Joseph E. Baggott

Supplementation with folic acid during methotrexate therapy for rheumatoid arthritis. A double-blind, placebo-controlled trial.

The folic acid antagonist methotrexate (n-10-methyl-aminopterin) is useful in low doses (2.5 to 20 mg/wk) for treating chronic inflammatory diseases [1-7]. Many trials have established the efficacy of methotrexate in rheumatoid arthritis [7-13]. Compared with other disease-modifying drugs, methotrexate has the highest probability of drug continuation at 10 years. Dose response-related toxic effects have been reported in 30% to 90% of patients given methotrexate [13]. Toxic effects include gastrointestinal intolerance, hematologic abnormalities, alopecia, hepatotoxicity, and pulmonary toxicity [14-22]. Some side effects of methotrexate administration, such as gastrointestinal intolerance, mimic complicated folate deficiency [23]. Folate deficiency occurs frequently in patients with rheumatoid arthritis; further, folate stores are decreased in patients with rheumatoid arthritis who take methotrexate, suggesting that impaired folate status is related to toxicity [24-26]. Folic acid supplementation has been reported anecdotally to lessen toxicity in patients receiving methotrexate treatment [27, 28]. In a 6-month, double-blind, placebo-controlled trial, 7 mg of folic acid weekly (1 mg/d or 2265 nmol/d) decreased methotrexate toxicity without affecting efficacy [29]. This was confirmed by Stewart and colleagues [30] in a retrospective chart review. Folinic acid (leucovorin, citrovorum factor) is a one-carbon-substituted, fully reduced folate that has also been administered during methotrexate therapy [31-36]. Low doses of the vitamin (1 to 7 mg/wk) have decreased methotrexate toxicity [35, 36]. Higher doses negate efficacy and lessen toxicity [31, 32]. Thus, the folinic acid dose may critically affect the efficacy of methotrexate therapy. The influence of the folic acid dose on methotrexate toxicity and efficacy remains controversial, and the effects of different doses of folic acid are not known [37, 38]. Some investigators argue that if toxic effects occur, the most rational approach is to reduce the dose of methotrexate rather than to provide folic acid supplements [37]. We designed a larger and longer study to evaluate different doses of folic acid, assuming that toxicity could be reduced without changing the efficacy of methotrexate. Methods Participants Patients aged 19 to 78 years who fulfilled the American College of Rheumatologys revised criteria for rheumatoid arthritis consented to participate in the trial [39]. Enrollment criteria included rheumatoid arthritis diagnosed more than 6 months previously, onset after the age of 16 years, and at least three of the following signs or symptoms: 3 or more swollen joints, 6 or more tender joints, at least 45 minutes of morning stiffness, and a Westergren erythrocyte sedimentation rate 28 mm/h. Referring rheumatologists and the principal investigator did the screening. Exclusion criteria included serious concomitant medical illnesses, liver enzyme levels twice the upper limit of normal, leukocyte counts less than 3.5 109/L or platelet counts less than 150 109/L, and use of methotrexate within the past 6 months. Gold salts were stopped for at least 10 days before the trial. This short washout period mirrors actual rheumatology practice. Patients remained under the care of their rheumatologists, abstained from alcohol use, did not become pregnant, and received stable doses of aspirin and nonsteroidal anti-inflammatory drugs. If prednisone was taken at entry, the dose could not exceed 10 mg/d. Hydroxychloroquine therapy was allowed during the study. Study Design Figure 1 shows the trial design. To maintain the double-blind status of the trial, the statistician carried out the randomization using a computer program in which the algorithm was transparent and a coded vial number represented the treatment assignment. Patients were assigned to treatment groups by a sequential treatment assignment process designed to balance the sample with respect to baseline features, including age, sex, folate-containing vitamin use, rheumatoid factor status, and prednisone use [40]. Patients agreed to discontinue therapy with folate-containing vitamins during the trial. Rheumatoid factor was considered positive if the level was more than 30 IU/mL or if the titer was more than 1:160. Patients received either visually identical placebo or 5 mg (low-dose folic acid group) or 27.5 mg folic acid (high-dose folic acid group) each week, prepared by the Hospital Investigational Drug Service. Spectrophotometric analysis indicated that the mean SD folic acid content was 1 0.15 mg (2.3 0.3 mol) and 5.5 0.3 mg (12.5 0.7 mol) per capsule in the low-dose and high-dose folic acid groups, respectively. Lederle Laboratories provided the methotrexate (Rheumatrex; Pearl River, New York), which was started in a median oral dose of 16.5 mol (7.5 mg) per week and increased in 5.5-mol (2.5 mg) increments at the rheumatologists discretion. Methotrexate was taken either in an undivided or a divided dose (that is, every 12 hours for three doses). The methotrexate dosing regimens were identical among the study groups. Folic acid supplements were given 5 days per week when methotrexate was not ingested. Compliance with the regimen was reinforced using a digital reminder cap (Counter Cap; Senetics, Boulder, Colorado). All participants and investigators were blinded to vitamin capsule content until the study was complete. Figure 1. Study design for the double-blind, placebo-controlled trial. Clinical Assessment Patients were evaluated immediately before methotrexate initiation at a mean of 13, 26, 39, and 53 weeks (Figure 1). Each patient was assessed by the same physician-nutritionist (SLM). Two research assistants (JSA or WHV) did the joint evaluations. In most cases, patients were examined by the same observer throughout the study. The joint counts for tenderness and swelling were not significantly different between the two observers (P = 0.6 for tenderness; P = 0.9 for swelling). The following variables were evaluated at each visit: 1) number of the 58 diarthrodial joints with swelling; 2) number of the 60 joints with tenderness on pressure or with pain on passive motion (or both); 3) joint swelling and tenderness indices, expressed as a sum, with each joint graded for swelling as 0 (none), 1 (mild), 2 (moderate), or 3 [severe]; 4) mean grip strength (three measurements) for both hands expressed in mm Hg; 5) duration of morning stiffness expressed in hours; 6) patients and physicians global assessments of disease activity graded as 0 (asymptomatic), 1 (mild), 2 (moderate), 3 (severe), or 4 (very severe); 7) current medications and doses; 8) a 1-day dietary recall using the Minnesota Nutrition Data System software, Food Database version 6A, Nutrient Database version F21 [41]; 9) eight activities of daily living assessed and averaged at baseline and at 12 months using the modified Health Assessment Questionnaire and scored on a scale of 1 (no difficulty) to 4 (unable to perform) [42]; and 10) presence, duration, intensity, and severity of toxic effects at every follow-up visit and every 3 weeks by telephone interview (done by SLM). Laboratory Assessments At the initial visit, complete blood cell count, Westergren erythrocyte sedimentation rate, liver enzyme (aspartate amino transferase and alkaline phosphatase), rheumatoid factor by nephelometry, and serum creatinine values were obtained. The complete blood cell count, creatinine, and liver function tests were repeated at all visits. If laboratory values were obtained more frequently than stipulated in the protocol, they were recorded and became part of the toxicity score. Blood for plasma and erythrocyte folate levels, using a methotrexate-resistant Lactobacillus casei microbiological assay, was drawn at all visits [43]. At baseline, blood was drawn for a vitamin panel (plasma and erythrocyte folate, vitamins B12, A, C, and E, carotene, riboflavin, thiamin, pyridoxine) [44-51]. If patients had abnormal values for any of the vitamins, other than folate, the abnormality was treated with single vitamin supplements. Radiographic Assessment Hand and wrist radiographs taken within 6 months of entering the trial were assessed by one of the rheumatologists (GSA) without knowledge of study status. Joint erosions and space narrowing were determined by the modified method of Sharp [52]. Results were expressed as the mean raw scores for joint erosion and joint space narrowing. Toxicity: Frequency and Severity Toxic effects and discontinuation of therapy with study medications due to toxicity were considered primary outcomes. We determined a toxicity score, modified from the one previously used, for each patient at each visit or until methotrexate therapy was discontinued [29] (Appendix). Efficacy We determined patient response to treatment using a modification of the criteria used in our previous folic acid supplementation trial and by others [8, 29, 53]. We defined marked improvement in the joint swelling index and the joint tenderness and pain index as a net decrease of 50% or more in value at any follow-up visit compared with visit 1. We defined moderate improvement as a 30% to 49% net decrease in the index, and no change as the index value remaining within 30% of the original value. We defined worsening as an increase in the index value of 30% or more. We examined the effects of folic acid supplementation on changes in physician and patient global assessments, grip strength, morning stiffness, erythrocyte sedimentation rate, findings of the modified Health Assessment Questionnaire, complete blood cell counts, blood folate levels, and dietary folate and vitamin B12 intake. Table 1. Demographic and Selected Clinical Characteristics of 79 Patients with Rheumatoid Arthritis* Statistical Analysis Sample Size and Analysis In our previous study, the mean toxicity scores for patients receiving low-dose folic acid and placebo recipients were 0.21 and 1.06, respectively, for

Homocysteine levels in patients with rheumatoid arthritis treated with low-dose methotrexate

Plasma homocysteine levels were determined in patients who participated in a randomized, double‐blind placebo‐controlled trial of folate supplementation (1 mg/day) during methotrexate therapy for rheumatoid arthritis. Plasma and red blood cell folate levels before methotrexate therapy were significantly negatively correlated with homocysteine levels. Homocysteine levels were not significantly correlated with the initial C1 index (an assay that measures the folate status of blood mononuclear cells) or the C1 index during methotrexate therapy. There was no significant difference in homocysteine levels between pre‐treatment and levels drawn at 3 or 6 months. Initial homocysteine levels were predictive of toxicities, such as gastrointestinal intolerance and elevations of liver enzymes in the placebo group. There was no significant correlation between occurrence of toxicity and initial homocysteine levels in the folic acid‐supplemented group. Homocysteine levels were not predictive of the efficacy of methotrexate therapy. We conclude that plasma homocysteine levels are correlated with plasma and red blood cell folate levels before methotrexate therapy but is not correlated with folate status in blood mononuclear cells.

Meta-analysis of cancer risk in folic acid supplementation trials

Several reports suggest that folate has a procarcinogenic effect. Folate has a unique role because its coenzymes are needed for de novo purine and thymine nucleotide biosynthesis. Antifolates, such as methotrexate, are used in cancer treatment. Using a meta-analysis weighted for the duration of folic acid (pteroylglutamic acid) supplementation, we analyzed the cancer incidence of six previously published large prospective folic acid-supplementation trials in men and women. These articles were carefully selected from over 1100 identified using PubMed search. Our analyses suggest that cancer incidences were higher in the folic acid-supplemented groups than the non-folic acid-supplemented groups (relative risk=1.21 [95% confidence interval: 1.05-1.39]). Folic acid-supplementation trials should be performed with careful monitoring of cancer incidence. Solid monitoring systems to detect side effects, including increase in cancer risk, should be established before the initiation of folic acid supplementation trials.

The effect of methotrexate and 7-hydroxymethotrexate on rat adjuvant arthritis and on urinary aminoimidazole carboxamide excretion

OBJECTIVE To study the efficacy, toxicity, and antifolate activities of 7-hydroxymethotrexate (7-OH-MTX) versus methotrexate (MTX) in the treatment of rat adjuvant-induced arthritis. METHODS Dose-dependent effects in rat adjuvant arthritis were determined by histologic and clinical examinations. Antifolate activity was determined by urinary levels of aminoimidazole carboxamide (AIC) as a marker for blockade of the folate-dependent enzyme, aminoimidazolecarboxamide ribotide transformylase (AICARTase). RESULTS MTX was 8 times more efficacious than 7-OH-MTX and resulted in higher urinary AIC levels. Increased urinary AIC levels were correlated with suppression of rat adjuvant arthritis regardless of the drug or dose level used. CONCLUSION The ability to metabolize MTX to 7-OH-MTX and the sensitivity of AICARTase to inhibition by 7-OH-MTX may at least partially account for the variability in response to MTX. Blocking of AICARTase may be important in the efficacy of these antifolates.

Methotrexate catabolism to 7‐hydroxymethotrexate in rheumatoid arthritis alters drug efficacy and retention and is reduced by folic acid supplementation

OBJECTIVE To assess the catabolism of methotrexate (MTX) to 7-hydroxy-MTX (7-OH-MTX) in patients with rheumatoid arthritis as well as the effect of folic acid and folinic acid on this catabolism. METHODS Urinary excretion of MTX and its catabolite, 7-OH-MTX, was measured in 2 24-hour urine specimens collected after MTX therapy. Urine samples were collected from patients after the sixth and seventh weekly doses of MTX. MTX and 7-OH-MTX concentrations were determined by high-performance liquid chromatography mass spectrometry. Swelling and pain/tenderness indices were used to measure symptoms before and at 6 and 7 weeks of therapy. Patients received either folic acid or folinic acid supplements (1 mg/day) from week 6 to week 7. RESULTS Folic acid inhibited aldehyde oxidase (AO), the enzyme that produces 7-OH-MTX, but folinic acid did not. Excretion of 7-OH-MTX (determined as a percentage of the dose of MTX or as mg 7-OH-MTX/gm creatinine) was not normally distributed (n=39). Patients with marked improvement in swelling and pain/tenderness indices had a lower mean 7-OH-MTX excretion level (P<0.05). Patients who received folic acid supplements had decreased 7-OH-MTX excretion (P=0.03). Relatively high 7-OH-MTX excretion was correlated with relatively high MTX excretion and with relatively low MTX retention in vivo (P<0.05) (n=35). CONCLUSION Our findings of a non-normal distribution of 7-OH-MTX excretion suggest that there are at least 2 phenotypes for this catabolism. Decreased 7-OH-MTX formation suggests folic acid inhibition of AO and a better clinical response, while increased 7-OH-MTX formation may interfere with MTX polyglutamylation and binding to enzymes and, therefore, may increase MTX excretion and decrease MTX retention and efficacy in vivo.

Homocysteine, Iron and Cardiovascular Disease: A Hypothesis

Elevated circulating total homocysteine (tHcy) concentrations (hyperhomocysteinemia) have been regarded as an independent risk factor for cardiovascular disease (CVD). However, several large clinical trials to correct hyperhomocysteinemia using B-vitamin supplements (particularly folic acid) have largely failed to reduce the risk of CVD. There is no doubt that a large segment of patients with CVD have hyperhomocysteinemia; therefore, it is reasonable to postulate that circulating tHcy concentrations are in part a surrogate marker for another, yet-to-be-identified risk factor(s) for CVD. We found that iron catalyzes the formation of Hcy from methionine, S-adenosylhomocysteine and cystathionine. Based on these findings, we propose that an elevated amount of non-protein-bound iron (free Fe) increases circulating tHcy. Free Fe catalyzes the formation of oxygen free radicals, and oxidized low-density lipoprotein is a well-established risk factor for vascular damage. In this review, we discuss our findings on iron-catalyzed formation of Hcy from thioethers as well as recent findings by other investigators on this issue. Collectively, these support our hypothesis that circulating tHcy is in part a surrogate marker for free Fe, which is one of the independent risk factors for CVD.

Binding of SPAAT, the 44‐residue C‐terminal peptide of α1‐antitrypsin, to proteins of the extracellular matrix

SPAAT (short piece of α1‐antitrypsin [AAT]), the 44‐residue C‐terminal peptide of AAT, was originally isolated from human placenta [Niemann et al. (1992): Matrix 12:233–241]. It was shown to be a competitive inhibitor of serine proteases [Niemann et al. (in press): Biochem Biophys Acta]. The binding of SPAAT to one or more proteins of the extracellular matrix (ECM) was initially suggested on the basis of its recovery from tissue residues following a series of extractions designed to remove easily solubilized proteins [Niemann et al. (1992): Matrix 12:233–241]. Our binding studies with the model ECMs, Matrigel and Amgel, suggested that SPAAT might be bound by a specific collagen type as well as one or more non‐collagenous ECM proteins. Individual ECM components were screened for their ability to bind SPAAT. When the four commonly occurring fiber‐forming collagens (types I, II, III, and V) were evaluated, type III was found to be preferred. In addition, although SPAAT bound to preformed type III collagen fibers in a concentration dependent fashion, it did not bind to type III collagen molecules undergoing fibril formation. This is consistent with a physiological mode of interaction between SPAAT and type III collagen in vivo. Of the non‐collagenous ECM macromolecules (laminin‐1, fibronectin, entactin, and heparan sulfate) tested, laminin‐1 was preferred. The binding of radiolabelled SPAAT to type III collagen and laminin‐1 was competitively inhibited by unlabelled SPAAT as well as an unrelated protein, human serum albumin (HSA), to establish binding specificity. The kinetics of the release of the bound radiolabelled SPAAT were also examined to substantiate the non‐covalent and reversible nature of this association. These results support the view that susceptible proteins of the ECM may actually be coated with SPAAT in vivo, possibly affording protection against inappropriate protease digestion. J. Cell. Biochem. 66:346–357, 1997.

Methotrexate therapy in rheumatoid arthritis patients diminishes lectin-induced mononuclear cell proliferation

SummaryMethotrexate (MTX) is an anti-folate drug used in cancer chemotherapy because of its anti-proliferative effects. However, it is unclear whether the anti-proliferative effects of MTX contribute to the efficacy of low-dose MTX in the treatment of rheumatoid arthritis (RA). To date, either no change or a paradoxical increase in lectin-induced proliferation has been observed in cultures of peripheral blood mononuclear cells (PBMC) from MTX-treated RA patients (RA+MTX). In these earlier studies, high folate-containing media and tritiated thymidine (3H-TdR) were used. Our studies were designed to test the hypothesis that the use of a culture medium with a low folate content along with tritiated deoxyuridine (3H-UdR) permits detection of diminished phytohemagglutinin (PHA)-induced proliferative responses of PBMC from RA+MTX. The data demonstrate decreased PHA-induced cellular proliferation of cultured PBMC from RA+MTX compared with controls. When comparing the PBMC proliferative responses in high vs low folate medium, a significantly greater increase (P<0.05) in proliferation occurs in the cells from RA+MTX cultured in the high folate medium. This suggests that an in vivo folate-deficient state of the cells from RA+MTX may be corrected in vitro when a high folate medium is used in culture. We conclude that the use of3H-UdR and a medium containing folate within the normal range of plasma folate levels eliminates artifacts associated with the use of high folate medium and3H-TdR, which obscures the anti-proliferative effect of MTX in RA patients.

Regulatory Role of Oxidized and Reduced Pteroylpolyglutamates

The conversion of the vitamin folk acid, pteroylglutamic acid (PteG,), to the biologically active intracellular folates requires two enzyme-mediated structural modifications: reduction of the pteridine ring to the 5, 6, 7 , 8 tetrahydro form by dihydrofolate reductase (reaction [2], FIG. l‘), and the sequential addition in gamma-peptide linkage of a varying number of glutamyl residues to the glutamic acid moiety of folic acid by folyl-poly-gamma-glutamate synthetase [3]. The biological role of the polyglutamyl chain has been the subject of much study since the advent of synthetic pteroylpolyglutamates in the late sixties.’ It is now unequivocally established that the polyglutamate chain is essential for the intracellular retention of folate~,~-’ and considerable evidence supports the hypothesis6v7 that changes in its length play a role in the regulation of onecarbon metabolism. The folates serve as coenzymes for one-carbon transfer reactions and as substrates for oxidation-reduction reactions that alter the oxidation state of folate-bound one-carbon fragments (summarized in FIG. 1 ) . These reactions are required for the de nouo biosynthesis of purines and thymidylate. They are therefore essential for RNA and DNA replication and for the synthesis of all purine-containing coenzymes. In addition, the folates provide de nouo generated methyl groups for the remethylation of homocysteine to methionine, an important step for the maintenance of adequate levels of S-adenosyl-methionine (SAM), the main coenzyme of numerous methyl-transfer reactions. These varied reactions comPete for the folates of the cell, which, with the exceptions of liver and kidney, occur in most tissues at rather low concentrations (0.5 to 2 P M ) , ~ often below the Michaelis constants (K,s) of some of the folate-requiring enzymes. This necessitates the presence of complex and as yet incompletely understood regulatory mechanisms. As a minimum, two conditions must be satisfied to validate the hypothesis that changes in the length of the poly-gamma-glutamyl chain of the folates play a role in the regulation of one-carbon fragment metabolism: one, the demonstration in vitro that the catalytic efficiency of folate-dependent enzymes changes in response to alterations in the length of their polyglutamyl cofactors and/or inhibitors; and two, changes in the polyglutamyl chain length occur in vivo under conditions that alter the steady-state of one-carbon metabolism.

Measurement of Erythrocyte Methotrexate Polyglutamate Levels: Ready for Clinical Use in Rheumatoid Arthritis?

Methotrexate (MTX) is one of the most commonly prescribed and most effective drugs for the treatment of rheumatoid arthritis (RA). Given the partial response of many patients and the side effect profile of the drug, there is considerable interest in identifying biomarkers to guide MTX therapy in RA. Upon entering cells, MTX is polyglutamated. Measuring MTX polyglutamate (MTX PG) levels in circulating red blood cells has been proposed as an objective measure to help optimize MTX therapy in RA. Data are conflicting with regard to the clinical utility of MTX PG measurements as a predictor of the efficacy or toxicity of low-dose MTX effects in RA. Should large, randomized clinical trials of this assay show consistent, reproducible, long-term correlations between MTX PG levels and efficacy or toxicity, this test could become a prominent tool for clinicians to optimize the use of MTX in treating RA.