Methotrexate (MTX) is an antifolate with a chemical structure similar to that of folic acid and folinic acid (5-formyl-tetrahydrofolic acid) (Figure 1). Administered in low dosages (7.5–25 mg/week), MTX inhibits a number of folate-dependent metabolic steps, including a very potent inhibition of dihydrofolate reductase (DHFR), which reduces folic acid to dihydrofolic acid and to tetrahydrofolate. Once absorbed, MTX is metabolized to polyglutamate derivatives, which have a greater ability to inhibit other folate-dependent enzymes, such as thymidylate synthetase, and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase, as compared with MTX (1).
The mechanism of action of low-dose MTX in the treatment of rheumatoid arthritis (RA) is unclear. Proposed mechanisms of action of MTX in RA have centered on adenosine-mediated antiinflammatory effects (2–8). Inhibition of AICAR transformylase causes increased levels of AICAR, with subsequent inhibition of AMP deaminase and adenosine deaminase (ADA) (1). MTX therapy then results in an increase in extracellular adenosine concentration. Adenosine binds to many transmembrane-spanning adenosine surface receptors (e.g., A1, A2α, A2β, and A3) (3). As a result of binding to A2 and A3 receptors, lymphocyte proliferation is inhibited and a cytokine environment favorable for lowering inflammation is fostered (3). However, the importance of adenosine-mediated mechanisms has recently been questioned in rat adjuvant-induced arthritis, where there was no attenuation of antiarthritic effect when 3 adenosine antagonists were combined with MTX (9).
We hypothesized that the effect of MTX in the treatment of RA is due to the inhibition of AICAR transformylase, a folate-dependent enzyme that catalyzes the last step in the de novo biosynthesis of inosine monophosphate (Figure 2). The resulting accumulation of AICA riboside inhibits ADA, interfering with normal adenosine metabolism. This inhibition of folate metabolism should increase urinary excretion of both adenosine and aminoimidazole-carboxamide (a catabolite of the riboside). We further hypothesized that if the MTX efficacy is driven by AICAR transformylase inhibition, the folic acid supplementation should not substantially alter urinary levels of AICA and adenosine, whereas folinic acid supplementation should attenuate the accumulation of these compounds. These results would occur because the folic acid supplement would be trapped as folic acid by the potent MTX inhibition of DHFR and therefore would not increase the pool of tetrahydrofolates (Figure 2). In contrast, folinic acid bypasses MTX inhibition of DHFR and is readily metabolized to 10-formyl-tetrahydrofolate, a cofactor used by the transformylase, and will readily replete the pool of metabolically active tetrahydrofolates (Figure 2). Thus, MTX inhibition of the transformylase would be substantially relieved by folinic acid supplementation and only marginally affected by folic acid supplementation. Therefore, we hypothesized that folinic acid supplementation during MTX therapy would reduce both urinary AICA and adenosine excretion more than folic acid supplementation.
Figure 2. Effect of methotrexate (MTX) on folate and purine metabolism. AICAR = 5-aminoimidazole-4-carboxamide ribonucleotide; T'ase = transformylase; IMP = inosine monophosphate; AICA = 5-aminoimidazole-4-carboxamide; ADA = adenosine deaminase; DHFR = dihydrofolate reductase. Broken lines with encircled minus signs indicate inhibition of enzymes by MTX and AICA riboside. Encircled arrows indicate an increase (↑) or a decrease (↓) in metabolite levels when MTX inhibits DHFR or AICAR transformylase and when AICA riboside inhibits ADA. Note that folinic acid, but not folic acid, can bypass MTX inhibition of DHFR and replete pools of 10-formyl-tetrahydrofolate.
Download figure to PowerPoint
PATIENTS AND METHODS
- Top of page
- PATIENTS AND METHODS
The Institutional Review Board of the University of Alabama at Birmingham (UAB) approved this protocol. Forty-one patients, ages 18–85 years, who had not been taking any folate supplements and who were to start MTX de novo gave informed consent to participate in the trial. Enrollment criteria included RA according to the American College of Rheumatology (formerly, the American Rheumatism Association) revised criteria (10), disease duration of 6 months or more, onset after 16 years of age, a Westergren erythrocyte sedimentation rate ≥28 mm/hour, and at least 3 of the following signs and symptoms: 3 or more swollen joints, 6 or more tender joints, and at least 45 minutes of morning stiffness. Exclusion criteria included serious concomitant medical illnesses, abnormal findings on hepatitis serologies, liver enzyme levels twice the upper limit of normal, leukocyte counts <3.5 × 109/liter or platelet counts <150 × 109/liter, use of MTX within the past 3 months, and current folic acid supplementation. Prednisone dosages did not exceed 10 mg/day, and patients abstained from alcohol use, practiced adequate contraception, and received stable doses of aspirin and/or nonsteroidal antiinflammatory drugs during the protocol, as prescribed by their treating rheumatologist. None of the patients were receiving concurrent biologic therapies.
Patients were admitted to the Pittman General Clinical Research Center (GCRC) at UAB for 3 24-hour visits over a 7-week period. Patients were enrolled from June 1997 to September 2001. The study design is shown in Figure 3.
Visit 1 (baseline).
At the first GCRC admission, the patients were not receiving MTX therapy. A 24-hour urine collection to determine AICA and adenosine levels was performed. Blood samples were drawn when the patients were fasting, and plasma and red blood cell (RBC) levels of folate and vitamin B12 were determined. If the vitamin B12 level was <200 pg/ml, patients were treated, in consultation with the referring rheumatologist, with 1,000 μg of vitamin B12 intramuscularly for 3 days prior to enrollment. Joint counts and indices for pain/tenderness and swelling were completed in the early morning of the visit by a trained registered nurse at the GCRC. The patient's assessment of pain and the physician's and patient's assessments of disease activity were determined using 10-cm visual analog scales, and the modified Health Assessment Questionnaire was also completed (11). A dietary history for the prior 24 hours was taken at the time of admission to the GCRC.
After the initial 24-hour urine collection was performed, MTX was started orally at a dosage of 7.5–10 mg/week based on the instructions of the referring rheumatologist. Patients were instructed to take their MTX dose 1 day per week. One patient was dropped from the protocol because he stopped taking the MTX after visit 1 to start biologic therapy.
Visit 2 (MTX therapy).
On the day that the patients were to take their sixth dose of MTX, they were readmitted to the GCRC. A second 24-hour urine collection to determine AICA and adenosine levels was begun when taking this dose of MTX. Blood samples were collected when the patients were fasting to determine plasma and RBC levels of folate; a complete blood cell count with differential cell count and a blood chemistry profile were also performed. Joint counts and scores and a dietary recall were completed as in visit 1.
Randomization to folic acid or folinic acid supplementation.
At visit 2, as shown in Figure 3, all patients were randomized to receive either 5 mg of folic acid or 5 mg of (6R,S)-folinic acid per day. This daily supplementation protocol would sustain high blood levels of folate. Randomization was stratified by rheumatoid factor status (positive or negative). The capsules were made by the Investigational Drug Service at UAB and were identical in appearance; therefore, the patient and the investigator were blinded to the form of the vitamin. Patients were instructed to take the first capsule after completion of the second 24-hour urine collection, take 1 capsule daily, and save the last capsule to be taken with MTX (the seventh dose) at the time of readmission 1 week later (i.e., visit 3).
Visit 3 (MTX and folate supplementation).
On the day that the patients were to take their seventh dose of MTX, they were readmitted to the GCRC. A third 24-hour urine collection to determine AICA and adenosine levels was begun at the time of the seventh weekly dose of MTX and the last folic acid or folinic acid supplement. Blood work, joint assessments, and a dietary recall were completed as in visit 1.
Blood was collected into tubes containing EDTA, and plasma and RBCs were separated within 1 hour and frozen at −70°C until assayed. All 24-hour urine volumes were measured, and a urine sample, adjusted to pH 2.5–3.5, was stored at −70°C.
Urinary AICA levels were determined by a colorimetric assay (12) after extraction (12, 13). Urinary adenosine levels were determined by a radioimmunocompetition assay (14). AICA and adenosine were normalized for creatinine excretion (kit 555; Sigma, St. Louis, MO). The coefficient of variation (CV) of these assays was ∼10%.
Plasma and RBC levels of folic acid were determined by a 96-well microtiter assay using MTX-resistant Lactobacillus casei (15). Vitamin B12 was measured by a radioactive competitive assay (kit 191-1041; Bio-Rad, Hercules, CA). The CV of these assays was ∼20%. Plasma homocysteine was determined by high-performance liquid chromatography (16). The CV of this assay was ∼10%.
The primary outcome was the changes in urinary AICA and adenosine levels before versus after MTX therapy and the effects of folic acid and folinic acid supplementation on these levels. Secondary outcome measures included the course of plasma and RBC levels of folate and homocysteine during the 3 visits.
Quantitative variables are expressed as the mean ± SD, and qualitative variables are expressed as frequencies and percentages. Baseline demographic characteristics of the study groups were compared using Fisher's exact test. Study group and visit comparisons were performed for primary and secondary outcome measures; these comparisons were performed using a mixed-model repeated-measures analysis of variance, assuming an unstructured covariance matrix. Tukey's test was then performed to determine which specific pairs of means were significantly different. Urinary AICA and adenosine excretion levels were log-transformed prior to these statistical analyses; this was done since logAICA and logadenosine followed a normal distribution. Changes from visit 2 to visit 3 for AICA, adenosine, plasma and RBC folate, and plasma homocysteine levels followed an approximately normal distribution; therefore, these changes were assessed using the standard 2-group t-test; when the variances were unequal between the 2 study groups, Satterthwaite's method was used. Specific cross-sectional comparisons, such as baseline comparisons of quantitative variables between study groups, were performed in a similar manner.
All pairwise correlations and their corresponding significance levels were obtained by Pearson's correlation analysis. These correlations were obtained only for logAICA versus percentage change (from visit 1 to visit 2) in the joint swelling count, swelling score, pain/tenderness joint count, and pain/tenderness score, and for logadenosine versus percentage change in the same measures. All of these variables followed a normal distribution. Correlations were also obtained for logAICA versus RBC folate. Wilcoxon's rank sum test was used for comparisons of the cumulative MTX dose between the 2 study groups. Results at visit 1 are not provided separately for folic acid and folinic acid supplementation groups since supplementation did not begin until visit 2. All statistical tests were 2-sided and were performed at a 5% significance level (i.e., α = 0.05). All statistical analyses were performed with SAS software (version 9.0; SAS Institute, Cary, NC).
- Top of page
- PATIENTS AND METHODS
Our data confirm the results from a previous study showing that MTX therapy causes levels of homocysteine to increase and that folate supplementation lowers homocysteine levels (18).
The relative levels of folate in both plasma and RBCs in our population, measured by an MTX-resistant microbiologic assay, were unexpected. We have previously shown that the MTX-resistant microbiologic assay provides lower levels of folate in plasma and RBCs than does a radioimmunoprecipitation assay (19). The baseline RBC levels of folate may now be relatively higher than in our previous experience because of food folate fortification (20). Our previous study measured folate levels during MTX therapy in patients recruited before 1998 and prior to food folate fortification becoming effective.
MTX is currently the most widely used disease-modifying antirheumatic drug for RA either as monotherapy or in combination with sulfasalazine and/or hydroxychloroquine and infliximab (and less commonly, with cyclosporine A, leflunomide, and other biologic therapies) (21–29). There is debate about the mechanism of action of MTX. We have hypothesized that antifolate antagonism is important in its mechanism of action (8). The fact that weekly MTX is effective suggests that polyglutamates of MTX affect enzymes in addition to dihydrofolate reductase, the traditional target of MTX (1). Consistent with our hypothesis, excessive folinic acid supplementation during MTX therapy can negate the activity of MTX (30), while more moderate supplementation of folic and folinic acid does not alter efficacy and lowers toxicity (31–37). It has recently been shown that folate supplementation lowers rates of hepatotoxicity and is associated with patients achieving a final dosage of MTX >15 mg/week (38, 39). Folinic acid is the widely used and effective antidote to counteract the excessive antifolate effects of MTX in cancer chemotherapy or accidental MTX overdose (40). The RBC levels of folate at visit 3 in the folinic acid supplementation group were higher, probably because folinic acid, and not folic acid, can be metabolized to 5-methyltetrahydrofolate without the action of dihydrofolate reductase.
Cronstein et al have hypothesized that inhibition of AICAR transformylase causes a release of adenosine (2–5). The antiinflammatory effects of MTX in culture appear to be mediated by A2 receptors, with increased adenosine release in the presence of neutrophils (2). The released adenosine inhibits neutrophil adhesion to connective tissue cells. Consistent with this hypothesis, the addition of 2 nonselective adenosine receptor antagonists (theophylline and caffeine) to MTX therapy in rat adjuvant-induced arthritis markedly reduced the efficacy of MTX (41). A question remaining is whether the total efficacy of MTX can be explained by its interference with adenosine metabolism.
We have previously shown in MTX-treated patients with psoriasis that MTX therapy increases both 24-hour AICA and adenosine excretion (42). Adenosine concentrations have been reported to be elevated in the cerebrospinal fluid of children treated for leukemia (43), and adenosine plasma levels have been shown to peak ∼2 hours after the administration of 7.5 mg of MTX (44). We elected to collect 24-hour urine samples to document both adenosine and AICA excretion in vivo. We acknowledge that a 24-hour urinary excretion of these metabolites might not mirror their blood or synovial levels but, on average, would demonstrate the effects of MTX and MTX plus a folate on purine metabolism.
Our results suggest that the effects of MTX in RA are more related to changes in AICA than to adenosine metabolism. These results are consistent with those reported by Andersson (9), where the addition of 3 adenosine antagonists (8-p-sulfophenyltheophyllamine, 3,7-dimethyl-1-propargylxanthine, and 8-cyclopentyl-1,3-dipropylxanthine) potentiated instead of lowered the efficacy of MTX in rat adjuvant-induced arthritis. This leaves open the possibility that AICAR transformylase inhibition works by mechanisms other than adenosine accumulation. Two possible mechanisms may operate together. First, since AICA increases with MTX therapy, purine nucleotide biosynthesis is to some extent blocked. Blockage of this fundamental metabolic pathway may be immunosuppressive in and of itself. Smolenska et al (45) reported that the whole blood concentrations of both hypoxanthine and uric acid decreased within 2 hours after a 7.5-mg dose of MTX, while adenosine concentrations remained constant. This suggests that purine nucleotide biosynthesis is rapidly blocked by MTX. Second, AICA or its metabolites might be immunotoxic substances. It may be difficult to adequately separate these 2 effects of MTX therapy since they occur together.
The effects of 2 folate supplements on AICA excretion suggests that our hypothesis is correct. Folinic acid, a potent antidote to the antifolate activity of MTX, is able to reduce urinary AICA excretion to relatively low levels (Table 3). As noted previously, folinic acid supplements can reverse the efficacy of MTX in RA (30). In contrast, folic acid, a weaker MTX antidote, is unable to match the urinary AICA-lowering capacity of folinic acid (Table 3). It is possible that the majority of the folic acid dose remains as such because of inhibition of dihydrofolate reductase by MTX (Figure 2). Adenosine excretion was not substantially affected by either supplement. The correlation of the log of urinary AICA excretion with improvements with joint swelling counts and scores are also consistent with our hypothesis. We speculate that AICA itself (or its metabolites) may be an immunosuppressive agent.
Finally, although it is tempting to try to demonstrate that a pharmaceutical agent has 1 or 2 mechanisms of action, in reality this may always be an oversimplification of biology and medicine. Purine nucleotide biosynthesis de novo and the pathways of purine nucleotide, nucleoside, and base interconversions and salvage are fundamental metabolic processes. Relatively small blockages at many steps in these pathways may have a highly synergistic effect on immune cell function.