Numerous new antifolate drugs have been developed in an attempt to overcome the potential mechanisms of tumor cell resistance to methotrexate, which can include decreased drug transport into cells; decreased polyglutamation, leading to increased drug efflux from cells; decreased drug affinity for folate-dependent enzymes; mutations of dihydrofolate reductase (DHFR), a key enzyme required for the maintenance of adequate intracellular reduced folate levels that is inhibited by methotrexate; and increased expression of the DHFR protein. Promising antifolate compounds undergoing clinical testing as anticancer agents include trimetrexate (which was recently approved by the FDA for the treatment of Pneumocystis carinii pneumonia), edatrexate, piritrexim, Tomudex, and lometrexol. The mechnisms of action, dosage, pharmacokinetics, clinical toxicity, and antitumor activity of these drugs are profiled.
Antifolates, such as methotrexate, were one of the earliest classes of drugs developed for clinical use in cancer chemotherapy. Today, methotrexate is still used extensively in the treatment of human leukemia, breast cancer, head and neck cancer, choriocarcinoma, osteosarcoma, and lymphoma. Antifolate compounds also have important clinical utility outside the realm of oncology, in the treatment of such diverse diseases as rheumatoid arthritis, psoriasis, bacterial and plasmodial infections, and opportunistic infections associated with AIDS.
Currently, numerous promising antineoplastic antifolate drugs are in clinical development, and many of these agents have interesting and unique mechanisms of action. An understanding of the cellular pharmacology of these agents will help the practicing clinician effectively use these new drugs as they become available for the treatment of human malignancies.
Mechanism of Action
The most widely used and best understood antifolate in cancer therapy is methotrexate, which differs from the essential vitamin, folic acid, by having an amino group substituted for a hydroxyl at the 4-position on the pteridine ring (Figure 1). This change transforms the enzyme substrate into a tight-binding inhibitor of dihydrofolate reductase (DHFR), a key enzyme required to maintain adequate intracellular levels of reduced folates .
Dihydrofolate reductase is critically important because folate molecules are biochemically active only in their fully reduced form as tetrahydrofolates. The tetrahydrofolates are essential cofactors that donate one-carbon groups in the enzymatic biosynthesis of thymidylate and purine nucleotide precursors for DNA synthesis (Figure 2). One reduced folate, 5,10-methylenetetrahydrofolate, participates in the reaction catalyzed by the enzyme thymidylate synthase (TS), which converts deoxyuridylate (dUMP) into thymidylate (dTMP). As a consequence of this reaction, 5,10-methylenetetrahydrofolate undergoes oxidation to dihydrofolate, which must then be reduced by DHFR back to tetrahydrofolate in order to replenish the intracellular reduced-folate pools.
Another reduced folate, 10-formyltetrahydrofolate, serves as a one-carbon donor for the reactions catalyzed by glycinamide ribonucleotide (GAR) and aminoimidazole carboxamide ribonucleotide (AICAR) transformylases. These enzymes are involved in the de novo biosynthesis of purine nucleotides. Thus, inhibition of DHFR by antifolates can lead ultimately to the decreased production of several essential precursors for DNA synthesis.
Both methotrexate and naturally occurring folate compounds can undergo intracellular metabolism to polyglutamate derivatives. These reactions, catalyzed by the enzyme folylpolyglutamyl synthase (FPGS), attach up to six glutamate residues to the pteridine ring, which help trap these molecules within the cell by decreasing their efflux. Methotrexate polyglutamates are also potent direct inhibitors of DHFR, as well as other folate-dependent enzymes, such as TS and GAR and AICAR transformylases. Furthermore, DHFR inhibition leads to the accumulation of dihydrofolate polyglutamates within the cell, which can directly inhibit the folate-dependent enzymes involved in the synthesis of thymidylate and purine nucleotides [2,3]. Thus, inhibition of DNA synthesis by the antifolates is a multifactorial process, resulting from the partial depletion of the intracellular reduced folate pool and from the direct inhibition of folate-dependent enzymes.
Administration of exogenous reduced folates, such as leucovorin calcium, to methotrexate-treated nonmalignant cells efficiently replenishes the reduced folate pool and directly competes with the drug-induced inhibition of folate-dependent enzymes. This is the biochemical rationale for the clinical use of leucovorin rescue to prevent severe toxicity in high-dose methotrexate chemotherapy regimens.
Ultimately, the depletion of thymidylate and purine nucleotide cofactors for DNA synthesis leads to a cessation of DNA synthesis, but it is not clear whether this action alone is enough to induce cell death. Lethal DNA damage resulting from a drug-induced lack of essential nucleotides may occur because of ineffective DNA repair, or because of misincorporation of uracil deoxynucleotides into DNA. Additional studies on this important issue are clearly necessary. Furthermore, the relative importance of the inhibition of thymidylate or purine nucleotide synthesis in the generation of methotrexate-induced cytotoxicity has yet to be defined.
Influx into Cells--At least two distinct, energy-dependent, carrier-mediated transport systems are responsible for the uptake of methotrexate into mammalian cells . The classic reduced-folate carrier system, which transports reduced folates, such as 5-methyltetrahydrofolate, and antifolates, such as methotrexate, has affinity constants in the micromolar range, and it is a relatively less efficient transporter of folic acid. A second carrier system that utilizes the hydrophobic membrane-associated binding protein, the human folate receptor (hFR), has a higher affinity (nanomolar range) for folic acid and reduced folates than it does for methotrexate. Some tumors, such as human ovarian cancers, overexpress the hFR on their cell surface.
The exact contribution of these two transport pathways to the uptake of methotrexate in clinical cancer chemotherapy is an area of intensive research. However, methotrexate resistance resulting from the decreased activity of one or both of these transport systems has been demonstrated in vitro, suggesting that transport deficiencies may be clinically important . More lipophilic antifolates, such as trimetrexate and piritrexim, are not substrates for these carrier-mediated folate transport systems, and can enter cells by either passive diffusion or by other transport mechanisms. Cell lines that are resistant to methotrexate because of decreased transport generally retain their sensitivity to these more lipophilic antifolate agents .
Efflux from Cells--Efflux of methotrexate from the cell is also mediated by several different transport systems, some of which are clearly distinct from the influx systems. Methotrexate efflux is not associated with the P-glycoprotein, multidrug resistance (MDR) system that has been described for numerous other antineoplastic agents. However, drug efflux is heavily influenced by the degree of methotrexate polyglutamation. As mentioned previously, both normal and malignant cells contain the enzyme FPGS, which can add glutamyl groups in a gamma peptide linkage to the pteridine ring in naturally occurring folates and in some antifolate drugs as well. This reaction serves two important functions: First, it facilitates the accumulation of intracellular folates by converting them into large anions that are less readily transported out of the cell. Second, polyglutamation enhances the affinity of methotrexate for several folate-dependent enzymes, including TS and AICAR transformylase.
Polyglutamation of methotrexate occurs more slowly compared to naturally occurring folates; however, the resulting methotrexate polyglutamates have extremely long intracellular half-lives, and can be detected in some tissues several months following a single drug administration. The accumulation of methotrexate polyglutamates in normal tissues, such as the liver, reduces the natural polyglutamation of endogenous folates, and may account for the chronic hepatotoxicity associated with methotrexate therapy . In addition, the selective nature of methotrexate cytotoxicity may result, in part, from the increased polyglutamation of methotrexate in cancer cells compared to normal tissues. The decreased ability to polyglutamate antifolates also appears to be an important mechanism of clinical drug resistance .
Binding to DHFR--Methotrexate is a competitive inhibitor of DHFR that binds noncovalently to this enzyme at the same binding site as the normal substrate, dihydrofolate. This interaction also depends on the intracellular concentration of reduced nicotinamide dinucleotide phosphate (NADPH), which is a normal cofactor for DHFR. Point mutations both inside and outside of the enzyme's active site have been identified that decrease the binding affinity of DHFR for methotrexate . Thus, mutations in the DHFR enzyme are another potential mechanism of resistance to antifolates.
Sensitivity to methotrexate cytotoxicity is highly dependent on the absolute amount of DHFR enzyme within the cell. Both human tumors and cancer cell lines that have increased levels of DHFR due to gene amplification are relatively resistant methotrexate . More subtle mechanisms may also exist that allow cells to acutely increase DHFR expression in response to antifolate treatment.
The expression of DHFR appears to be controlled at least partially by the binding of the DHFR protein to its own mRNA (Figure 3) . This binding of DHFR prevents further synthesis of the DHFR protein, resulting in an autoregulatory negative feedback loop. Excess normal substrate, such as dihydrofolate, or inhibitors, such as methotrexate, interfere with the binding of the DHFR protein to its mRNA and block this negative feedback loop, thereby allowing the synthesis of DHFR protein to continue unimpeded. This permits normal cellular function to be maintained, even in the presence of inhibitors such as methotrexate or other antifolates. The importance of this mechanism in the development of clinical antifolate resistance must be defined.
For a detailed discussion of the pharmacokinetics of methotrexate, including specific dosage and scheduling information, readers are referred to reference 1.
1. Allegra CJ: Antifolates, in Chabner BA, Collins JM (eds): Cancer Chemotherapy: Principles & Practice, pp 110-153. Philadelphia, Lippincott, 1990.
2. Allegra CJ, Fine RL, Drake JC, et al: The effect of methotrexate on intracellular folate pools in human MCF-7 breast cancer cells: Evidence for direct inhibition of purine synthesis. J Biol Chem 261:6478-6485, 1986.
3. Allegra CJ, Hoang K, Yeh GC, et al: Evidence for direct inhibition of de novo purine synthesis in human MCF-7 breast cells as a principal mode of metabolic inhibition by methotrexate. J Biol Chem 262:13520-13526, 1987
4. Chu E, Takimoto CH: Antimetabolites, in DeVita VT, Hellman S, Rosenberg SA (eds): Cancer: Principles & Practice of Oncology, 4th Ed, pp 358-374. Philadelphia, Lippincott, 1993.
5. Marshall JL, DeLap RJ: Clinical pharmacokinetics and pharmacology of trimetrexate. Clin Pharmacokinet 26:190-200, 1994.
6. Li WW, Lin JT, Tong WP, et al: Mechanisms of natural resistance to antifolates in human soft tissue sarcomas. Cancer Res 52:1434-1438, 1992.
7. Curt CA, Carney DN, Cowan KH, et al: Unstable methotrexate resistance in human small-cell carcinoma is associated with double minute chromosomes. N Engl J Med 308:199-202, 1983.
8. Chu E, Takimoto CH, Voeller D, et al: Specific binding of human dihydrofolate reductase protein to dihydrofolate reductase messenger RNA in vitro. Biochemistry 32:4756-4760, 1993.
9. Fleming GF, Schilsky RL: Antifolates: The next generation. Semin Oncol 19:707-719, 1992.
10. Arkin H, Ohnuma T, Kamen BA, et al: Multidrug resistance in a human leukemic cell line selected for resistance to trimetrexate. Cancer Res 49:6556-6561, 1989.
11. Fry DW, Besserer JA: Characterization of trimetrexate transport in human lymphoblastoid cells and development of impaired influx as a mechanism of resistance to lipophilic antifolates. Cancer Res 48:6986-6991, 1988.
12. Rogers P, Allegra CJ, Murphy RF, et al: Bioavailability of oral trimetrexate in patients with acquired immunodeficiency syndrome. Antimicrob Agents Chemother 32:324-326, 1988.
13. Weis RB, James WD, Major WB, et al: Skin reactions induced by trimetrexate, an analog of methotrexate. Invest New Drugs 4:159-163, 1986.
14. Duch DS, Edelstein MP, Nichol CA: Inhibition of histamine metabolic enzymes and elevation of histamine levels in tissues by lipid soluble anticancer folate antagonists. Mol Pharmacol 18:100-104, 1980.
15. Robert F: Trimetrexate as a single agent in patients with advanced head and neck cancer. Semin Oncol 15:22-26, 1988.
16. Maroun J: Clinical response to trimetrexate as sole therapy for non small cell lung cancer. Semin Oncol 15:17-21, 1988.
17. Fossella FV, Winn RJ, Holoye PY, et al: Phase II trial of trimetrexate for unresectable or metastatic non-small cell bronchogenic carcinoma. Invest New Drugs 10:331-335, 1992.
18. Gesme DH, Jett JR, Schreffler DD, et al: A randomized phase II trial of amonafide or trimetrexate in patients with advanced non-small cell lung cancer: A trial of the North Central Cancer Treatment Group. Cancer 71:2723-2726, 1993.
19. Kris MG, D'Acquisto RW, Gralla RJ, et al: Phase II trial of trimetrexate in patients with stage III and IV non-small-cell lung cancer. Am J Clin Oncol 12:24-26, 1989.
20. Witte RS, Elson P, Khandakar J, et al: An Eastern Cooperative Oncology Group phase II trial of trimetrexate in the treatment of advanced urothelial carcinoma. Cancer 73:688-691, 1994.
21. Scher HI, Curley T, Geller N, et al: Trimetrexate in prostatic cancer: Preliminary observations on the use of prostate-specific antigen and acid phosphatase as a marker in measurable hormone-refractory disease. J Clin Oncol 8:1830-1838, 1990.
22. Eisenhauer EA, Wierzbicki R, Knowling M, et al: Phase II trials of trimetrexate in advanced adult soft tissue sarcoma. Ann Oncol 2:689-690, 1991.
23. Licht JD, Gonin R, Antman KH: Phase II trial of trimetrexate in patients with advanced soft-tissue sarcoma. Cancer Chemother Pharmacol 28:223-225, 1991.
24. Leiby JM: Trimetrexate: A phase 2 study in previously treated patients with metastatic breast cancer. Semin Oncol 15:27-31, 1988.
25. Dawson NA, Costanza ME, Korzun AH, et al: Trimetrexate in untreated and previously treated patients with metastatic breast cancer: A Cancer and Leukemia Group B study. Med Pediatr Oncol 19:283-288, 1991.
26. Alberts AS, Falkson G, Badata M, et al: Trimetrexate in advanced carcinoma of the esophagus. Invest New Drugs 6:319-321, 1988.
27. Falkson G, Ryan LM, Haller DB: Phase II trial for the evaluation of trimetrexate in patients with inoperable squamous carcinoma of the esophagus. Am J Clin Oncol 15:433-435, 1992.
28. Vogelzang NJ, Weissman LB, Herndon JE, et al: Trimetrexate in malignant mesothelioma: A Cancer and Leukemia Group B Phase II study. J Clin Oncol 12:1436-1442, 1994.
29. Witte RS, Elson P, Bryan GT, et al: Trimetrexate in advanced renal cell carcinoma. Invest New Drugs 10:51-54, 1992.
30. Weiss GR, Liu PY, O'Sullivan J, et al: A randomized phase II trial of trimetrexate or didemnin B for the treatment of metastatic or recurrent squamous carcinoma of the uterine cervix: A Southwest Oncology Group trial. Gynecol Oncol 45:303-306, 1992.
31. Carlson RW, Doroshow JH, Odujinrin OO, et al: Trimetrexate in locally advanced or metastatic adenocarcinoma of the pancreas: A phase II study of the Northern California Oncology Group. Invest New Drugs 8:387-389, 1990.
32. Ajani JA, Abbruzzese JL, Faintuch JS, et al: A phase II study of trimetrexate therapy for metastatic colorectal carcinoma. Cancer Invest 8:619-621, 1990.
33. Odujinrin O, Goldberg D, Doroshow J, et al: Treatment of metastatic malignant melanoma with trimetrexate: A phase II study. Med Pediatr Oncol 18:49-52, 1990.
34. Iscoe NA, Eisenhauer EA, Bodurtha AJ: Phase II study of trimetrexate in malignant melanoma: A NCI of Canada Clinical Trials Group study. Invest New Drugs 8:121-123, 1990.
35. Caincross JG, Eisenhauer EA, Macdonald DR, et al: Phase II study of trimetrexate in recurrent anaplastic glioma: National Cancer Institute of Canada Clinical Trials Group study. Can J Neurol Sci 17:21-23, 1990.
36. Romanini A, Li WW, Colofiore JR, et al: Leucovorin enhances cytotoxicity of trimetrexate/fluorouracil, but not methotrexate/fluorouracil in CCRF-CEM cells. J Natl Cancer Inst 84:1033-1038, 1992.
37. Conti JA, Kemeny N, Seiter K, et al: Trial of sequential trimetrexate, fluorouracil, and high-dose leucovorin in previously treated patients with gastrointestinal ca. J Clin Oncol 12:695-700, 1994.
38. Hudes GR, LaCreta F, Walczak J, et al: Pharmacokinetic study of trimetrexate in combination with cisplatin. Cancer Res 51:3080-3087, 1991.
39. Mattson K, Maasilta P, Tammilehto L, et al: Trimetrexate and cyclophosphamide for metastatic inoperable non small cell lung cancer. Semin Oncol 15:31-37, 1988.
40. Allegra CJ, Chabner BA, Tuazon CU, et al: Trimetrexate for the treatment of Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome. N Engl J Med 317:978-985, 1987.
41. Grant SC, Kris MG, Young CW, et al: Edatrexate, an antifolate with antitumor activity: A review. Cancer Invest 11:36-45, 1993.
42. Green MD, Sherman P, Zalcberg J: Phase II study of 10-EdAM in patients with squamous cell ca of the head and neck, previously untreated with chemo. Invest New Drugs 10:21-34, 1992.
43. Schornagel JH, Verweij J, de Mulder PH, et al: A phase II trial of 10-ethyl-10-deaza-aminopterin, a novel antifolate, in patients with advanced and/or recurrent squamous cell carcinoma of the head and neck: The EORTC Head and Neck Cancer Cooperative Group. Ann Oncol 3:223-226, 1992.
44. Vandenberg TA, Pritchard KI, Eisenhauer EA, et al: Phase II study of weekly edatrexate as first-line chemotherapy for metastatic breast cancer: A National Cancer Institute of Canada Clinical Trials Group study. J Clin Oncol 11:1241-1244, 1993.
45. Schornagel JH, van der Vegt S, Verweij J, et al: Phase II study of edatrexate in chemotherapy-naive patients with metastatic breast cancer. Ann Oncol 3:549-552, 1992.
46. Booser DJ, Dye CA, Clements SB, et al: Edatrexate (10-EDAM) for metastatic breast cancer: Phase II study. Proc Am Soc Clin Oncol 13:109, 1994.
47. Perez EA, Whitall D, Hesketh PJ: Phase I trial of biweekly edatrexate in metastatic breast cancer. Proc Am Soc Clin Oncol 13:59, 1994.
48. Shum KY, Kris MG, Gralla RJ, et al: Phase II study of 10-ethyl-10-deaza-aminopterin in patients with stage III and IV non-small-cell-lung cancer. J Clin Oncol 6:446-450, 1988.
49. Souhami RL, Rudd RM, Spiro SG, et al: Phase II study of edatrexate in stage III and IV non-small-cell lung cancer. Cancer Chemother Pharmacol 30:465-468, 1992.
50. Lee JS, Libshitz HI, Murphy WK, et al: Phase II study of 10-ethyl-10-deaza-aminopterin for stage IIIB or IV non-small-cell lung cancer. Invest New Drugs 8:299-304, 1990.
51. Yokoyama A, Kinameri K, Kurita Y, et al: Phase II study of 10EDAM in non small cell lung cancer. Proc Am Soc Clin Oncol 13:358, 1994.
52. Casper ES, Christman KL, Schwart GK, et al: Edatrexate in patients with soft tissue sarcoma: Activity in malignant fibrous histiocytoma. Cancer 72:766-770, 1993.
53. Moore D, Pazdur R, Bready B, et al: Phase II trial of edatrexate in hepatocellular carcinoma. Proc Am Soc Clin Oncol 12:216, 1993.
54. Moore DF, Pazdur R, Abbruzzese JL, et al: Phase II trial of edatrexate in patients with advanced pancreatic adenocarcinoma. Ann Oncol 5:286-287, 1994.
55. Wiesenfeld M, Jett JR, Su JQ, et al: Phase II trial of edatrexate in small cell lung cancer. Cancer 73:1189-1193, 1994.
56. Kemeny N, Israel K, O'Hehir M, et al: Phase II trial of 10-EDAM in patients with advanced colorectal carcinoma. Am J Clin Oncol 13:42-44, 1990.
57. Schultz PK, Liebertz C, Kelly WK, et al: Post-therapy change in prostatic-specific antigen levels as a clinical trial endpoint in hormone-refractory prostatic cancer: A trial with 10-ethyl-deaza-aminopterin. Urology 44:237-241, 1994.
58. Verma S, Quirt IC, Eisenhauer EA, et al: A phase II study of weekly edatrexate in metastatic melanoma: An NCI of Canada Clinical Trials Group study. Ann Oncol 4:254-255, 1993.
59. Casper ES, Schwartz GK, Johnson B, et al: Phase II trial of edatrexate in patients with advanced pancreatic adenocarcinoma. Invest New Drugs 10:313-316, 1992.
60. Grunberg SM, Spears CP, Natale R, et al: Phase I evaluation of high-dose edatrexate with leucovorin rescue. Proc Am Soc Clin Oncol 13:146, 1994.
61. Pisters KMW, Tyson LB, Bertino JR, et al: High dose edatrexate with oral leucovorin rescue: A phase I trial. Proc Am Soc Clin Oncol 13:364, 1994.
62. Adamson PC, Balis FM, Miser J, et al: Pediatric phase I trial and pharmacokinetic study of piritrexim administered orally on a 5 day schedule. Cancer Res 50:4464-4467, 1990.
63. Adamson PC, Balis FM, Miser J, et al: Pediatric phase I trial, pharmacokinetic study, and limited sampling strategy for piritrexim administered on a low-dose, intermittent schedule. Cancer Res 52:521-524, 1992.
64. de Wit R, Kaye SB, Roberts JT, et al: Oral piritrexim, an effective treatment for metastatic urothelial cancer. Br J Cancer 67:388-390, 1993.
65. Uen WC, Huang AT, Mennel R, et al: A phase II study of piritrexim in patients with advanced squamous head and neck cancer. Cancer 69:1008-1011, 1992.
66. Degardin M, Domenge C, Cappelaere P, et al: Phase II piritrexim study in recurrent and/or metastatic head and neck cancer. Proc Am Soc Clin Oncol 11:244, 1992.
67. Feun LG, Gonzales R, Savaraj N, et al: Phase II trial of piritrexim in metastatic melanoma using intermittent, low-dose administration. J Clin Oncol 9:464-467, 1991.
68. Schiesel JD, Carabasi M, Magill G, et al: Oral piritrexim, a phase II study in patients with advanced soft tissue sarcoma. Invest New Drugs 10:97-98, 1992.
69. de Vries EG, Gietema JA, Workman P, et al: A phase II and pharmacokinetic study with oral piritrexim for metastatic breast cancer. Br J Cancer 68:641-644, 1993.
70. Vokes EE, Haraf DJ, McEvilly JM, et al: Neoadjuvant PFL augmented by methotrexate and piritrexim followed by concomitant chemoradiotherapy for advanced head and neck cancer. Ann Oncol 3:79-81, 1992.
71. Vokes EE, Dimery IW, Jacobs CD, et al: A phase II study of piritrexim in combination with methotrexate in recurrent and metastatic head and neck cancer. Cancer 67:2253-2257, 1991.
72. Jackman AL, Taylor GA, Gibson W, et al: ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent inhibitor of L1210 tumor cell growth in vitro and in vivo. Cancer Res 51:5579-5586, 1991.
73. Clarke SJ, Ward J, de Boer M, et al: Phase I study of the new thymidylate synthase inhibitor Tomudex (ZD1694). Ann Oncol 5:240, 1994.
74. Sorenson JM, Jordan E, Grem JL, et al: Phase I trial of ZD1694 (Tomudex), a direct inhibitor of thymidylate synthase. Ann Oncol 5:241, 1994.
75. Zalcberg J, Cunningham D, Van Cutsem E, et al: Good antitumour activity of the new thymidylate synthase inhibitor Tomudex (ZD1694) in colorectal cancer. Ann Oncol 5:243, 1994.
76. Cunningham D, Zalcberg J, Francois E, et al: Tomudex (ZD1694) a new thymidylate synthase inhibitor with good antitumor activity in advanced colorectal cancer. Proc Am Soc Clin Oncol 13:199, 1994.
76. Smith IE, Spielmann M, Bonneterre J, et al: Tomudex (ZD1694), a new thymidylate synthase inhibitor with antitumour activity in breast cancer. Ann Oncol 5:242, 1994.
78. Pazdur R, Casper ES, Meropol NJ, et al: Phase II trial of Tomudex (ZD1694), a thymidylate synthase inhibitor in advanced pancreatic cancer. Proc Am Soc Clin Oncol 13:207, 1994.
79. Burris H, Von Hoff D, Bowen K, et al: A phase II trial of ZD1694, a novel thymidylate synthase inhibitor in patients with advanced non-small cell lung cancer. Ann Oncol 5:244, 1994.
80. Gore M, Earl H, Cassidy J, et al: Phase II study of Tomudex (ZD1694) in refractory ovarian cancer. Ann Oncol 5:245, 1994.
81. Beardsley GP, Moroson BA, Taylor EC, et al: A new folate antimetabolite, 5,10-dideaza-5,6,7,8-tetrahydrofolate is a potent inhibitor of de novo purine synthesis. J Biol Chem 164:328-333, 1989.
82. Pizzorno G, Sololoski JA, Cashmore AR, et al: Intracellular metabolism of 5,10-dideazatetrahydrofolic acid in human leukemia cell lines. Mol Pharmacol 39:85-89, 1991.
83. Ray MS, Muggia FM, Leichman CG, et al: Phase I study of (6R)-5,10-dideazatetrahydrofolate: A folate antimetabolite inhibitory to de novo purine synthesis. J Natl Cancer Inst 85:1154-1159, 1993.
84. Pagani O, Sessa C, deJong J, et al: Phase I studies of lometrexol (DDATHF) given in combination with leucovorin. Proc Am Soc Clin Oncol 11:109, 1992.
85. Rinaldi DA, Burris HA, Dorr FA, et al: A phase I evaluation of the novel thymidylate synthase inhibitor, LY231514, in patients with advanced solid tumors. Proc Am Soc Clin Oncol 13:159, 1994.
86. Clendeninn NJ, Peterkin JJ, Webber S, et al: AG-331, a "non-classical" lipophilic thymidylate synthase inhibitor for the treatment of solid tumors. Ann Oncol 5:246, 1994.
87. Rafi I, Taylor GA, Balmanno K, et al: A phase I study of the novel antifolate 3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)-quinazolone dihydrochloride (AG337) given by a 24-hour intravenous continuous infusion. Ann Oncol 5:238, 1994.