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From: The Rheumatologist, November 2011

A&R Abstracts: Methotrexate

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Want to know more about methotrexate? Check out these abstracts from Arthritis & Rheumatism and log on to to read the full articles.


Increased sensitivity to apoptosis induced by methotrexate is mediated by jun N-terminal kinase.

(Arthritis Rheum. 2011;63:2606-2616.)


Objective: Low-dose methotrexate (MTX) is an effective therapy for rheumatoid arthritis (RA), yet its mechanism of action is incompletely understood. The aim of this study was to explore the induction of apoptosis by MTX.

Methods: Flow cytometry was performed to assess changes in the levels of intracellular proteins, reactive oxygen species (ROS), and apoptosis. Quantitative polymerase chain reaction was performed to assess changes in the transcript levels of select target genes in response to MTX.

Results: MTX did not directly induce apoptosis but rather “primed” cells for markedly increased sensitivity to apoptosis via either mitochondrial or death receptor pathways, by a JNK-dependent mechanism. Increased sensitivity to apoptosis was mediated, at least in part, by MTX-dependent production of ROS, JNK activation, and JNK-dependent induction of genes whose protein products promote apoptosis. Supplementation with tetrahydrobiopterin blocked these MTX-induced effects. Patients with RA who were receiving low-dose MTX therapy expressed elevated levels of the JNK target gene, jun.

Conclusion: Our results support a model whereby MTX inhibits reduction of dihydrobiopterin to tetrahydrobiopterin, resulting in increased production of ROS, increased JNK activity, and increased sensitivity to apoptosis. The finding of increased jun levels in patients with RA receiving low-dose MTX supports the notion that this pathway is activated by MTX in vivo and may contribute to the efficacy of MTX in inflammatory disease.


Reversal of the antiinflammatory effects of methotrexate by the nonselective adenosine receptor antagonists theophylline and caffeine. Evidence that the antiinflammatory effects of methotrexate are mediated via multiple adenosine receptors in rat adjuvant arthritis.

(Arthritis Rheum. 2000;43:656-663.)


Objective: Weekly low-dose methotrexate (MTX) remains the mainstay of second-line therapy for rheumatoid arthritis (RA). We have previously reported that adenosine, acting at specific receptors on inflammatory cells, mediates the antiinflammatory effects of MTX in both in vitro and in vivo models of acute inflammation, but the mechanism by which MTX suppresses the chronic inflammation of arthritis remains controversial. The present study was undertaken to further investigate the means by which adenosine mediates the antiinflammatory effects of MTX.

Methods: The effects of two nonselective adenosine receptor antagonists, theophylline and caffeine, were examined, using the rat adjuvant arthritis model of RA. These agents were given alone and in conjunction with MTX, and arthritis severity was assessed clinically, radiologically, and histologically. Since rodent adenosine A3 receptors are not blocked by theophylline, selective A1, A2A, and A2B receptor antagonists were tested as well.

Results: Control animals developed severe arthritis, which was markedly attenuated by weekly treatment with MTX (0.75 mg/kg/week). Neither theophylline alone nor caffeine alone (each at 10 mg/kg/day) significantly affected the severity of the arthritis, but both agents markedly reversed the effect of MTX as measured by a severity index, hindpaw swelling, and hindpaw ankylosis. Radiographic and histologic analyses confirmed these observations. Neither A1, A2A, nor A2B receptor antagonists affected the capacity of MTX to ameliorate inflammation in adjuvant arthritis.

Conclusion: These results provide strong evidence that adenosine mediates the antiinflammatory effects of MTX in this model of RA. Moreover, the findings suggest that abstinence from caffeine, a ubiquitous food additive and medication, may enhance the therapeutic effects of MTX in RA.


A clinical pharmacogenetic model to predict the efficacy of methotrexate monotherapy in recent-onset rheumatoid arthritis.

(Arthritis Rheum. 2007;56:1765-1775.)


Objective: To develop a clinical pharmacogenetic model to predict the efficacy of methotrexate (MTX) in rheumatoid arthritis (RA).

Methods: Two hundred five patients with newly diagnosed RA and active disease were treated with MTX (initiated at a dosage of 7.5 mg/week and increased to 15 mg/week after four weeks) and folic acid (1 mg/day). If the Disease Activity Score (DAS) was >2.4 at three months, the dosage of MTX was increased up to 25 mg/week. Twenty-four baseline variables possibly influencing disease state and drug response were selected. In addition, 17 polymorphisms in 13 genes related to the MTX mechanism of action, purine and pyrimidine synthesis, were determined. Factors were compared between responders (defined as patients with a DAS ≤2.4 at six months) and nonresponders. In case of differences, a stepwise selection procedure identified the predictors for response. A clinical score was designed by simplifying regression coefficients of the independent variables. Cutoff levels were chosen based on the clinical score, and positive and negative response rates were calculated. An evaluation of the model was performed in a second group of patients.

Results: The model for MTX efficacy consisted of sex, rheumatoid factor and smoking status, the DAS, and four polymorphisms in the AMPD1, ATIC, ITPA, and MTHFD1 genes. This prediction model was transformed into a scoring system ranging from 0 to 11.5. Scores of ≤3.5 had a true positive response rate of 95%. Scores of ≥6 had a true negative response rate of 86%. Sixty percent of the patients were categorized as either responders or nonresponders, whereas 32% of the patients were categorized using a nongenetic model. Evaluation of the model in 38 additional patients with RA supported the results.

Conclusion: This study established a model for predicting the efficacy of MTX in patients with RA. This pharmacogenetic model may lead to better-tailored initial treatment decisions in patients with RA.


Relationship between genetic variants in the adenosine pathway and outcome of methotrexate treatment in patients with recent-onset rheumatoid arthritis.

(Arthritis Rheum. 2006;54:2830-2839.)


Objective: Among patients with rheumatoid arthritis (RA), there is a high degree of interindividual variability in the degree of response to methotrexate (MTX) treatment. This study was undertaken to explore polymorphisms in genes contributing to antiinflammatory adenosine release as novel predictors of MTX treatment outcome.

Methods: In 205 patients with newly diagnosed RA, five polymorphisms in five genes coding for enzymes related to the release of adenosine were analyzed. All patients received standardized MTX treatment (up to 25 mg per week orally), combined with folic acid. MTX efficacy was evaluated by the Disease Activity Score (DAS) and compared among genotypes. The association between MTX-related adverse events and genotype was also assessed. The following polymorphisms were determined: AMPD1 34C>T, ATIC 347C>G, ITPA 94C>A, MTR 2756A>G, and MTRR 66A>G. When significant differences were found by chi-square analysis, odds ratios (ORs) and 95% confidence intervals were calculated.

Results: Patients carrying the AMPD1 34T allele, ATIC 347CC, or ITPA 94CC were more likely to have a good clinical response, as defined by a DAS of ≤2.4 (OR [95% confidence interval] 2.1 [1.0–4.5], 2.5 [1.3–4.7], and 2.7 [1.1–8.1], respectively). The likelihood of a good clinical response was increased if patients possessed all 3 favorable genotypes (OR 27.8 [95% confidence interval 3.2–250]). Regarding toxicity, only ATIC G allele carriers experienced a greater frequency of adverse events (OR 2.0 [95% confidence interval 1.1–3.7]).

Conclusion: Polymorphisms in the AMPD1, ATIC, and ITPA genes are associated with good clinical response to MTX treatment. These findings indicate that genotyping may help in the identification of patients who will benefit most from MTX treatment and may assist clinicians in making treatment decisions regarding patients with recent-onset RA.


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