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Folate status and the safety profile of antifolates
Folate Status and the Safety Profile of Antifolates Hilary Calvert Throughout the history of cancer chemotherapy, the control of drug-related toxicity has been a major concern. Antifolates such as methotrexate also have a reputation for sporadic and unpredictable toxicity. Pretreatment levels of plasma or red cell folate have not been found to be useful in predicting which patients will develop toxicity. During the phase II development of pemetrexed, the plasma levels of homocysteine and methylmalonic acid were studied as sensitive surrogate markers for folate and vitamin B12 status, respectively. These were found to be strongly correlated with the subsequent development of serious drug-related toxicities (myelosuppression, diarrhea, mucosal toxicity, and infection), suggesting that toxicity was related to relative folate deficiency in some cancer patients. A policy of nutritional supplementation was introduced and led to a marked reduction in toxicity and the abolition of treatment-related deaths with apparent preservation of anticancer activity. Semin Oncol 29 (suppl 5):3-7. Copyright 2002, Elsevier Science (USA). All rights reserved.
F
OR EVERY antifolate drug introduced into clinical practice, folic acid or its reduced derivatives have been found to be capable of modulating the toxicity and/or efficacy. This article reviews folic acid and drug interactions for two licensed drugs (methotrexate and raltitrexed) and two investigational agents (lometrexol and pemetrexed [Alimta, LY231514; Eli Lilly and Co, Indianapolis, IN]). In the case of pemetrexed, the study of plasma vitamin metabolite levels (homocysteine and methylmalonic acid) has provided insight into the interaction of nutritional status with chemotherapy and has allowed the development of innovative treatment protocols. METHOTREXATE
In the late 1940s, Dr Sidney Farber first noted that folic acid levels were low in children with leukemia, an observation that showed the role of folic acid in proliferating cells and led to the development of the first antifolates, aminopterin and methotrexate.1 These drugs were the first to induce remissions in childhood leukemia but were also found to be capable of causing serious toxicities. Indeed, some investigators attributed their antileukemic effects directly to their toxicity.2 Nevertheless, the therapeutic effects of methotrexate in both leukemia and solid tumors led to expansion of its use for many indications in cancer Seminars in Oncology, Vol 29, No 2, Suppl 5 (April), 2002: pp 3-7
treatment and a consequent interest in the sporadic toxic events induced by the drug. Severe methotrexate toxicity consisting of myelosuppression with significant neutropenia and gastrointestinal toxicity with symptoms of diarrhea and mucosal damage have been reported sporadically on most methotrexate schedules. When complicated by gram-negative sepsis, it frequently resulted in drug-related death.3 The clinical use of methotrexate generated a huge interest in folate metabolism and an understanding of how methotrexate interacts with folate.4 Methotrexate is a folate analogue that acts by inhibiting the folate-metabolizing enzyme dihydrofolate reductase. Because it competes with natural folates both for cellular uptake and for the formation of polyglutamates (the natural forms of intracellular folate retained within the cell), it might be expected that the pretreatment folate status of the patient would predict the subsequent toxicity of the drug. However, studies in cancer patients failed to identify plasma or red cell folate levels as useful predictors of methotrexate toxicity,5 although animal studies showed a protective effect of pretreatment folate supplementation.6 The discovery that administration of folinic acid could rescue the patient from the toxic effects of methotrexate7 provided a partial solution to the problem of sporadic toxicity and led to the development of techniques for predicting and preventing toxicity in patients who had received high-dose methotrexate by monitoring the blood level of methotrexate 72 hours after treatment.3
From the Department of Oncology, University of Newcastle Upon Tyne, Newcastle Upon Tyne, England. Dr Calvert has received research grant support and honoraria from, and has served as a consultant to Eli Lilly and Co. Address reprint requests to Hilary Calvert, MBBS, PhD, Department of Oncology, University of Newcastle Upon Tyne, Newcastle Upon Tyne, UK NE4 6BE. 0093-7754/01/2806T003$35.00/0 Copyright 2002, Elsevier Science (USA). All rights reserved. 0093-7754/02/2902-0503$35.00/0 doi:10.1053/sonc.2002.30761 3
4
HILARY CALVERT
Folate-Based Thymidylate Synthase Inhibitors Antifolates designed to inhibit thymidylate synthase (TS) were specifically developed with the objective of improving the spectrum and reducing the toxicity associated with methotrexate. The first of these to enter clinical trials was CB 3717 (N10-propargyl-5,8-dideazafolic acid).8 Although this compound showed activity in breast and ovarian cancers,9,10 it was withdrawn from clinical development because of the sporadic occurrence of severe toxicity (myelosuppression, gastrointestinal, and renal). Of 173 patients in phase II trials, there were eight treatment-related deaths. These were hard to predict on the basis of the pretreatment factors that might predispose to toxicity and were examined at the time; however, none of these variables were valuable in predicting which patients would develop toxicity. Raltitrexed was developed as a more soluble follow-up compound11 and has significant activity in colon cancer,12 an indication for which it has been licensed in some countries. Although the problem with sporadic drug-related serious toxicities has been reduced, it has not been eliminated. Four drug-related deaths were reported in a large phase II trial of 176 patients with colon cancer.12 Drug-related deaths, characterized by myelosuppression and diarrhea leading to sepsis, were also reported in two randomized studies in which raltitrexed was compared with 5-fluorouracil (5-FU)/ folinic acid combinations, although deaths were also reported in the 5-FU arm in one of these studies.13 The second, a 3-way trial, was terminated when an excess number of deaths was noted in the treatment arm compared with either of two control arms.14 GLYCINAMIDE RIBONUCLEOTIDE FORMYLTRANSFERASE INHIBITORS
Although there was little evidence relating the pretreatment folate status of patients to subsequent toxicity in these studies, the development of another class of antifolates showed a significant effect of folate supplementation on the toxicity of the drug. Lometrexol, an antifolate known specifically to inhibit glycinamide ribonucleotide formyltransferase, was found to be poorly tolerated in initial phase I trials.15-17 Doses were far lower than those expected from preclinical studies. The adverse effects of myelosuppression and gastroin-
testinal toxicity were cumulative and slow to abate, making it impractical to continue development of the compound. However, preclinical data showed that the toxicity of lometrexol in mice was markedly dependent on the folate content of the diet and that folate-supplemented mice could withstand a markedly higher dose of the drug. The therapeutic index was determined in groups of mice bearing the C3H mammary tumor that received varying levels of folate supplementation. In mice with no supplementation, the drug was toxic and the therapeutic index was poor, whereas in mice receiving a very high level of supplementation the antitumor activity of the drug was reduced. However, a good therapeutic index was observed in mice receiving a range of intermediate doses.18 Subsequently, phase I clinical trials were designed on the basis of these data, which showed that up to 10-fold higher doses of lometrexol could be given to patients receiving folate supplements, with significantly reduced toxicities.19,20 Therapeutic responses were observed in these studies. PEMETREXED SAFETY PROFILE
Pemetrexed is a potent inhibitor of TS, glycinamide ribonucleotide formyltransferase, and dihydrofolate reductase in vitro. End-product reversal studies suggest that although TS is the main target at low 50% inhibitory concentrations (IC50) of the drug, the other targets are affected at slightly higher levels.21 The levels necessary to inhibit the additional loci are greatly exceeded in the plasma of patients receiving the drug,22 and the patterns of cross-resistance in antifolate-resistant cell lines differ for pemetrexed and raltitrexed.23 These observations have led to the belief that pemetrexed is effectively acting as a multitargeted antifolate24 and consequently has been referred to as MTA. Pemetrexed is undergoing active phase II and III clinical evaluation.25 During the early stages of the phase II trials, a number of drug-related deaths characterized by myelosuppression, diarrhea, and sepsis were recorded. The clear rationale for a poor pretreatment folate status affecting the tolerance of pemetrexed led to a search for a more sensitive method to establish the folate status of patients. Homocysteine has been established as such a marker26 because of the role of the folate-dependent methionine synthetase (MS) folate metabolism (see Fig 1 in the article by by Calvert and Bunn in this supplement). Methionine synthetase
FOLATE STATUS AND SAFETY OF ANTIFOLATES
converts homocysteine to methionine using the methyl group from 5-methyltetrahydrofolate. Methionine is involved in a number of cellular methylation reactions that regenerate homocysteine. Thus, any reduction in the function of the folate pool can lead to an accumulation of homocysteine, which is measurable in the plasma. Methionine synthetase is also dependent on vitamin B12, which is essential for its catalytic function. Thus, an elevated plasma level of homocysteine can indicate a functional deficiency of B12 or folate, which may not be apparent by measurement of levels of the vitamins themselves. B12 deficiency is also characterized by an elevated level of methylmalonic acid, resulting from decreased activity of the second human vitamin B12– dependent enzyme, methylmalonyl Co-A mutase. Thus, measurement of both vitamin homocysteine and methylmalonic acid will allow determination of the relative roles of vitamin B12 and folate in causing the elevation. Homocysteine and methylmalonic acid levels were measured in 139 patients participating in phase II studies of pemetrexed. Grade 4 neutropenia (n ⫽ 21) was significantly correlated with pretreatment albumin and homocysteine levels, and grade 4 thrombocytopenia (n ⫽ 8) was highly predicted by homocysteine levels. If the pretreatment homocysteine level was more than 10 mol/L, grade 4 neutropenia occurred in 75% of patients during the first cycle.27 There was a strong correlation of elevated homocysteine levels and subsequent development of toxicity, but there was no clear cut-off homocysteine level that would predict all cases of severe toxicity. A policy of supplementing all patients with folic acid (350 to 1,000 g/d continuously) and vitamin B12 (1,000 g/ 9 weeks) before the start of treatment with pemetrexed was undertaken.28 Vitamin B12 supplementation was also given because elevated methylmalonic acid levels were also found to be correlated with severe toxicities.28 Data on 246 patients who did not receive vitamin supplementation were compared with those of 78 patients in whom the supplementation was given. There were marked reductions in drug-related death (from 5% to 0%), grade 4 neutropenia (from 32% to 2.6%), grade 4 thrombocytopenia (from 8% to 0%), grade 3 to 4 diarrhea (from 6% to 2.6%), and grade 3 to 4 mucositis (from 5% to 1.3%; Table 1).28 There is currently little published information concerning the therapeutic activity in patients receiving folate
5
Table 1. Effect of Pretreatment Folate and Vitamin B12 Supplementation on the Toxicities of Pemetrexed
Toxicity Grade 4 neutropenia Grade 4 hematologic toxicity ⫹ grade 3 or 4 nonhematologic toxicity Grade 4 thrombocytopenia Grade 3 or 4 mucositis Grade 3 or 4 diarrhea Grade 4 neutropenia ⫹ grade 3 or 4 mucositis Grade 4 neutropenia ⫹ grade 3 or 4 diarrhea Grade 4 neutropenia ⫹ grade 3 or 4 infection Drug-related death
Before After Supplementation Supplementation (%) (%) 32
2.6
37 8 5 6
6.4 0 1.3 2.6
3
0
3
0
2 5
0 0
Data from Bunn et al.28
supplementation, but in a phase II study in gastric cancer,29 six patients were initially treated without folate supplementation. There were three drugrelated deaths, and one patient withdrew from treatment because of toxicity. None of these patients responded. Subsequently, 21 patients received folic acid, 5 mg/d, for 2 days before and 5 days after pemetrexed treatment. There were no drug-related deaths, and six partial responses were reported. DISCUSSION AND CONCLUSIONS
The occurrence of unpredictable toxicity has been a feature of the use of many antifolate drugs since they were introduced into clinical practice. It is reasonable to expect that patients with poor pretreatment folate status would be more likely to suffer toxicity because natural folates can compete with antifolates for cellular uptake and polyglutamation and potentially at the target enzyme site. However, it has been difficult to show any predictive value for pretreatment plasma or red cell folate levels. Plasma folate levels probably are poorly predictive because they do not reflect the intracellular levels of folate, which are crucial for the folate-dependent reactions. Red cell levels, although a measure of intracellular folate, also may not reflect the levels in the crucial proliferating tissues because red cells are not proliferating and
6
will have been released from the bone marrow up to 100 days before sampling. However, homocysteine, as a sensitive surrogate for functional folate status, has proved significantly predictive of the toxicity of pemetrexed. To date, there are no published reports of the utility of homocysteine in predicting the toxicities of other antifolates. The use of a standard folate and vitamin B12 supplement has resulted in a marked reduction in the toxicity of pemetrexed, with apparent elimination of the occasional life-threatening events. One possible explanation for this is that the expansion of the intracellular folate pools in normal tissues prevents accumulation of persistent intracellular pools of pemetrexed polyglutamates. Nolatrexed is a nonclassical antifolate that is structurally precluded from forming polyglutamates. Phase I and II trials have been performed,30,31and little variation in the toxicity experienced by different patients has been observed. This finding is in contrast to the variable and sporadic toxicities observed with agents capable of forming polyglutamates. Although it is clear that vitamin supplementation has a beneficial effect on reducing the toxicity of pemetrexed, thus far there are fewer data concerning clinical activity in supplemented patients. However, a number of considerations suggest that the efficacy of the drug is not compromised. In a study in mice, folic acid supplementation was shown to preserve the antitumor activity of pemetrexed while reducing toxicity.32 A meta-analysis of randomized trials of methotrexate with or without folate supplementation in the treatment of rheumatoid arthritis showed that vitamin supplementation reduced the toxicity of methotrexate while the therapeutic effect was maintained.33 The level of dietary folate intake varies from country to country and tends to be higher in the United States because of supplementation of foods with folate, but there is no suggestion that the activity seen in phase II trials of pemetrexed is dependent on country. In conclusion, the use of a low-level dietary supplement of folic acid together with vitamin B12 injections has led to a marked improvement in the safety profile of pemetrexed, making it a welltolerated drug. To date, there is no evidence that efficacy is decreased and no reason to believe that it will be. It appears that the technique of vitamin supplementation will benefit the development of the drug, allowing better-tolerated single-agent
HILARY CALVERT
treatment and probably permitting a broader range of combination treatments with other myelosuppressive agents. REFERENCES 1. Farber S, Diamon LK, Mercer RD, et al: Temporary remissions in acute leukaemia in children produced by folic acid antagonist 4-amino pteroyl-glutamic acid (aminopterin). N Engl J Med 238:787-793, 1948 2. Barnard RD, Freeman MD: Reversal of aminopterin toxicity by water-soluble cuprodihyrdoporphyrins (chloresium). Am J Digest Dis 21:163-169, 1954 3. Stoller RG, Hande KR, Jacobs SA, et al: Use of plasma pharmacokinetics to predict and prevent methotrexate toxicity. N Engl J Med 297:630-634, 1977 4. Goldman ID, Matherly LH: The cellular pharmacology of methotrexate. Pharmacol Ther 28:77-102, 1985 5. Schroder H, Clausen N, Ostergard E, et al: Folic acid supplements in vitamin tablets: A determinant of hematological drug tolerance in maintenance therapy of childhood acute lymphoblastic leukemia. Pediatr Hematol Oncol 3:241-247, 1986 6. Mead JAR, Venditti JM, Schrecker AW, et al: The effect of reduced derivatives of folic acid on the toxicity and antileukaemic effect of methotrexate in mice. Biochem Pharmacol 12:371-383, 1973 7. Sullivan RD, Miller E, Sykes P: Antimetabolite-metabolite combination in cancer chemotherapy. Effects of intraarterial methotrexate-intramuscular citrovorum factor therapy in human cancer. Cancer 12:1248-1262, 1959 8. Calvert AH, Alison DL, Harland SJ, et al: A phase I evaluation of the quinazoline antifolate thymidylate synthase inhibitor N10-propargyl-5,8-dideazafolic acid. J Clin Oncol 4:1245-1252, 1986 9. Cantwell BM, Macaulay V, Harris AL, et al: Phase II study of the antifolate N10-propargyl-5,8-dideazafolic acid (CB 3717) in advanced breast cancer. Eur J Cancer Clin Oncol 24:733-736, 1988 10. Hughes A, Calvert H: An update on thymidylate synthase inhibitors. Ann Oncol 10:1137-1139, 1999 11. Clarke SJ, Hanwell J, de Boer M, et al: Phase I trial of ZD1694, a new folate-based thymidylate synthase inhibitor, in patients with solid tumors. J Clin Oncol 14:1495-1503, 1996 12. Zalcberg JR, Cunningham D, Van Cutsem E, et al: ZD1694: A novel thymidylate synthase inhibitor with substantial activity in the treatment of patients with advanced colorectal cancer. Tomudex Colorectal Study Group. J Clin Oncol 14:716-721, 1996 13. Cunningham D, Zalcberg JR, Rath U, et al: Final results of a randomised trial comparing ’Tomudex’ (raltitrexed) with 5-fluorouracil plus leucovorin in advanced colorectal cancer. “Tomudex” Colorectal Cancer Study Group. Ann Oncol 7:961-965, 1996 14. Maughan TS, James RD, Kerr D, et al: Preliminary results of a multicentre randomised trial comparing 3 chemotherapy regimens (de Gramont, Lokich and raltitrexed) in metastatic colorectal cancer. Proc Am Soc Clin Oncol 18: 262a, 1999 (abstr 1007) 15. Nelson R, Butler F, Dugan W Jr, et al: Phase I clinical
FOLATE STATUS AND SAFETY OF ANTIFOLATES
trial of LY264618 (dideazatetrahydrofolic acid, DDATHF). Proc Am Soc Clin Oncol 9:76, 1990 (abstr) 16. 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 17. Young C, Currie V, Baltzer L, et al: Phase I and clinical pharmacologic study of LY264618, 5-10-dideazatetrahydrofolate. Proc Am Assoc Cancer Res 31:177, 1990 (abstr) 18. Alati T, Worzalla JF, Shih C, et al: Augmentation of the therapeutic activity of lometrexol [(6-R)5,10- dideazatetra hydrofolate] by oral folic acid. Cancer Res 56:2331-2335, 1996 19. Roberts JD, Poplin EA, Tombes MB, et al: Weekly lometrexol with daily oral folic acid is appropriate for phase II evaluation. Cancer Chemother Pharmacol 45:103-110, 2000 20. Laohavinij S, Wedge SR, Lind MJ, et al: A phase I clinical study of the antipurine antifolate lometrexol (DDATHF) given with oral folic acid. Invest New Drugs 14: 325-335, 1996 21. Shih C, Chen VJ, Gossett LS, et al: LY231514, a pyrrolo[2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes. Cancer Res 57:1116-1123, 1997 22. Ouellet D, Periclou AP, Johnson RD, et al: Population pharmacokinetics of pemetrexed disodium (ALIMTA) in patients with cancer. Cancer Chemother Pharmacol 46:227-234, 2000 23. Schultz RM, Chen VJ, Bewley JR, et al: Biological activity of the multitargeted antifolate, MTA (LY231514), in human cell lines with different resistance mechanisms to antifolate drugs. Semin Oncol 26:68-73, 1999 (suppl 2) 24. Calvert H: MTA, a novel multitargeted antifolate, for preclinical to phase I and beyond: Summary and conclusions. Semin Oncol 26:105-108, 1999 (suppl 2)
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25. O’Dwyer PJ, Nelson K, Thornton DE: Overview of phase II trials of MTA in solid tumors. Semin Oncol 26:99-104, 1999 (suppl 6) 26. Savage DG, Lindenbaum J, Stabler SP, et al: Sensitivity of serum methylmalonic acid and total homocysteine determinations for diagnosing cobalamin and folate deficiencies. Am J Med 96:239-246, 1994 27. Niyikiza C, Baker S, Johnson R, et al: Relationship of vitamin metabolite profile, drug exposure, and other patient characteristics to toxicity. Ann Oncol 9:609P, 1998 (suppl 4) 28. Bunn P, Paoletti P, Niyikiza C, et al: Vitamin B12 and folate reduce toxicity of Alimta (pemetrexed disodium, LY231514, MTA), a novel antifolate/antimetabolite. Proc Am Assoc Cancer Res 20:76a, 2001 (abstr 300) 29. Celio L, Bajetta E, Buzzoni R, et al: Efficacy and toxicity of pemetrexed disodium (Alimta) with folic acid (FA) in gastric cancer. Proc Am Assoc Cancer Res 20:166a, 2001 (abstr 660) 30. Hughes AN, Rafi I, Griffin MJ, et al: Phase I studies with the nonclassical antifolate nolatrexed dihydrochloride (AG337, THYMITAQ) administered orally for 5 days. Clin Cancer Res 5:111-118, 1999 31. Stuart K, Tessitore J, Rudy J, et al: A phase II trial of nolatrexed dihydrochloride in patients with advanced hepatocellular carcinoma. Cancer 86:410-414, 1999 32. Worzalla JF, Shih C, Schultz RM: Role of folic acid in modulating the toxicity and efficacy of the multitargeted antifolate, LY231514. Anticancer Res 18:3235-3239, 1998 33. Ortiz Z, Shea B, Suarez-Almazor ME, et al: The efficacy of folic acid and folinic acid in reducing methotrexate gastrointestinal toxicity in rheumatoid arthritis. A metaanalysis of randomized controlled trials. J Rheumatol 25:36-43, 1998
F
OR EVERY antifolate drug introduced into clinical practice, folic acid or its reduced derivatives have been found to be capable of modulating the toxicity and/or efficacy. This article reviews folic acid and drug interactions for two licensed drugs (methotrexate and raltitrexed) and two investigational agents (lometrexol and pemetrexed [Alimta, LY231514; Eli Lilly and Co, Indianapolis, IN]). In the case of pemetrexed, the study of plasma vitamin metabolite levels (homocysteine and methylmalonic acid) has provided insight into the interaction of nutritional status with chemotherapy and has allowed the development of innovative treatment protocols. METHOTREXATE
In the late 1940s, Dr Sidney Farber first noted that folic acid levels were low in children with leukemia, an observation that showed the role of folic acid in proliferating cells and led to the development of the first antifolates, aminopterin and methotrexate.1 These drugs were the first to induce remissions in childhood leukemia but were also found to be capable of causing serious toxicities. Indeed, some investigators attributed their antileukemic effects directly to their toxicity.2 Nevertheless, the therapeutic effects of methotrexate in both leukemia and solid tumors led to expansion of its use for many indications in cancer Seminars in Oncology, Vol 29, No 2, Suppl 5 (April), 2002: pp 3-7
treatment and a consequent interest in the sporadic toxic events induced by the drug. Severe methotrexate toxicity consisting of myelosuppression with significant neutropenia and gastrointestinal toxicity with symptoms of diarrhea and mucosal damage have been reported sporadically on most methotrexate schedules. When complicated by gram-negative sepsis, it frequently resulted in drug-related death.3 The clinical use of methotrexate generated a huge interest in folate metabolism and an understanding of how methotrexate interacts with folate.4 Methotrexate is a folate analogue that acts by inhibiting the folate-metabolizing enzyme dihydrofolate reductase. Because it competes with natural folates both for cellular uptake and for the formation of polyglutamates (the natural forms of intracellular folate retained within the cell), it might be expected that the pretreatment folate status of the patient would predict the subsequent toxicity of the drug. However, studies in cancer patients failed to identify plasma or red cell folate levels as useful predictors of methotrexate toxicity,5 although animal studies showed a protective effect of pretreatment folate supplementation.6 The discovery that administration of folinic acid could rescue the patient from the toxic effects of methotrexate7 provided a partial solution to the problem of sporadic toxicity and led to the development of techniques for predicting and preventing toxicity in patients who had received high-dose methotrexate by monitoring the blood level of methotrexate 72 hours after treatment.3
From the Department of Oncology, University of Newcastle Upon Tyne, Newcastle Upon Tyne, England. Dr Calvert has received research grant support and honoraria from, and has served as a consultant to Eli Lilly and Co. Address reprint requests to Hilary Calvert, MBBS, PhD, Department of Oncology, University of Newcastle Upon Tyne, Newcastle Upon Tyne, UK NE4 6BE. 0093-7754/01/2806T003$35.00/0 Copyright 2002, Elsevier Science (USA). All rights reserved. 0093-7754/02/2902-0503$35.00/0 doi:10.1053/sonc.2002.30761 3
4
HILARY CALVERT
Folate-Based Thymidylate Synthase Inhibitors Antifolates designed to inhibit thymidylate synthase (TS) were specifically developed with the objective of improving the spectrum and reducing the toxicity associated with methotrexate. The first of these to enter clinical trials was CB 3717 (N10-propargyl-5,8-dideazafolic acid).8 Although this compound showed activity in breast and ovarian cancers,9,10 it was withdrawn from clinical development because of the sporadic occurrence of severe toxicity (myelosuppression, gastrointestinal, and renal). Of 173 patients in phase II trials, there were eight treatment-related deaths. These were hard to predict on the basis of the pretreatment factors that might predispose to toxicity and were examined at the time; however, none of these variables were valuable in predicting which patients would develop toxicity. Raltitrexed was developed as a more soluble follow-up compound11 and has significant activity in colon cancer,12 an indication for which it has been licensed in some countries. Although the problem with sporadic drug-related serious toxicities has been reduced, it has not been eliminated. Four drug-related deaths were reported in a large phase II trial of 176 patients with colon cancer.12 Drug-related deaths, characterized by myelosuppression and diarrhea leading to sepsis, were also reported in two randomized studies in which raltitrexed was compared with 5-fluorouracil (5-FU)/ folinic acid combinations, although deaths were also reported in the 5-FU arm in one of these studies.13 The second, a 3-way trial, was terminated when an excess number of deaths was noted in the treatment arm compared with either of two control arms.14 GLYCINAMIDE RIBONUCLEOTIDE FORMYLTRANSFERASE INHIBITORS
Although there was little evidence relating the pretreatment folate status of patients to subsequent toxicity in these studies, the development of another class of antifolates showed a significant effect of folate supplementation on the toxicity of the drug. Lometrexol, an antifolate known specifically to inhibit glycinamide ribonucleotide formyltransferase, was found to be poorly tolerated in initial phase I trials.15-17 Doses were far lower than those expected from preclinical studies. The adverse effects of myelosuppression and gastroin-
testinal toxicity were cumulative and slow to abate, making it impractical to continue development of the compound. However, preclinical data showed that the toxicity of lometrexol in mice was markedly dependent on the folate content of the diet and that folate-supplemented mice could withstand a markedly higher dose of the drug. The therapeutic index was determined in groups of mice bearing the C3H mammary tumor that received varying levels of folate supplementation. In mice with no supplementation, the drug was toxic and the therapeutic index was poor, whereas in mice receiving a very high level of supplementation the antitumor activity of the drug was reduced. However, a good therapeutic index was observed in mice receiving a range of intermediate doses.18 Subsequently, phase I clinical trials were designed on the basis of these data, which showed that up to 10-fold higher doses of lometrexol could be given to patients receiving folate supplements, with significantly reduced toxicities.19,20 Therapeutic responses were observed in these studies. PEMETREXED SAFETY PROFILE
Pemetrexed is a potent inhibitor of TS, glycinamide ribonucleotide formyltransferase, and dihydrofolate reductase in vitro. End-product reversal studies suggest that although TS is the main target at low 50% inhibitory concentrations (IC50) of the drug, the other targets are affected at slightly higher levels.21 The levels necessary to inhibit the additional loci are greatly exceeded in the plasma of patients receiving the drug,22 and the patterns of cross-resistance in antifolate-resistant cell lines differ for pemetrexed and raltitrexed.23 These observations have led to the belief that pemetrexed is effectively acting as a multitargeted antifolate24 and consequently has been referred to as MTA. Pemetrexed is undergoing active phase II and III clinical evaluation.25 During the early stages of the phase II trials, a number of drug-related deaths characterized by myelosuppression, diarrhea, and sepsis were recorded. The clear rationale for a poor pretreatment folate status affecting the tolerance of pemetrexed led to a search for a more sensitive method to establish the folate status of patients. Homocysteine has been established as such a marker26 because of the role of the folate-dependent methionine synthetase (MS) folate metabolism (see Fig 1 in the article by by Calvert and Bunn in this supplement). Methionine synthetase
FOLATE STATUS AND SAFETY OF ANTIFOLATES
converts homocysteine to methionine using the methyl group from 5-methyltetrahydrofolate. Methionine is involved in a number of cellular methylation reactions that regenerate homocysteine. Thus, any reduction in the function of the folate pool can lead to an accumulation of homocysteine, which is measurable in the plasma. Methionine synthetase is also dependent on vitamin B12, which is essential for its catalytic function. Thus, an elevated plasma level of homocysteine can indicate a functional deficiency of B12 or folate, which may not be apparent by measurement of levels of the vitamins themselves. B12 deficiency is also characterized by an elevated level of methylmalonic acid, resulting from decreased activity of the second human vitamin B12– dependent enzyme, methylmalonyl Co-A mutase. Thus, measurement of both vitamin homocysteine and methylmalonic acid will allow determination of the relative roles of vitamin B12 and folate in causing the elevation. Homocysteine and methylmalonic acid levels were measured in 139 patients participating in phase II studies of pemetrexed. Grade 4 neutropenia (n ⫽ 21) was significantly correlated with pretreatment albumin and homocysteine levels, and grade 4 thrombocytopenia (n ⫽ 8) was highly predicted by homocysteine levels. If the pretreatment homocysteine level was more than 10 mol/L, grade 4 neutropenia occurred in 75% of patients during the first cycle.27 There was a strong correlation of elevated homocysteine levels and subsequent development of toxicity, but there was no clear cut-off homocysteine level that would predict all cases of severe toxicity. A policy of supplementing all patients with folic acid (350 to 1,000 g/d continuously) and vitamin B12 (1,000 g/ 9 weeks) before the start of treatment with pemetrexed was undertaken.28 Vitamin B12 supplementation was also given because elevated methylmalonic acid levels were also found to be correlated with severe toxicities.28 Data on 246 patients who did not receive vitamin supplementation were compared with those of 78 patients in whom the supplementation was given. There were marked reductions in drug-related death (from 5% to 0%), grade 4 neutropenia (from 32% to 2.6%), grade 4 thrombocytopenia (from 8% to 0%), grade 3 to 4 diarrhea (from 6% to 2.6%), and grade 3 to 4 mucositis (from 5% to 1.3%; Table 1).28 There is currently little published information concerning the therapeutic activity in patients receiving folate
5
Table 1. Effect of Pretreatment Folate and Vitamin B12 Supplementation on the Toxicities of Pemetrexed
Toxicity Grade 4 neutropenia Grade 4 hematologic toxicity ⫹ grade 3 or 4 nonhematologic toxicity Grade 4 thrombocytopenia Grade 3 or 4 mucositis Grade 3 or 4 diarrhea Grade 4 neutropenia ⫹ grade 3 or 4 mucositis Grade 4 neutropenia ⫹ grade 3 or 4 diarrhea Grade 4 neutropenia ⫹ grade 3 or 4 infection Drug-related death
Before After Supplementation Supplementation (%) (%) 32
2.6
37 8 5 6
6.4 0 1.3 2.6
3
0
3
0
2 5
0 0
Data from Bunn et al.28
supplementation, but in a phase II study in gastric cancer,29 six patients were initially treated without folate supplementation. There were three drugrelated deaths, and one patient withdrew from treatment because of toxicity. None of these patients responded. Subsequently, 21 patients received folic acid, 5 mg/d, for 2 days before and 5 days after pemetrexed treatment. There were no drug-related deaths, and six partial responses were reported. DISCUSSION AND CONCLUSIONS
The occurrence of unpredictable toxicity has been a feature of the use of many antifolate drugs since they were introduced into clinical practice. It is reasonable to expect that patients with poor pretreatment folate status would be more likely to suffer toxicity because natural folates can compete with antifolates for cellular uptake and polyglutamation and potentially at the target enzyme site. However, it has been difficult to show any predictive value for pretreatment plasma or red cell folate levels. Plasma folate levels probably are poorly predictive because they do not reflect the intracellular levels of folate, which are crucial for the folate-dependent reactions. Red cell levels, although a measure of intracellular folate, also may not reflect the levels in the crucial proliferating tissues because red cells are not proliferating and
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will have been released from the bone marrow up to 100 days before sampling. However, homocysteine, as a sensitive surrogate for functional folate status, has proved significantly predictive of the toxicity of pemetrexed. To date, there are no published reports of the utility of homocysteine in predicting the toxicities of other antifolates. The use of a standard folate and vitamin B12 supplement has resulted in a marked reduction in the toxicity of pemetrexed, with apparent elimination of the occasional life-threatening events. One possible explanation for this is that the expansion of the intracellular folate pools in normal tissues prevents accumulation of persistent intracellular pools of pemetrexed polyglutamates. Nolatrexed is a nonclassical antifolate that is structurally precluded from forming polyglutamates. Phase I and II trials have been performed,30,31and little variation in the toxicity experienced by different patients has been observed. This finding is in contrast to the variable and sporadic toxicities observed with agents capable of forming polyglutamates. Although it is clear that vitamin supplementation has a beneficial effect on reducing the toxicity of pemetrexed, thus far there are fewer data concerning clinical activity in supplemented patients. However, a number of considerations suggest that the efficacy of the drug is not compromised. In a study in mice, folic acid supplementation was shown to preserve the antitumor activity of pemetrexed while reducing toxicity.32 A meta-analysis of randomized trials of methotrexate with or without folate supplementation in the treatment of rheumatoid arthritis showed that vitamin supplementation reduced the toxicity of methotrexate while the therapeutic effect was maintained.33 The level of dietary folate intake varies from country to country and tends to be higher in the United States because of supplementation of foods with folate, but there is no suggestion that the activity seen in phase II trials of pemetrexed is dependent on country. In conclusion, the use of a low-level dietary supplement of folic acid together with vitamin B12 injections has led to a marked improvement in the safety profile of pemetrexed, making it a welltolerated drug. To date, there is no evidence that efficacy is decreased and no reason to believe that it will be. It appears that the technique of vitamin supplementation will benefit the development of the drug, allowing better-tolerated single-agent
HILARY CALVERT
treatment and probably permitting a broader range of combination treatments with other myelosuppressive agents. REFERENCES 1. Farber S, Diamon LK, Mercer RD, et al: Temporary remissions in acute leukaemia in children produced by folic acid antagonist 4-amino pteroyl-glutamic acid (aminopterin). N Engl J Med 238:787-793, 1948 2. Barnard RD, Freeman MD: Reversal of aminopterin toxicity by water-soluble cuprodihyrdoporphyrins (chloresium). Am J Digest Dis 21:163-169, 1954 3. Stoller RG, Hande KR, Jacobs SA, et al: Use of plasma pharmacokinetics to predict and prevent methotrexate toxicity. N Engl J Med 297:630-634, 1977 4. Goldman ID, Matherly LH: The cellular pharmacology of methotrexate. Pharmacol Ther 28:77-102, 1985 5. Schroder H, Clausen N, Ostergard E, et al: Folic acid supplements in vitamin tablets: A determinant of hematological drug tolerance in maintenance therapy of childhood acute lymphoblastic leukemia. Pediatr Hematol Oncol 3:241-247, 1986 6. Mead JAR, Venditti JM, Schrecker AW, et al: The effect of reduced derivatives of folic acid on the toxicity and antileukaemic effect of methotrexate in mice. Biochem Pharmacol 12:371-383, 1973 7. Sullivan RD, Miller E, Sykes P: Antimetabolite-metabolite combination in cancer chemotherapy. Effects of intraarterial methotrexate-intramuscular citrovorum factor therapy in human cancer. Cancer 12:1248-1262, 1959 8. Calvert AH, Alison DL, Harland SJ, et al: A phase I evaluation of the quinazoline antifolate thymidylate synthase inhibitor N10-propargyl-5,8-dideazafolic acid. J Clin Oncol 4:1245-1252, 1986 9. Cantwell BM, Macaulay V, Harris AL, et al: Phase II study of the antifolate N10-propargyl-5,8-dideazafolic acid (CB 3717) in advanced breast cancer. Eur J Cancer Clin Oncol 24:733-736, 1988 10. Hughes A, Calvert H: An update on thymidylate synthase inhibitors. Ann Oncol 10:1137-1139, 1999 11. Clarke SJ, Hanwell J, de Boer M, et al: Phase I trial of ZD1694, a new folate-based thymidylate synthase inhibitor, in patients with solid tumors. J Clin Oncol 14:1495-1503, 1996 12. Zalcberg JR, Cunningham D, Van Cutsem E, et al: ZD1694: A novel thymidylate synthase inhibitor with substantial activity in the treatment of patients with advanced colorectal cancer. Tomudex Colorectal Study Group. J Clin Oncol 14:716-721, 1996 13. Cunningham D, Zalcberg JR, Rath U, et al: Final results of a randomised trial comparing ’Tomudex’ (raltitrexed) with 5-fluorouracil plus leucovorin in advanced colorectal cancer. “Tomudex” Colorectal Cancer Study Group. Ann Oncol 7:961-965, 1996 14. Maughan TS, James RD, Kerr D, et al: Preliminary results of a multicentre randomised trial comparing 3 chemotherapy regimens (de Gramont, Lokich and raltitrexed) in metastatic colorectal cancer. Proc Am Soc Clin Oncol 18: 262a, 1999 (abstr 1007) 15. Nelson R, Butler F, Dugan W Jr, et al: Phase I clinical
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