin DS, Stolfi RL, Sawyer RC, Young CW (1985) Application of biochemical modulation with therapeutically inactive modulating agents in clinical trials of cancer chemotherapy. Cancer Treat Rep 69: 421–423

  18. 18.

    Martin DS, Stolfi RL, Colofiore JR (1988) Failure of high-dose leucovorin to improve the therapeutic index of the maximally tolerated dose of 5-fluorouracil: a murine study with clinical relevance. J Natl Cancer Inst 80: 496–501

  19. 19.

    Milano G, Etienne MC, Cassuto-Viguier E, Thyss A, Santini J, Frenay M, Renee N, Schneider M, Demard F (1992) Influence of sex and age on fluorouracil clearance. J Clin Oncol 10: 1171–1175

  20. 20.

    Miller AB, Hoogstraten B, Staquet M, Winkeler A (1981) Reporting results of cancer treatment. Cancer 47: 207–214

  21. 21.

    Moran RG, Scanlon KL (1991) Schedule-dependent enhancement of the cytotoxicity of fluoropyrimidines to human carcinoma cells in the presence of folinic acid. Cancer Res 51: 4618–4623

  22. 22.

    Newman EM, Straw JA, Doroshow JH (1989) Pharmacokinetics of diastereoisomers of (6R,S)-folinic acid (leucovorin) in humans during constant high-dose intravenous infusion. Cancer Res 49: 5755–5760

  23. 23.

    O'Connell MJ (1989) A phase III trial of 5-fluorouracil and leucovorin in the treatment of advanced colorectal cancer. Cancer 63: 1026–1030

  24. 24.

    O'Dwyer PJ (1994) Strategies for circumventing clinical resistance to antimetabolites. In: Goldstein LJ, Ozols RF (eds) Drug resistance, (Cancer treatment and research series, vol 3) Kluwer (in press)

  25. 25.

    O'Dwyer PJ, Paul AR, Walczak J, Weiner LM, Litwin S, Comis RL (1990) Phase II study of biochemical modulation of 5-fluorouracil by low-dose PALA in patients with colorectal cancer. J Clin Oncol 8: 1497–1503

  26. 26.

    O'Dwyer PJ, Hudes GR, Colofiore J, Walczak J, Hoffman J, LaCreta FP, Comis RL, Martin DS, Ozols RF (1991) Phase I trial of fluorouracil modulation byN-phosphonacetyl-l-aspartate and 6-methylmercaptopurine riboside: optimization of 6-methylmercaptopurine riboside dose and schedule through biochemical analysis of sequential tumor biopsy specimens. J Natl Cancer Inst 83:1235–1240

  27. 27.

    Petrelli N, Herrera L, Rustum Y, Burke P, Creaven P, Stulc J, Emrich LJ, Mittelman A (1987) A prospective randomized trial of 5-fluorouracil versus 5-fluorouracil and high-dose leucovorin versus 5-fluorouracil and methotrexate in previously untreated patients with advanced colorectal carcinoma. J Clin Oncol 5: 1559–1565

  28. 28.

    Petrelli N, Douglass HO, Herrera L, Russell D, Stablein DM, Brucker HW, Mayer RJ, Schinella R, Green MD, Muggia FM, Megibow A, Greenwald ES, Bukowski RM, Harris J, Levin B, Gaynor E, Loutfi A, Kalser MH, Barkin JS, Benedetto P, Woolley PV, Nauta R, Weaver DW, Leichman LP (1989) The modulation of fluorouracil with leucovorin in metastatic colorectal carcinoma: a prospective randomized phase III trial. J Clin Oncol 7:1419–1426

  29. 29.

    Poon MA, O'Connell MJ, Moertel CG, Wieand HS, Cullian SA, Everson LK, Krook JE, Mailliard JA, Laurie JA, Tschetter LK, Wiesenfeld M (1989) Biochemical modulation of fluorouracil: evidence of significant improvement of survival and quality of life in patients with advanced colorectal carcinoma. J Clin Oncol 7:1407–1417

  30. 30.

    Poon MA, O'Connell MJ, Wieand HS, Krook JE, Gerstner JB, Tschetter LK, Levitt R, Kardinal CG, Mailliard JA (1991) Biochemical modulation of fluorouracil with leucovorin: confirmatory evidence of improved therapeutic efficacy in advanced colorectal cancer. J Clin Oncol 9:1967–1972

  31. 31.

    Santi DV, McHenry CA, Sommer H (1974) Mechanism of interaction of thymidylate snythetase with 5-fluorodeoxyuridylate. Biochemistry 13:471–481

  32. 32.

    Santini J, Milano G, Thyss A, Renee N, Viens P, Ayela P, Schneider M, Demard F (1989) 5-FU therapeutic monitoring and dose adjustment leads to an improved therapeutic index in head and neck cancer. Br J Cancer 59:287–290

  33. 33.

    Speigelman S, Sawyer R, Nayak R, Ritzi E, Stolfi R, Martin D (1980) Improving the anti-tumor activity of 5-fluorouracil by increasing its incorporation into RNA via metabolic modulation. Proc Natl Acad Sci USA 77:4966–4970

  34. 34.

    Stetson PL, Shukla UA, Ensminger WD (1985) Sensitive high-performance liquid chromatographic method for the determination of 5-fluorouracil in plasma. J Chromatogr 344:385–390

  35. 35.

    Yin MB, Zakrzewski SF, Hakala MT (1983) Relationship of cellular folate cofactor pools to the activity of 5-fluorouracil. Mol Pharmacol 23:190–197

  36. 36.

    Zhang Z-G, Rustum YM (1991) Effects of diastereoisomers of 5-formyltetrahydrofolate on cellular growth, sensitivity to 5-fluoro-2′-deoxyuridine and methylenetetrahydrofolate polyglutamate levels in HCT-8 cells. Cancer Res 51:3476–3481

  37. 37.

    Zhang Z-G, Harstick A, Rustum YM (1992) Modulation of fluoropyrimidines: role of dose and schedule of leucovorin administration. Semin Oncol 19:10–15

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Correspondence to Alexandre Hageboutros.

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Supported in part by NCI CA06927, NCI CA38053, and an appropriation from the Commonwealth of Pennsylvania

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Hageboutros, A., Rogatko, A., Newman, E.M. et al. Phase I study of phosphonacetyl-l-aspartate, 5-fluorouracil, and leucovorin in patients with advanced cancer. Cancer Chemother. Pharmacol. 35, 205–212 (1995). https://doi.org/10.1007/BF00686549

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Key words

  • 5-FU
  • Biochemical modulation