Folic Acid Supplementation During Methotrexate Immunosuppression

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ORIGINAL ARTICLE Folic acid supplementation during methotrexate immunosuppression is not associated with early toxicity, riskof acute graft-versus-host disease or relapse following hematopoietic transplantation

K Robien1,2,3, MM Schubert4, Y Yasui5, P Martin4, R Storb4, JD Potter1,6 and CM Ulrich1,6

1Cancer Prevention Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 2Division of Epidemiology and Community Health, University of Minnesota, Minneapolis, MN, USA; 3University of Minnesota Cancer Center, Minneapolis, MN, USA; 4Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 5Department of Public Health Sciences, University of Alberta, Edmonton, AB, Canada and 6Department of Epidemiology, University of Washington, Seattle, WA, USA

Methotrexate (MTX) is used as an immunosuppressive Introduction agent for acute graft-versus-host disease (GVHD) prophylaxis following hematopoietic cell transplantation Limited evidence currently exists to guide practice regard- (HCT). Concerns that folate intake may impair MTX ing folate intake or supplementation during methotrexate effectiveness or selectively rescue leukemic cells have led (MTX) treatment in oncology settings. Although the to variations in clinical practice regarding supplemental nutrient folate plays a vital role in hematopoiesis and cell folic acid during MTX administration. A retrospective, repair processes following myeloablative treatment regi- observational study was undertaken to determine the mens, concerns that folate may impair MTX effectiveness association between folic acid intake (days 0–18 post or selectively rescue cancer cells have led some clinicians to transplant) and MTX toxicity and efficacy following restrict supplemental folic acid during MTX administra- HCT. The study population consisted of 311 adult tion.1,2 An unusual nationwide intravenous multivitamin patients who received a myeloablative HCT for chronic shortage in the United States from late 1996 through the myelogenous leukemia, all four scheduled doses of MTX, end of 1998 allowed a unique opportunity to evaluate the and did not require leucovorin rescue. Multiple linear effect of variation in supplemental folic acid intake regression models were used to assess the relationships on MTX toxicity and treatment effectiveness following between folic acid intake (days 0–18 post-HCT) and oral hematopoietic cell transplantation (HCT). Methotrexate is mucositis index (OMI) scores, time to engraftment and commonly used as an immunosuppressive agent for pro- riskof detectable acute GVHD. No statistically signifi- phylaxis against acute graft-versus-host disease (GVHD) cant differences in mean OMI scores, time to engraft- immediately following HCT, and toxicities include ment, riskof acute GVHD, days to acute GVHD, riskof mucositis and myelosuppression.3–5 relapse or survival were observed when comparing patients The majority of HCT patients receive parenteral nutrition taking, on average, o400 (14%), 400 (58%) or 4400 lg as their primary source of nutrients, including folate, during (28%) folic acid per day. Our results suggest that the early post-transplant period. During the nationwide concurrent folic acid supplementation does not change intravenous multivitamin shortage, recommendations from MTX effectiveness or toxicity in this patient population. the FDA, the Centers for Disease Control and Prevention, Bone Marrow Transplantation (2006) 37, 687–692. and the American Society for Parenteral and Enteral doi:10.1038/sj.bmt.1705303; published online 27 February Nutrition were to limit the frequency of intravenous multi- 2006 vitamin infusions to three times per week, and to use Keywords: folic acid; methotrexate; hematopoietic cell oral supplements for patients able to absorb at least 50% of transplantation; chronic myelogenous leukemia their nutrients enterally.6 In addition to these guidelines, our center added 1 mg folic acid daily as an individual supplement to each patient’s parenteral nutrition solution. Thus, patients were receiving as much as 1400 mg folic acid/day (usual amount was 400 mg folic acid/day) during Correspondence: Dr K Robien, Division of Epidemiology and Commu- nity Health, University of Minnesota, 1300 S 2nd Street, Suite 300, both parenteral nutrition support and oral multivitamin Minneapolis, MN 55454, USA. supplements, effectively resulting in greater folic acid E-mails: [email protected]; [email protected] supplementation during this period of vitamin ‘shortage’. Presented as an oral and poster presentation at the 13th International Folic acid, the synthetic monoglutamic form of folate Symposium on the Chemistry and Biology of Pteridines and Folates, Egmond aan Zee, the Netherlands, 2005. used in vitamin supplements and fortified foods, must be Received 13 October 2005; revised 19 December 2005; accepted 22 reduced to tetrahydrofolate by the enzyme dihydrofolate December 2005; published online 27 February 2006 reductase in order to be metabolically active within the cell Folic acid during methotrexate immunosuppression K Robien et al 688 and act as a methyl group donor for nucleotide synthesis. oral mucosal changes (atrophy, erythema, ulceration, As most of the antifolate agents specifically block the pseudomembranous ulcerations and edematous changes) action of dihydrofolate reductase, we hypothesized that and consists of 34 items each scaled from 0 to 3 (normal to folic acid is not likely to be metabolically active during severe).12 Each HCT patient at the FHCRC is examined for antifolate treatment and should not affect treatment- oral mucositis every 2–3 days during the early post- related toxicity or outcomes. transplant period by a trained examiner. Results are recorded on a standardized machine-readable form, from which the OMI is computed. Oral mucositis following Materials and methods HCT is generally noted to peak between days 7 and 11, and resolve by days 18–21 post transplant.4,12 Therefore, Study design and patient population outcomes identified for analyses were mean OMI days Subjects in this retrospective cohort study were patients 6–12 (patients with at least two assessments during receiving allogeneic HCT at the Fred Hutchinson Cancer this period) and mean OMI days 1–18 (patients with Research Center (FHCRC) between 1992 and 2002 who: at least four assessments during this period). The number (1) had a diagnosis of CML in chronic or accelerated phase of days post transplantation to reach 500 granulocytes/ml before transplant, (2) were 18 years of age or older at and 20 000 platelets/ml were obtained from the patient the time of transplant and (3) received myeloablative database. conditioning regimens with either cyclophosphamide/total body irradiation (CyTBI) or busulfan/cyclophosphamide (BuCy), as previously described.7,8 None of the patients Outcome data received marrow or peripheral blood stem cell infusions Data on acute GVHD, relapse and survival were collected that were T-cell depleted. Additional eligibility criteria from the center’s patient database. The FHCRC Long- restricted the study cohort to patients who received Term Follow-Up program maintains contact with patients cyclosporine and all four scheduled doses of MTX and their referring physicians after discharge from the (15 mg/m2 on day 1; 10 mg/m2 on days 3, 6 and 11 post center, and contributes information on relapse and death to transplant) for GVHD prophylaxis following previously the center’s patient database. Relapse was defined by described protocols,9 and did not require leucovorin rescue. cytogenetic (X5 metaphases positive for the Philadelphia This study was approved by the FHCRC Institutional chromosome at any point in time or the presence of any Review Board, and all patients provided informed consent Philadelphia chromosome on two successive cytogenetic to use medical records for research. evaluations at least 6 months apart13) or hematologic criteria. Relapse and survival data were locked for analysis Data collection on July 11, 2005. Medical records and patient databases were used to abstract study data for each study participant. Data Statistical analysis collected included height, weight, body surface area, body The data were stratified by average daily supplemental folic mass index (kg/m2), age, sex, race, time from diagnosis to acid intake, between days 0 and 18 post transplant, into transplant, conditioning regimen, source of hematopoietic those receiving less than, equal to or more than the US- progenitor cells (bone marrow vs peripheral blood), recommended dietary intake of 400 mg/day.14 Multiple average busulfan concentration at steady state, donor linear regression models were used to determine the relationship, donor sex, date of HCT, number of geometric mean OMI scores, time to engraftment (platelets MTX doses and total dose received post transplant and and granulocytes) and days to developing acute GVHD leucovorin administration for rescue from MTX toxicity. (among those who developed acute GVHD) for each of the Data on folic acid intake from parenteral solutions and supplemental folic acid intake categories, adjusting for age, oral multivitamin supplements were abstracted from the year of transplant, conditioning regimen and donor medication administration records for days 0–18 post relationship (related vs unrelated). Pairwise comparisons transplant. This time frame was chosen because most and trend tests were used to test for differences between the patients were hospitalized for this period of time, and group geometric means. medication administration records were available for Multivariable logistic regression analyses were used to documentation of folic acid intake. Furthermore, patients assess the relationship between supplemental folic acid typically begin to engraft10 and oral mucositis begins to intake and risk of detectable acute GVHD. Multivariable resolve4,11 around day 18 following full myeloablative Cox regression analyses were used to assess the relationship treatment regimens. between supplemental folic acid intake and risk of relapse To verify reproducibility of chart abstraction data, 13 and survival. Individuals with an average supplemental charts were selected randomly and re-abstracted. Complete folic acid intake of 400 mg/day were used as the reference agreement between the two abstractions was confirmed. group for both the logistic and Cox regression analyses. Potential confounding factors (including year of transplant, Toxicity data stage of CML at time of transplant, conditioning regimen, Severity of oral mucositis and days to granulocyte and donor relationship, age and sex) were selected for inclusion platelet engraftment were used as indicators of MTX in the various models if they had either been previously toxicity. Oral mucositis was measured using the oral shown to be modulators of the given outcome15–17 or mucositis index (OMI), which assesses clinically evident altered the regression coefficients of supplemental folic acid

Bone Marrow Transplantation Folic acid during methotrexate immunosuppression K Robien et al 689 intake by at least 10%. Source of hematopoietic progenitor Table 1 Characteristics of the study population cells and time from diagnosis to transplant were considered n ¼ 311 as potential confounding factors, but did not signiﬁcantly n (%) alter the means or regression coefﬁcients, and thus, were not included in the models. Analyses were performed using Demographics 7 a SAS version 8.2 (SAS Institute Inc., Cary, NC, USA). Age (years) 40.5 9.1 (18–67) Sex Male 183 (59) Results Female 128 (41)

Race A total of 311 patients met eligibility criteria for the study. White 273 (88) Characteristics of the study population are described in Non-white 38 (12) Table 1. Nearly all patients (n ¼ 302, 97%) received Weight (kg) 80.2717.1 (41.2–132.1)a parenteral nutrition as their primary source of nutrients Height (cm) 172.479.6 (146.1–195.5)a 2 7 a (more than half of their daily caloric intake) during the Body mass index (kg/m ) 26.9 5.2 (16.2–53.6) study period. Inclusion of patients (n ¼ 9) who did not Transplant information require parenteral nutrition support did not alter the Stage ofCML at transplant overall trends in the observed associations. Chronic 271 (87) Average supplemental folic acid intake between days 0 Accelerated 40 (13) 7 a and 18 post transplant varied from 0 to 1400 mg/day, with a Time from diagnosis to transplant (days) 427.1 576.4 (24–5342) mean of 567 mg/day. Most patients (n ¼ 166, 58%) averaged Conditioning regimen 400 mg/day, with 14% (n ¼ 57) receiving less than 400 mg/ Cytoxan/total body irradiation 160 (51) day and 28% (n ¼ 88) receiving more than 400 mg/day on Busulfan/cytoxan 151 (49) average. Forty-seven individuals (15%) did not receive any Donor relationship folic acid supplementation between days 0 and 18 post Related donors 156 (50) transplantation (Table 1). HLA-matched 148 (95) No statistically significant differences in mean OMI HLA-mismatched 8 (5) scores, time to engraftment or days to acute GVHD were Unrelated 155 (50) observed when comparing patients taking, on average, Source ofhematopoietic progenitor cells o400 (14%), 400 (58%) or 4400 mg (28%) supplemental Bone marrow 297 (96) folic acid per day (Table 2). Compared with individuals Peripheral blood 14 (4) receiving an average of 400 mg folic acid per day (reference group), no significant variation in risk of acute GVHD, Folic acid intake days 0–18 post transplant o400 mg/day 57 (14) relapse or survival was observed for those receiving more 400 mg/day 166 (58) than or less than 400 mg folic acid per day. No clear trends 4400 mg/day 88 (28) were observed for the relationship between increasing folic acid intake and OMI scores or days to 500 granulocytes/ml. Outcome data Mean OMI scores (days 1–18) 17.079.4 (1.3–54.4)a Although not statistically significant, a trend toward Mean days to 500 granulocytes 21.674.3 (12–51)a delayed platelet recovery (days to 20 000 platelets) was Mean days to 20 000 platelets 20.8710.1 (9–85)a observed for increasing folic acid intake (P-trend ¼ 0.14) Mean days to acute GVHD 23.8714.5 (6–96)a (Table 2). Acute graft-versus-host disease No detectable acute GVHD 55 (18) Grade 1 28 (9) Grade 2 181 (58) Discussion Grade 3 41 (13) Grade 4 6 (2) To our knowledge, this is the first study to evaluate the Relapsed as of July 11, 2005 52 (17) effects of early post-transplant folic acid supplementation Surviving as of July 11, 2005 231 (74) on toxicity and outcomes in the HCT population. We have Abbreviations: GVHD ¼ graft-versus-host disease; OMI ¼ oral mucositis previously reported that pre-transplant multivitamin sup- index. plement use (before MTX administration) was associated aMean7standard deviation (range). with lower mean OMI scores in this cohort;16 however, detailed information on duration of supplement use or actual composition of the individual supplement was not available for our previous analysis. The current study folic acid supplementation during MTX therapy does not focused on the effect of folic acid supplementation during appear to negate treatment effects and may, in fact, be MTX treatment, and utilized medication administration beneficial. records to compile actual folic acid intake data over a Mead and colleagues18 found that mice given folic acid specific post-transplant period (days 0–18 post transplant). (25 mg/kg/day) 1 h before or at the same time as the MTX Our findings are in keeping with two studies in mice18,19 and (0.75 or 1.5 mg/kg) had significantly longer median survival an observational study of children receiving high-dose times than mice given 20 mg/kg/day folinic acid (leucovorin, MTX for acute lymphocytic leukemia,1 which suggest that 5-formyltetrahydrofolate, a downstream product of the

Bone Marrow Transplantation Folic acid during methotrexate immunosuppression K Robien et al 690 Table 2 Geometric means or risk estimates for various outcomes by folic acid intake

Average folic acid days 0–18

o400 mg 400 mg 4400 mg n ¼ 57 n ¼ 166 n ¼ 88

Mean OMI days 6–12a 14.7 (11.7–18.5) 18.0 (15.8–20.4) 14.1 (10.9–18.2) Mean OMI days 1–18a 13.5 (10.9–16.6) 14.6 (12.9–16.4) 13.2 (9.8–17.9) Mean days to 500 granulocytesa 21.1 (20.0–22.2) 21.6 (20.9–22.2) 20.6 (19.7–21.5) Mean days to 20 000 plateletsa 18.1 (16.3–20.0) 19.3 (18.2–20.5) 20.0 (18.4–21.7) Mean days to acute GVHDb 20.7 (17.7–24.2) 20.2 (18.4–22.1) 20.3 (17.9–23.1) Risk of acute GVHDc 1.0 (0.5–2.2) 1.0 (reference) 1.2 (0.5–2.7) Hazard of relapsed 0.9 (0.4–1.9) 1.0 (reference) 1.2 (0.6–2.6) Survivale 0.9 (0.5–1.6) 1.0 (reference) 0.9 (0.5–1.5)

Abbreviations: GVHD ¼ graft-versus-host disease; OMI ¼ oral mucositis index. aAdjusted for age, year of transplant and conditioning regimen. bAmong patients who developed acute GVHD. Adjusted for age, year of transplant and donor relationship (related vs unrelated). cOdds ratio. Adjusted for age, year of transplant and donor relationship (related vs unrelated). dHazard ratio. Adjusted for stage of CML at time of transplant (chronic or accelerated phase), year of transplant and age at transplant. eHazard ratio. Adjusted for stage of CML at time of transplant (chronic or accelerated phase), age at transplant and donor relationship (related vs unrelated).

dihydrofolate reductase enzyme) or mice that did not none of the children were folate deficient based on receive MTX. They also reported that mice given folic acid erythrocyte folate levels, and suggested that folic acid did not appear to suffer life-threatening toxicity until the should not be given during MTX treatment. However, MTX dose reached 2.5 mg/kg. Although not directly because erythrocytes have a half-life of approximately 120 comparable to our current study owing to significantly days, erythrocyte folate levels at 8 weeks after beginning higher MTX doses, the findings of Mead et al. suggest that treatment would reflect both pre-treatment folate status concurrent folic acid does not significantly alter MTX and folate status after MTX exposure. They also reported toxicity, whereas folinic acid does mitigate MTX toxicity. no difference in erythrocyte MTX levels between patient Similarly, Parchure et al.19 found that mice given a large who received folic acid supplements and those who did not, dose of folic acid (62.5 mg/kg) at the same time as MTX suggesting that folic acid supplementation did not compete (8.7 mg/kg) had a statistically significant increase in median with MTX for uptake into cells. The study did not evaluate survival time compared with mice given MTX without folic the effect of folic acid supplementation on treatment acid and mice given folic acid either 12 or 24 h before MTX outcomes, and dietary folate intake was not considered as administration. The authors proposed several hypotheses a potential effect modifier. that might explain their findings. The first was that the folic Studies on the effect of oral folic acid supplementation acid becomes trapped within the cell as dihydrofolate, during MTX treatment in the rheumatoid arthritis (RA) which later is reduced to THF once MTX has been cleared population, where MTX doses are more comparable to and dihydrofolate reductase activity is regenerated, thus those given following HCT, have generally found improved sparing marginally viable normal tissues that otherwise drug tolerance and minimal interference with drug effec- would have experienced significant toxicity. Their second tiveness.20,21 In a meta-analysis, Ortiz et al.22 found a hypothesis was that folic acid and MTX compete for renal statistically significant reduction in mucosal and gastro- excretion, and folic acid may retard elimination of MTX, intestinal side effects for RA patients receiving MTX with thus increasing the amount of time tumor cells are exposed folic acid supplementation (odds ratio ¼ 0.21, 95% con- to the drug. Although MTX is given at much lower doses fidence interval: 0.1–0.4) compared with individuals who for its immunosuppressive properties rather than its did not receive supplementation. Patients receiving folinic antineoplastic properties in the HCT setting, if this second acid supplementation reported less toxicity, but also hypothesis were true, we might have expected to see some reported a more modest improvement in joint tenderness. evidence of a decreased risk of relapse among individuals Current recommendations from the European rheumato- who received greater amounts of supplemental folic acid; logy community call for prescription of 5 mg folic acid per this was not seen. day orally in patients who are taking MTX for the A cross-sectional observational study of children with treatment of RA23 or evaluation of dietary folate intake acute lymphocytic leukemia receiving maintenance chemo- and supplementation as needed.24 therapy with high-dose MTX (20 mg/m2 per week) and The FDA recently amended its requirements for adult 6-mercaptopurine1 is the only human study to evaluate the parenteral multivitamin products, increasing the require- effect of folic acid supplementation before and continuing ment for folic acid from 400 to 600 mg per unit dose.25 Our through MTX treatment. At 8 weeks after beginning study suggests that this increase in standard parenteral folic therapy, slightly more than half of the patients (54%, 25 acid supplementation will have no detrimental effects on of 46) had been taking folic acid supplements (75–200 mg/ the HCT patient population. day) for at least 3 months, whereas 46% (21 of 46) had not A limitation of this study is that the multivitamin received any supplementation. The researchers found that shortage leading to the variation in folic acid intake

Bone Marrow Transplantation Folic acid during methotrexate immunosuppression K Robien et al 691 occurred for all patients undergoing HCT during a 5 Cutler C, Li S, Kim HT, Laglenne P, Szeto KC, Hoffmeister L specified time period. The time dependency of this variation et al. Mucositis after allogeneic hematopoietic stem cell in intake might have masked the effects of other transplantation: a cohort study of methotrexate- and non- unidentified, time-dependent factors contributing to treat- methotrexate-containing graft-versus-host disease prophylaxis ment-related toxicity and outcome such as improvements regimens. Biol Blood Marrow Transplant 2005; 11: 383–388. in supportive care, although this would be an odd 6 Alloju M, Ehrinpreis MN. Shortage of intravenous multivitamin solution in the United States. N Engl J Med 1997; 337: coincidence. 54; author reply -5. Another limitation is the lack of accurate, quantitative 7 Clift RA, Buckner CD, Thomas ED, Bensinger WI, Bowden data on pre-transplant supplemental folic acid intake, R, Bryant E et al. Marrow transplantation for chronic myeloid dietary folate intake throughout the pre- and post- leukemia: a randomized study comparing cyclophosphamide transplant period or measurements of pre-transplant folate and total body irradiation with busulfan and cyclophos- status as indicated by erythrocyte folate levels or plasma phamide. Blood 1994; 84: 2036–2043. homocysteine levels. It is likely that pre-transplant folate 8 Hansen JA, Gooley TA, Martin PJ, Appelbaum F, Chauncey intake and/or folate status has a greater effect on MTX TR, Clift RA et al. Bone marrow transplants from unrelated response during the early post-transplant period, as donors for patients with chronic myeloid leukemia. N Engl J Med 1998; 338: 962–968. suggested by our previous report.16 Protocols for more 9 Storb R, Deeg HJ, Whitehead J, Appelbaum F, Beatty P, recently developed antifolate drugs, including peme- Bensinger W et al. Methotrexate and cyclosporine compared 26–28 29 trexed and lometrexol, call for optimizing the with cyclosporine alone for prophylaxis of acute graft versus patient’s folate status with folic acid supplementation host disease after marrow transplantation for leukemia. N Engl before treatment and between treatment cycles in attempts JMed1986; 314: 729–735. to minimize treatment-related toxicity in normal tissues. 10 Riley RS, Idowu M, Chesney A, Zhao S, McCarty J, Lamb LS In summary, supplementing folic acid up to 1400 mg/day et al. Hematologic aspects of myeloablative therapy and bone did not mitigate MTX toxicity or treatment outcome in this marrow transplantation. J Clin Lab Anal 2005; 19: 47–79. HCT population. Prospective studies, which include 11 Woo SB, Sonis ST, Monopoli MM, Sonis AL. A longitudinal assessment of pre-transplant folate status, are needed to study of oral ulcerative mucositis in bone marrow transplant Cancer confirm these findings within the HCT population, as well recipients. 1993; 72: 1612–1617. 12 Schubert MM, Williams BE, Lloid ME, Donaldson G, as other oncology patient populations, especially those Chapko MK. Clinical assessment scale for the rating of oral receiving high-dose MTX. mucosal changes associated with bone marrow transplantation. Development of an oral mucositis index. Cancer 1992; 69: 2469–2477. Acknowledgements 13 Radich JP, Gehly G, Gooley T, Bryant E, Clift RA, Collins S et al. Polymerase chain reaction detection of the BCR-ABL fusion transcript after allogeneic marrow transplantation for This project would not have been possible without the support chronic myeloid leukemia: results and implications in 346 of colleagues from the Fred Hutchinson Cancer Research patients. Blood 1995; 85: 2632–2638. Center, Seattle, WA, USA including clinical data support from 14 Institute of Medicine. Folate. Dietary Reference Intakes for Gary Schoch, and clinical nutrition data from the Seattle Cancer Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Niacin, Care Alliance Clinical Nutrition Staff. And, we are extremely Vitamin B12, Pantothenic acid, Biotin, and Choline. Institute grateful to the patients who consented to participate in this, and of Medicine, Subcommittee of Folate, Other B Vitamins, and other, research studies. 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