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  • Review Article
  • Published:

Mechanism of action of methotrexate in rheumatoid arthritis, and the search for biomarkers

Nature Reviews Rheumatology volume 12, pages 731–742 (2016)Cite this article

Subjects

Key Points

  • Methotrexate shows good efficacy in a proportion of patients: 40% of treated patients with rheumatoid arthritis achieve an ACR50 response

  • The mechanism of action of methotrexate has not fully been defined, however potentiation of adenosine signalling carries the most robust data

  • Pharmacokinetic parameters, particularly intracellular methotrexate polyglutamation, show some association with disease activity, although they cannot yet be used to predict treatment response

  • An exploration of the expression and polymorphisms of genes encoding molecules linked to proposed mechanisms of methotrexate action is underway to identify methotrexate-responsive signatures

  • At present, no robust markers or predictive models exist for methotrexate responsiveness in RA

Abstract

The treatment and outcomes of patients with rheumatoid arthritis (RA) have been transformed over the past two decades. Low disease activity and remission are now frequently achieved, and this success is largely the result of the evolution of treatment paradigms and the introduction of new therapeutic agents. Despite the rapid pace of change, the most commonly used drug in RA remains methotrexate, which is considered the anchor drug for this condition. In this Review, we describe the known pharmacokinetic properties and putative mechanisms of action of methotrexate. Consideration of the pharmacodynamic perspective could inform the development of biomarkers of responsiveness to methotrexate, enabling therapy to be targeted to specific groups of patients. Such biomarkers could revolutionize the management of RA.

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Figure 1: Pharmacodynamics of methotrexate in RA.
Figure 2: Potential mechanisms of action of low-dose methotrexate in rheumatoid arthritis.
Figure 3: Potential role of methotrexate in depletion of the nucleotide pool.
Figure 4: Proposed mechanism by which methotrexate increases adenosine signalling.
Figure 5: Role of dyhydrofolate reductase (DHFR) in the generation of methyl donors.
Figure 6: Metabolism of pterins.

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References

  1. Haraoui, B. & Pope, J. Treatment of early rheumatoid arthritis: concepts in management. Semin. Arthritis Rheum. 40, 371–388 (2011).

    PubMed  Google Scholar 

  2. Visser, K. & van der Heijde, D. Optimal dosage and route of administration of methotrexate in rheumatoid arthritis: a systematic review of the literature. Ann. Rheum. Dis. 68, 1094–1099 (2009).

    CAS  PubMed  Google Scholar 

  3. Gubner, R., August, S. & Ginsberg, V. Therapeutic suppression of tissue reactivity. II. Effect of aminopterin in rheumatoid arthritis and psoriasis. Am. J. Med. Sci. 221, 176–182 (1951).

    CAS  PubMed  Google Scholar 

  4. Weinblatt, M. E. et al. Efficacy of low-dose methotrexate in rheumatoid arthritis. N. Engl. J. Med. 312, 818–822 (1985).

    CAS  PubMed  Google Scholar 

  5. Williams, H. J. et al. Comparison of low-dose oral pulse methotrexate and placebo in the treatment of rheumatoid arthritis. A controlled clinical trial. Arthritis Rheum. 28, 721–730 (1985).

    CAS  PubMed  Google Scholar 

  6. Lopez-Olivo, M. A. et al. Methotrexate for treating rheumatoid arthritis. Cochrane Database Syst. Rev. 6, CD000957 (2014).

    Google Scholar 

  7. Kavanaugh, A. et al. Clinical, functional and radiographic consequences of achieving stable low disease activity and remission with adalimumab plus methotrexate or methotrexate alone in early rheumatoid arthritis: 26-week results from the randomised, controlled OPTIMA study. Ann. Rheum. Dis. 72, 64–71 (2013).

    CAS  PubMed  Google Scholar 

  8. Detert, J. et al. Induction therapy with adalimumab plus methotrexate for 24 weeks followed by methotrexate monotherapy up to week 48 versus methotrexate therapy alone for DMARD-naive patients with early rheumatoid arthritis: HIT HARD, an investigator-initiated study. Ann. Rheum. Dis. 72, 844–850 (2013).

    CAS  PubMed  Google Scholar 

  9. Horslev-Petersen, K. et al. Adalimumab added to a treat-to-target strategy with methotrexate and intra-articular triamcinolone in early rheumatoid arthritis increased remission rates, function and quality of life. The OPERA Study: an investigator-initiated, randomised, double-blind, parallel-group, placebo-controlled trial. Ann. Rheum. Dis. 73, 654–661 (2014).

    CAS  PubMed  Google Scholar 

  10. O'Dell, J. R. et al. Validation of the methotrexate-first strategy in patients with early, poor-prognosis rheumatoid arthritis: results from a two-year randomized, double-blind trial. Arthritis Rheum. 65, 1985–1994 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Bathon, J. M. et al. A comparison of etanercept and methotrexate in patients with early rheumatoid arthritis. N. Engl. J. Med. 343, 1586–1593 (2000).

    CAS  PubMed  Google Scholar 

  12. Klareskog, L. et al. Therapeutic effect of the combination of etanercept and methotrexate compared with each treatment alone in patients with rheumatoid arthritis: double-blind randomised controlled trial. Lancet 363, 675–681 (2004).

    CAS  PubMed  Google Scholar 

  13. Goekoop-Ruiterman, Y. P. et al. Clinical and radiographic outcomes of four different treatment strategies in patients with early rheumatoid arthritis (the BeSt study): a randomized, controlled trial. Arthritis Rheum. 52, 3381–3390 (2005).

    CAS  PubMed  Google Scholar 

  14. Breedveld, F. C. et al. The PREMIER study: a multicenter, randomized, double-blind clinical trial of combination therapy with adalimumab plus methotrexate versus methotrexate alone or adalimumab alone in patients with early, aggressive rheumatoid arthritis who had not had previous methotrexate treatment. Arthritis. Rheum. 54, 26–37 (2006).

    CAS  PubMed  Google Scholar 

  15. Soubrier, M. et al. Evaluation of two strategies (initial methotrexate monotherapy versus its combination with adalimumab) in management of early active rheumatoid arthritis: data from the GUEPARD trial. Rheumatology 48, 1429–1434 (2009).

    CAS  PubMed  Google Scholar 

  16. Tak, P. P. et al. Inhibition of joint damage and improved clinical outcomes with rituximab plus methotrexate in early active rheumatoid arthritis: the IMAGE trial. Ann. Rheum. Dis. 70, 39–46 (2011).

    CAS  PubMed  Google Scholar 

  17. de Jong, P. H. et al. Induction therapy with a combination of DMARDs is better than methotrexate monotherapy: first results of the tREACH trial. Ann. Rheum. Dis. 72, 72–78 (2013).

    CAS  PubMed  Google Scholar 

  18. Emery, P. et al. Golimumab, a human anti-tumor necrosis factor monoclonal antibody, injected subcutaneously every 4 weeks in patients with active rheumatoid arthritis who had never taken methotrexate: 1-year and 2-year clinical, radiologic, and physical function findings of a phase III, multicenter, randomized, double-blind, placebo-controlled study. Arthritis Care Res. (Hoboken) 65, 1732–1742 (2013).

    CAS  Google Scholar 

  19. Takeuchi, T. et al. Adalimumab, a human anti-TNF monoclonal antibody, outcome study for the prevention of joint damage in Japanese patients with early rheumatoid arthritis: the HOPEFUL 1 study. Ann. Rheum. Dis. 73, 536–543 (2014).

    CAS  PubMed  Google Scholar 

  20. Nam, J. L. et al. A randomised controlled trial of etanercept and methotrexate to induce remission in early inflammatory arthritis: the EMPIRE trial. Ann. Rheum. Dis. 73, 1027–1036 (2014).

    CAS  PubMed  Google Scholar 

  21. Emery, P. et al. Evaluating drug-free remission with abatacept in early rheumatoid arthritis: results from the phase 3b, multicentre, randomised, active-controlled AVERT study of 24 months, with a 12-month, double-blind treatment period. Ann. Rheum. Dis. 74, 19–26 (2015).

    CAS  PubMed  Google Scholar 

  22. Visentin, M., Zhao, R. & Goldman, I. D. The antifolates. Hematol. Oncol. Clin. North Am. 26, 629–648 (2012).

    PubMed  PubMed Central  Google Scholar 

  23. Whittle, S. L. & Hughes, R. A. Folate supplementation and methotrexate treatment in rheumatoid arthritis: a review. Rheumatology 43, 267–271 (2004).

    CAS  PubMed  Google Scholar 

  24. Salliot, C. & van der Heijde, D. Long-term safety of methotrexate monotherapy in patients with rheumatoid arthritis: a systematic literature research. Ann. Rheum. Dis. 68, 1100–1104 (2009).

    CAS  PubMed  Google Scholar 

  25. Hazlewood, G. S. et al. Methotrexate monotherapy and methotrexate combination therapy with traditional and biologic disease modifying antirheumatic drugs for rheumatoid arthritis: abridged Cochrane systematic review and network meta-analysis. BMJ 353, i1777 (2016).

    PubMed  PubMed Central  Google Scholar 

  26. Hamilton, R. A. & Kremer, J. M. Why intramuscular methotrexate may be more efficacious than oral dosing in patients with rheumatoid arthritis. Br. J. Rheum. 36, 86–90 (1997).

    CAS  Google Scholar 

  27. Pichlmeier, U. & Heuer, K. U. Subcutaneous administration of methotrexate with a prefilled autoinjector pen results in a higher relative bioavailability compared with oral administration of methotrexate. Clin. Exp. Rheumatol. 32, 563–571 (2014).

    PubMed  Google Scholar 

  28. Hoekstra, M. et al. Bioavailability of higher dose methotrexate comparing oral and subcutaneous administration in patients with rheumatoid arthritis. J. Rheumatol. 31, 645–648 (2004).

    CAS  PubMed  Google Scholar 

  29. Herman, R. A., Veng-Pedersen, P., Hoffman, J., Koehnke, R. & Furst, D. E. Pharmacokinetics of low-dose methotrexate in rheumatoid arthritis patients. J. Pharm. Sci. 78, 165–171 (1989).

    CAS  PubMed  Google Scholar 

  30. Lebbe, C., Beyeler, C., Gerber, N. J. & Reichen, J. Intraindividual variability of the bioavailability of low dose methotrexate after oral administration in rheumatoid arthritis. Ann. Rheum. Dis. 53, 475–477 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Schiff, M. H., Jaffe, J. S. & Freundlich, B. Head-to-head, randomised, crossover study of oral versus subcutaneous methotrexate in patients with rheumatoid arthritis: drug-exposure limitations of oral methotrexate at doses ≥15 mg may be overcome with subcutaneous administration. Ann. Rheum. Dis. 73, 1549–1551 (2014).

    CAS  PubMed  Google Scholar 

  32. Hoekstra, M. et al. Splitting high-dose oral methotrexate improves bioavailability: a pharmacokinetic study in patients with rheumatoid arthritis. J. Rheumatol. 33, 481–485 (2006).

    CAS  PubMed  Google Scholar 

  33. Wegrzyn, J., Adeleine, P. & Miossec, P. Better efficacy of methotrexate given by intramuscular injection than orally in patients with rheumatoid arthritis. Ann. Rheum. Dis. 63, 1232–1234 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Desmoulin, S. K., Hou, Z., Gangjee, A. & Matherly, L. H. The human proton-coupled folate transporter: biology and therapeutic applications to cancer. Cancer Biol. Ther. 13, 1355–1373 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Edno, L. et al. Total and free methotrexate pharmacokinetics in rheumatoid arthritis patients. Ther. Drug Monit. 18, 128–134 (1996).

    CAS  PubMed  Google Scholar 

  36. Seideman, P., Beck, O., Eksborg, S. & Wennberg, M. The pharmacokinetics of methotrexate and its 7-hydroxy metabolite in patients with rheumatoid arthritis. Br. J. Clin. Pharmacol. 35, 409–412 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Bressolle, F., Bologna, C., Kinowski, J. M., Sany, J. & Combe, B. Effects of moderate renal insufficiency on pharmacokinetics of methotrexate in rheumatoid arthritis patients. Ann. Rheum. Dis. 57, 110–113 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Fotoohi, A. K. et al. Gene expression profiling of leukemia T-cells resistant to methotrexate and 7-hydroxymethotrexate reveals alterations that preserve intracellular levels of folate and nucleotide biosynthesis. Biochem. Pharmacol. 77, 1410–1417 (2009).

    CAS  PubMed  Google Scholar 

  39. Baggott, J. E., Morgan, S. L. & Vaughn, W. H. Differences in methotrexate and 7-hydroxymethotrexate inhibition of folate-dependent enzymes of purine nucleotide biosynthesis. Biochem. J. 300, 627–629 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Baggott, J. E., Morgan, S. L. & Koopman, W. J. The effect of methotrexate and 7-hydroxymethotrexate on rat adjuvant arthritis and on urinary aminoimidazole carboxamide excretion. Arthritis Rheum. 41, 1407–1410 (1998).

    CAS  PubMed  Google Scholar 

  41. Fabre, G., Fabre, I., Matherly, L. H., Cano, J. P. & Goldman, I. D. Synthesis and properties of 7-hydroxymethotrexate polyglutamyl derivatives in Ehrlich ascites tumor cells in vitro. J. Biol. Chem. 259, 5066–5072 (1984).

    CAS  PubMed  Google Scholar 

  42. Nuernberg, B., Koehnke, R., Solsky, M., Hoffman, J. & Furst, D. E. Biliary elimination of low-dose methotrexate in humans. Arthritis Rheum. 33, 898–902 (1990).

    CAS  PubMed  Google Scholar 

  43. Bremnes, R. M., Slordal, L., Wist, E. & Aarbakke, J. Dose-dependent pharmacokinetics of methotrexate and 7-hydroxymethotrexate in the rat in vivo. Cancer Res. 49, 6359–6364 (1989).

    CAS  PubMed  Google Scholar 

  44. Sinnett, M. J., Groff, G. D., Raddatz, D. A., Franck, W. A. & Bertino, J. S. Jr. Methotrexate pharmacokinetics in patients with rheumatoid arthritis. J. Rheumatol. 16, 745–748 (1989).

    CAS  PubMed  Google Scholar 

  45. Godfrey, C., Sweeney, K., Miller, K., Hamilton, R. & Kremer, J. The population pharmacokinetics of long-term methotrexate in rheumatoid arthritis. Br. J. Clin. Pharmacol. 46, 369–376 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Koizumi, S., Curt, G. A., Fine, R. L., Griffin, J. D. & Chabner, B. A. Formation of methotrexate polyglutamates in purified myeloid precursor cells from normal human bone marrow. J. Clin. Invest. 75, 1008–1014 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Angelis-Stoforidis, P., Vajda, F. J. & Christophidis, N. Methotrexate polyglutamate levels in circulating erythrocytes and polymorphs correlate with clinical efficacy in rheumatoid arthritis. Clin. Exp. Rheumatol. 17, 313–320 (1999).

    CAS  PubMed  Google Scholar 

  48. Murakami, T. & Mori, N. Involvement of multiple transporters-mediated transports in mizoribine and methotrexate pharmacokinetics. Pharmaceuticals (Basel) 5, 802–836 (2012).

    CAS  Google Scholar 

  49. Baggott, J. E., Vaughn, W. H. & Hudson, B. B. Inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase, adenosine deaminase and 5′-adenylate deaminase by polyglutamates of methotrexate and oxidized folates and by 5-aminoimidazole-4-carboxamide riboside and ribotide. Biochem. J. 236, 193–200 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Jolivet, J., Schilsky, R. L., Bailey, B. D., Drake, J. C. & Chabner, B. A. Synthesis, retention, and biological activity of methotrexate polyglutamates in cultured human breast cancer cells. J. Clin. Invest. 70, 351–360 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Goodsell, D. S. The molecular perspective: methotrexate. Oncologist 4, 340–341 (1999).

    CAS  PubMed  Google Scholar 

  52. Quemeneur, L. et al. Differential control of cell cycle, proliferation, and survival of primary T lymphocytes by purine and pyrimidine nucleotides. J. Immunol. 170, 4986–4995 (2003).

    CAS  PubMed  Google Scholar 

  53. Budzik, G. P., Colletti, L. M. & Faltynek, C. R. Effects of methotrexate on nucleotide pools in normal human T cells and the CEM T cell line. Life Sci. 66, 2297–2307 (2000).

    CAS  PubMed  Google Scholar 

  54. Fairbanks, L. D. et al. Methotrexate inhibits the first committed step of purine biosynthesis in mitogen-stimulated human T-lymphocytes: a metabolic basis for efficacy in rheumatoid arthritis? Biochem. J. 342, 143–152 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Genestier, L. et al. Immunosuppressive properties of methotrexate: apoptosis and clonal deletion of activated peripheral T cells. J. Clin. Invest. 102, 322–328 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Nakajima, A., Hakoda, M., Yamanaka, H., Kamatani, N. & Kashiwazaki, S. Divergent effects of methotrexate on the clonal growth of T and B lymphocytes and synovial adherent cells from patients with rheumatoid arthritis. Ann. Rheum. Dis. 55, 237–242 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Hasko, G. & Cronstein, B. Regulation of inflammation by adenosine. Front. Immunol. 4, 85 (2013).

    PubMed  PubMed Central  Google Scholar 

  58. Johnson, H. & Lapin, C. 4-aminoimidazole-5-carboxamide excretion in acute leukemia. Med. Pediatr. Oncol. 5, 225–229 (1978).

    CAS  PubMed  Google Scholar 

  59. Baggott, J. E., Morgan, S. L., Sams, W. M. & Linden, J. Urinary adenosine and aminoimidazolecarboxamide excretion in methotrexate-treated patients with psoriasis. Arch. Dermatol. 135, 813–817 (1999).

    CAS  PubMed  Google Scholar 

  60. Moser, G. H., Schrader, J. & Deussen, A. Turnover of adenosine in plasma of human and dog blood. Am. J. Physiol. 256, C799–C806 (1989).

    CAS  PubMed  Google Scholar 

  61. Fredholm, B. B. et al. International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors — an update. Pharmacol. Rev. 63, 1–34 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Hasko, G., Linden, J., Cronstein, B. & Pacher, P. Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. Nat. Rev. Drug Discov. 7, 759–770 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Vincenzi, F. et al. A2A adenosine receptors are differentially modulated by pharmacological treatments in rheumatoid arthritis patients and their stimulation ameliorates adjuvant-induced arthritis in rats. PLoS ONE 8, e54195 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Varani, K. et al. A2A and A3 adenosine receptor expression in rheumatoid arthritis: upregulation, inverse correlation with disease activity score and suppression of inflammatory cytokine and metalloproteinase release. Arthritis Res. Ther. 13, R197 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Varani, K. et al. Normalization of A2A and A3 adenosine receptor up-regulation in rheumatoid arthritis patients by treatment with anti-tumor necrosis factor α but not methotrexate. Arthritis Rheum. 60, 2880–2891 (2009).

    CAS  PubMed  Google Scholar 

  66. Nguyen, D. K., Montesinos, M. C., Williams, A. J., Kelly, M. & Cronstein, B. N. TH1 cytokines regulate adenosine receptors and their downstream signaling elements in human microvascular endothelial cells. J. Immunol. 171, 3991–3998 (2003).

    PubMed  Google Scholar 

  67. Stamp, L. K. et al. Adenosine receptor expression in rheumatoid synovium: a basis for methotrexate action. Arthritis Res. Ther. 14, R138 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Bar-Yehuda, S. et al. The anti-inflammatory effect of A3 adenosine receptor agonists: a novel targeted therapy for rheumatoid arthritis. Expert Opin. Investig. Drugs 16, 1601–1613 (2007).

    CAS  PubMed  Google Scholar 

  69. Silverman, M. H. et al. Clinical evidence for utilization of the A3 adenosine receptor as a target to treat rheumatoid arthritis: data from a phase II clinical trial. J. Rheumatol. 35, 41–48 (2008).

    CAS  PubMed  Google Scholar 

  70. Cronstein, B. N., Eberle, M. A., Gruber, H. E. & Levin, R. I. Methotrexate inhibits neutrophil function by stimulating adenosine release from connective tissue cells. Proc. Natl Acad. Sci. USA 88, 2441–2445 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Cronstein, B. N., Naime, D. & Ostad, E. The antiinflammatory mechanism of methotrexate. Increased adenosine release at inflamed sites diminishes leukocyte accumulation in an in vivo model of inflammation. J. Clin. Investig. 92, 2675–2682 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Asako, H., Wolf, R. E. & Granger, D. N. Leukocyte adherence in rat mesenteric venules: effects of adenosine and methotrexate. Gastroenterology 104, 31–37 (1993).

    CAS  PubMed  Google Scholar 

  73. Delano, D. L. et al. Genetically based resistance to the antiinflammatory effects of methotrexate in the air-pouch model of acute inflammation. Arthritis Rheum. 52, 2567–2575 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Montesinos, M. C., Desai, A. & Cronstein, B. N. Suppression of inflammation by low-dose methotrexate is mediated by adenosine A2A receptor but not A3 receptor activation in thioglycollate-induced peritonitis. Arthritis Res. Ther. 8, R53 (2006).

    PubMed  PubMed Central  Google Scholar 

  75. Montesinos, M. C. et al. The antiinflammatory mechanism of methotrexate depends on extracellular conversion of adenine nucleotides to adenosine by ecto-5′-nucleotidase: findings in a study of ecto-5′-nucleotidase gene-deficient mice. Arthritis Rheum. 56, 1440–1445 (2007).

    CAS  PubMed  Google Scholar 

  76. Morabito, L. et al. Methotrexate and sulfasalazine promote adenosine release by a mechanism that requires ecto-5′-nucleotidase-mediated conversion of adenine nucleotides. J. Clin. Invest. 101, 295–300 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Morovic-Vergles, J., Culo, M. I., Gamulin, S. & Culo, F. Cyclic adenosine 5′-monophosphate in synovial fluid of rheumatoid arthritis and osteoarthritis patients. Rheumatol. Int. 29, 167–171 (2008).

    CAS  PubMed  Google Scholar 

  78. Bours, M. J. et al. Adenosine 5′-triphosphate infusions reduced disease activity and inflammation in a patient with active rheumatoid arthritis. Rheumatology 49, 2223–2225 (2010).

    PubMed  Google Scholar 

  79. Fletcher, J. M. et al. CD39+Foxp3+ regulatory T cells suppress pathogenic TH17 cells and are impaired in multiple sclerosis. J. Immunol. 183, 7602–7610 (2009).

    CAS  PubMed  Google Scholar 

  80. Loza, M. J., Anderson, A. S., O'Rourke, K. S., Wood, J. & Khan, I. U. T-cell specific defect in expression of the NTPDase CD39 as a biomarker for lupus. Cell. Immunol. 271, 110–117 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Komatsu, N. et al. Pathogenic conversion of Foxp3+ T cells into TH17 cells in autoimmune arthritis. Nat. Med. 20, 62–68 (2014).

    CAS  PubMed  Google Scholar 

  82. Li, N. et al. Increased apoptosis induction in CD4+CD25+ Foxp3+ T cells contributes to enhanced disease activity in patients with rheumatoid arthritis through Il-10 regulation. Eur. Rev. Med. Pharmacol. Sci. 18, 78–85 (2014).

    CAS  PubMed  Google Scholar 

  83. Peres, R. S. et al. Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis. Proc. Natl Acad. Sci. USA 112, 2509–2514 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Nesher, G., Mates, M. & Zevin, S. Effect of caffeine consumption on efficacy of methotrexate in rheumatoid arthritis. Arthritis Rheum. 48, 571–572 (2003).

    PubMed  Google Scholar 

  85. Montesinos, M. C. et al. 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. 43, 656–663 (2000).

    CAS  PubMed  Google Scholar 

  86. Benito-Garcia, E. et al. Dietary caffeine intake does not affect methotrexate efficacy in patients with rheumatoid arthritis. J. Rheumatol. 33, 1275–1281 (2006).

    CAS  PubMed  Google Scholar 

  87. Zakeri, Z. et al. Comparison of adenosine deaminase levels in serum and synovial fluid between patients with rheumatoid arthritis and osteoarthritis. Int. J. Clin. Exp. Med. 5, 195–200 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Zamani, B., Jamali, R. & Jamali, A. Serum adenosine deaminase may predict disease activity in rheumatoid arthritis. Rheumatol. Int. 32, 1967–1975 (2012).

    CAS  PubMed  Google Scholar 

  89. Andersson, S. E., Johansson, L. H., Lexmuller, K. & Ekstrom, G. M. Anti-arthritic effect of methotrexate: is it really mediated by adenosine? Eur. J. Pharm. Sci. 9, 333–343 (2000).

    CAS  PubMed  Google Scholar 

  90. Teramachi, J. et al. Adenosine abolishes MTX-induced suppression of osteoclastogenesis and inflammatory bone destruction in adjuvant-induced arthritis. Lab. Investig. 91, 719–731 (2011).

    CAS  PubMed  Google Scholar 

  91. Ottonello, L. et al. Delayed neutrophil apoptosis induced by synovial fluid in rheumatoid arthritis: role of cytokines, estrogens, and adenosine. Ann. NY Acad. Sci. 966, 226–231 (2002).

    CAS  PubMed  Google Scholar 

  92. Smolenska, Z., Kaznowska, Z., Zarowny, D., Simmonds, H. A. & Smolenski, R. T. Effect of methotrexate on blood purine and pyrimidine levels in patients with rheumatoid arthritis. Rheumatology 38, 997–1002 (1999).

    CAS  PubMed  Google Scholar 

  93. Egan, L. J., Sandborn, W. J., Mays, D. C., Tremaine, W. J. & Lipsky, J. J. Plasma and rectal adenosine in inflammatory bowel disease: effect of methotrexate. Inflamm. Bowel Dis. 5, 167–173 (1999).

    CAS  PubMed  Google Scholar 

  94. Yukioka, K. et al. Polyamine levels in synovial tissues and synovial fluids of patients with rheumatoid arthritis. J. Rheumatol. 19, 689–692 (1992).

    CAS  PubMed  Google Scholar 

  95. Nesher, G. & Moore, T. L. The in vitro effects of methotrexate on peripheral blood mononuclear cells. Modulation by methyl donors and spermidine. Arthritis Rheum. 33, 954–959 (1990).

    CAS  PubMed  Google Scholar 

  96. Nesher, G., Osborn, T. G. & Moore, T. L. In vitro effects of methotrexate on polyamine levels in lymphocytes from rheumatoid arthritis patients. Clin. Exp. Rheumatol. 14, 395–399 (1996).

    CAS  PubMed  Google Scholar 

  97. Nesher, G., Osborn, T. G. & Moore, T. L. Effect of treatment with methotrexate, hydroxychloroquine, and prednisone on lymphocyte polyamine levels in rheumatoid arthritis: correlation with the clinical response and rheumatoid factor synthesis. Clin. Exp. Rheumatol. 15, 343–347 (1997).

    CAS  PubMed  Google Scholar 

  98. Huang, C. et al. Ornithine decarboxylase prevents methotrexate-induced apoptosis by reducing intracellular reactive oxygen species production. Apoptosis 10, 895–907 (2005).

    CAS  Google Scholar 

  99. Smith, D. M., Johnson, J. A. & Turner, R. A. Biochemical perturbations of BW 91Y (3-deazaadenosine) on human neutrophil chemotactic potential and lipid metabolism. Int. J. Tissue React. 13, 1–18 (1991).

    CAS  PubMed  Google Scholar 

  100. Cronstein, B. N. Low-dose methotrexate: a mainstay in the treatment of rheumatoid arthritis. Pharmacol. Rev. 57, 163–172 (2005).

    CAS  PubMed  Google Scholar 

  101. Kim, Y. I., Logan, J. W., Mason, J. B. & Roubenoff, R. DNA hypomethylation in inflammatory arthritis: reversal with methotrexate. J. Lab. Clin. Med. 128, 165–172 (1996).

    CAS  Google Scholar 

  102. Karouzakis, E., Gay, R. E., Gay, S. & Neidhart, M. Increased recycling of polyamines is associated with global DNA hypomethylation in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum. 64, 1809–1817 (2012).

    CAS  PubMed  Google Scholar 

  103. Phillips, D. C., Woollard, K. J. & Griffiths, H. R. The anti-inflammatory actions of methotrexate are critically dependent upon the production of reactive oxygen species. Br. J. Pharmacol. 138, 501–511 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  104. Herman, S., Zurgil, N. & Deutsch, M. Low dose methotrexate induces apoptosis with reactive oxygen species involvement in T lymphocytic cell lines to a greater extent than in monocytic lines. Inflamm. Res. 54, 273–280 (2005).

    CAS  PubMed  Google Scholar 

  105. Crabtree, M. J., Hale, A. B. & Channon, K. M. Dihydrofolate reductase protects endothelial nitric oxide synthase from uncoupling in tetrahydrobiopterin deficiency. Free Radic. Biol. Med. 50, 1639–1646 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. Vasquez-Vivar, J. et al. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc. Natl Acad. Sci. USA 95, 9220–9225 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  107. Schoedon, G. et al. Biosynthesis and metabolism of pterins in peripheral blood mononuclear cells and leukemia lines of man and mouse. Eur. J. Biochem. 166, 303–310 (1987).

    CAS  PubMed  Google Scholar 

  108. Altindag, Z. Z., Sahin, G., Inanici, F. & Hascelik, Z. Urinary neopterin excretion and dihydropteridine reductase activity in rheumatoid arthritis. Rheumatol. Int. 18, 107–111 (1998).

    CAS  PubMed  Google Scholar 

  109. Kullich, W. Correlation of interleukin-2 receptor and neopterin secretion in rheumatoid arthritis. Clin. Rheumatol. 12, 387–391 (1993).

    CAS  PubMed  Google Scholar 

  110. Spurlock, C. F. III et al. Increased sensitivity to apoptosis induced by methotrexate is mediated by JNK. Arthritis Rheum. 63, 2606–2616 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  111. Spurlock, C. F. III et al. Methotrexate-mediated inhibition of nuclear factor κB activation by distinct pathways in T cells and fibroblast-like synoviocytes. Rheumatology 54, 178–187 (2014).

    PubMed  PubMed Central  Google Scholar 

  112. Sung, J. Y. et al. Methotrexate suppresses the interleukin-6 induced generation of reactive oxygen species in the synoviocytes of rheumatoid arthritis. Immunopharmacology 47, 35–44 (2000).

    CAS  PubMed  Google Scholar 

  113. Sigmundsdottir, H., Johnston, A., Gudjonsson, J. E., Bjarnason, B. & Valdimarsson, H. Methotrexate markedly reduces the expression of vascular E-selectin, cutaneous lymphocyte-associated antigen and the numbers of mononuclear leucocytes in psoriatic skin. Exp. Dermatol. 13, 426–434 (2004).

    CAS  PubMed  Google Scholar 

  114. Johnston, A., Gudjonsson, J. E., Sigmundsdottir, H., Ludviksson, B. R. & Valdimarsson, H. The anti-inflammatory action of methotrexate is not mediated by lymphocyte apoptosis, but by the suppression of activation and adhesion molecules. Clin. Immunol. 114, 154–163 (2005).

    CAS  PubMed  Google Scholar 

  115. Dolhain, R. J. et al. Methotrexate reduces inflammatory cell numbers, expression of monokines and of adhesion molecules in synovial tissue of patients with rheumatoid arthritis. Br. J. Rheumatol. 37, 502–508 (1998).

    CAS  PubMed  Google Scholar 

  116. Klimiuk, P. A., Fiedorczyk, M., Sierakowski, S. & Chwiecko, J. Soluble cell adhesion molecules (sICAM-1, sVCAM-1, and sE-selectin) in patients with early rheumatoid arthritis. Scand. J. Rheumatol. 36, 345–350 (2007).

    CAS  PubMed  Google Scholar 

  117. Cobankara, V. et al. Successful treatment of rheumatoid arthritis is associated with a reduction in serum sE-selectin and thrombomodulin level. Clin. Rheumatol. 23, 430–434 (2004).

    PubMed  Google Scholar 

  118. Sands, W. A., Martin, A. F., Strong, E. W. & Palmer, T. M. Specific inhibition of nuclear factor-κB-dependent inflammatory responses by cell type-specific mechanisms upon A2A adenosine receptor gene transfer. Mol. Pharmacol. 66, 1147–1159 (2004).

    CAS  PubMed  Google Scholar 

  119. Hassanian, S. M., Dinarvand, P. & Rezaie, A. R. Adenosine regulates the proinflammatory signaling function of thrombin in endothelial cells. J. Cell. Physiol. 229, 1292–1300 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  120. Miranda-Carus, M. E., Balsa, A., Benito-Miguel, M., Perez de Ayala, C. & Martin-Mola, E. IL-15 and the initiation of cell contact-dependent synovial fibroblast-T lymphocyte cross-talk in rheumatoid arthritis: effect of methotrexate. J. Immunol. 173, 1463–1476 (2004).

    CAS  PubMed  Google Scholar 

  121. Wijngaarden, S., van Roon, J. A., van de Winkel, J. G., Bijlsma, J. W. & Lafeber, F. P. Down-regulation of activating Fcγ receptors on monocytes of patients with rheumatoid arthritis upon methotrexate treatment. Rheumatology 44, 729–734 (2005).

    CAS  PubMed  Google Scholar 

  122. Cooper, D. L. et al. FcγRIIIa expression on monocytes in rheumatoid arthritis: role in immune-complex stimulated TNF production and non-response to methotrexate therapy. PLoS ONE 7, e28918 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. Barrera, P. et al. Circulating concentrations and production of cytokines and soluble receptors in rheumatoid arthritis patients: effects of a single dose methotrexate. Br. J. Rheumatol. 33, 1017–1024 (1994).

    CAS  PubMed  Google Scholar 

  124. Barrera, P. et al. Effect of methotrexate alone or in combination with sulphasalazine on the production and circulating concentrations of cytokines and their antagonists. Longitudinal evaluation in patients with rheumatoid arthritis. Br. J. Rheumatol. 34, 747–755 (1995).

    CAS  PubMed  Google Scholar 

  125. Gerards, A. H., de Lathouder, S., de Groot, E. R., Dijkmans, B. A. & Aarden, L. A. Inhibition of cytokine production by methotrexate. Studies in healthy volunteers and patients with rheumatoid arthritis. Rheumatology (Oxford) 42, 1189–1196 (2003).

    CAS  Google Scholar 

  126. Rudwaleit, M. et al. Response to methotrexate in early rheumatoid arthritis is associated with a decrease of T cell derived tumour necrosis factor α, increase of interleukin 10, and predicted by the initial concentration of interleukin 4. Ann. Rheum. Dis. 59, 311–314 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  127. Majumdar, S. & Aggarwal, B. B. Methotrexate suppresses NF-κB activation through inhibition of IκBα phosphorylation and degradation. J. Immunol. 167, 2911–2920 (2001).

    CAS  PubMed  Google Scholar 

  128. Mello, S. B., Barros, D. M., Silva, A. S., Laurindo, I. M. & Novaes, G. S. Methotrexate as a preferential cyclooxygenase 2 inhibitor in whole blood of patients with rheumatoid arthritis. Rheumatology 39, 533–536 (2000).

    CAS  PubMed  Google Scholar 

  129. Vergne, P. et al. Methotrexate and cyclooxygenase metabolism in cultured human rheumatoid synoviocytes. J. Rheumatol. 25, 433–440 (1998).

    CAS  PubMed  Google Scholar 

  130. Novaes, G. S., Mello, S. B., Laurindo, I. M. & Cossermelli, W. Low dose methotrexate decreases intraarticular prostaglandin and interleukin 1 levels in antigen induced arthritis in rabbits. J. Rheumatol. 23, 2092–2097 (1996).

    CAS  PubMed  Google Scholar 

  131. Leroux, J. L., Damon, M., Chavis, C., Crastes De Paulet, A. & Blotman, F. Effects of methotrexate on leukotriene and derivated lipoxygenase synthesis in polynuclear neutrophils in rheumatoid polyarthritis. Rev. Rheum. Mal. Osteoartic. 59, 587–591 (in French) (1992).

    CAS  Google Scholar 

  132. Fiedorczyk, M., Klimiuk, P. A., Sierakowski, S., Gindzienska-Sieskiewicz, E. & Chwiecko, J. Serum matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with early rheumatoid arthritis. J. Rheumatol. 33, 1523–1529 (2006).

    CAS  PubMed  Google Scholar 

  133. Seitz, M. & Dayer, J. M. Enhanced production of tissue inhibitor of metalloproteinases by peripheral blood mononuclear cells of rheumatoid arthritis patients responding to methotrexate treatment. Rheumatology 39, 637–645 (2000).

    CAS  PubMed  Google Scholar 

  134. Tchetverikov, I. et al. Leflunomide and methotrexate reduce levels of activated matrix metalloproteinases in complexes with α2 macroglobulin in serum of rheumatoid arthritis patients. Ann. Rheum. Dis. 67, 128–130 (2008).

    CAS  PubMed  Google Scholar 

  135. Bulatovic Calasan, M. et al. Methotrexate polyglutamates in erythrocytes are associated with lower disease activity in juvenile idiopathic arthritis patients. Ann. Rheum. Dis. 74, 402–407 (2013).

    Google Scholar 

  136. de Rotte, M. C. et al. Methotrexate polyglutamates in erythrocytes are associated with lower disease activity in patients with rheumatoid arthritis. Ann. Rheum. Dis. 74, 408–414 (2013).

    PubMed  Google Scholar 

  137. Stamp, L. K. et al. Effects of changing from oral to subcutaneous methotrexate on red blood cell methotrexate polyglutamate concentrations and disease activity in patients with rheumatoid arthritis. J. Rheumatol. 38, 2540–2547 (2011).

    CAS  PubMed  Google Scholar 

  138. Dervieux, T. et al. Pharmacogenetic and metabolite measurements are associated with clinical status in patients with rheumatoid arthritis treated with methotrexate: results of a multicentred cross sectional observational study. Ann. Rheum. Dis. 64, 1180–1185 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  139. Becker, M. L. et al. The effect of genotype on methotrexate polyglutamate variability in juvenile idiopathic arthritis and association with drug response. Arthritis Rheum. 63, 276–285 (2011).

    CAS  PubMed  Google Scholar 

  140. Stamp, L. K. et al. Methotrexate polyglutamate concentrations are not associated with disease control in rheumatoid arthritis patients receiving long-term methotrexate therapy. Arthritis Rheum. 62, 359–368 (2010).

    CAS  PubMed  Google Scholar 

  141. Dervieux, T., Weinblatt, M. E., Kivitz, A. & Kremer, J. M. Methotrexate polyglutamation in relation to infliximab pharmacokinetics in rheumatoid arthritis. Ann. Rheum. Dis. 72, 908–910 (2013).

    CAS  PubMed  Google Scholar 

  142. Jani, M. et al. Clinical utility of random anti-tumor necrosis factor drug-level testing and measurement of antidrug antibodies on the long-term treatment response in rheumatoid arthritis. Arthritis Rheumatol. 67, 2011–2019 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  143. Dervieux, T., Zablocki, R. & Kremer, J. Red blood cell methotrexate polyglutamates emerge as a function of dosage intensity and route of administration during pulse methotrexate therapy in rheumatoid arthritis. Rheumatology 49, 2337–2345 (2010).

    CAS  PubMed  Google Scholar 

  144. Dervieux, T. et al. Contribution of common polymorphisms in reduced folate carrier and γ-glutamylhydrolase to methotrexate polyglutamate levels in patients with rheumatoid arthritis. Pharmacogenetics 14, 733–739 (2004).

    CAS  PubMed  Google Scholar 

  145. Korell, J. et al. Comparison of intracellular methotrexate kinetics in red blood cells with the kinetics in other cell types. Br. J. Clin. Pharmacol. 77, 493–497 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  146. Blits, M. et al. Methotrexate normalizes up-regulated folate pathway genes in rheumatoid arthritis. Arthritis Rheum. 65, 2791–2802 (2013).

    CAS  PubMed  Google Scholar 

  147. O'Dell, J. R. et al. HLA-DRB1 typing in rheumatoid arthritis: predicting response to specific treatments. Ann. Rheum. Dis. 57, 209–213 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  148. Sharma, S. et al. Interaction of genes from influx–metabolism–efflux pathway and their influence on methotrexate efficacy in rheumatoid arthritis patients among Indians. Pharmacogenet. Genomics 18, 1041–1049 (2008).

    CAS  PubMed  Google Scholar 

  149. Sharma, S. et al. Purine biosynthetic pathway genes and methotrexate response in rheumatoid arthritis patients among north Indians. Pharmacogenet. Genomics 19, 823–828 (2009).

    CAS  PubMed  Google Scholar 

  150. Wessels, J. A. et al. Relationship between genetic variants in the adenosine pathway and outcome of methotrexate treatment in patients with recent-onset rheumatoid arthritis. Arthritis Rheum. 54, 2830–2839 (2006).

    CAS  PubMed  Google Scholar 

  151. Wessels, J. A. et al. A clinical pharmacogenetic model to predict the efficacy of methotrexate monotherapy in recent-onset rheumatoid arthritis. Arthritis Rheum. 56, 1765–1775 (2007).

    CAS  PubMed  Google Scholar 

  152. Fransen, J. et al. Clinical pharmacogenetic model to predict response of MTX monotherapy in patients with established rheumatoid arthritis after DMARD failure. Pharmacogenomics 13, 1087–1094 (2012).

    CAS  PubMed  Google Scholar 

  153. Dervieux, T. et al. Patterns of interaction between genetic and nongenetic attributes and methotrexate efficacy in rheumatoid arthritis. Pharmacogenet. Genomics 22, 1–9 (2012).

    CAS  PubMed  Google Scholar 

  154. Owen, S. A. et al. Genetic polymorphisms in key methotrexate pathway genes are associated with response to treatment in rheumatoid arthritis patients. Pharmacogenomics J. 13, 227–234 (2013).

    CAS  PubMed  Google Scholar 

  155. Aslibekyan, S. et al. Genetic variants associated with methotrexate efficacy and toxicity in early rheumatoid arthritis: results from the treatment of early aggressive rheumatoid arthritis trial. Pharmacogenomics J. 14, 48–53 (2014).

    CAS  PubMed  Google Scholar 

  156. Senapati, S. et al. Genome-wide analysis of methotrexate pharmacogenomics in rheumatoid arthritis shows multiple novel risk variants and leads for TYMS regulation. Pharmacogenet. Genomics 24, 211–219 (2014).

    CAS  PubMed  Google Scholar 

  157. Kung, T. N. et al. RFC1 80G>A is a genetic determinant of methotrexate efficacy in rheumatoid arthritis: a huge review and meta-analysis of observational studies. Arthritis Rheumatol. 66, 1111–1120 (2013).

    Google Scholar 

  158. Morgan, M. D. et al. MTHFR functional genetic variation and methotrexate treatment response in rheumatoid arthritis: a meta-analysis. Pharmacogenomics 15, 467–475 (2014).

    CAS  PubMed  Google Scholar 

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Acknowledgements

The authors' work is supported by the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre based at Newcastle Hospitals National Health Service (NHS) Foundation Trust and Newcastle University, UK. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

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  1. Institute of Cellular Medicine, Faculty of Medical Sciences, Framlington Place, NE2 4HH, Newcastle upon Tyne, UK

    Philip M. Brown, Arthur G. Pratt & John D. Isaacs

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P.M.B. researched the data for the article, and wrote the manuscript. All authors contributed substantially to discussions of the article content and to review or editing of the manuscript before submission.

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Brown, P., Pratt, A. & Isaacs, J. Mechanism of action of methotrexate in rheumatoid arthritis, and the search for biomarkers. Nat Rev Rheumatol 12, 731–742 (2016). https://doi.org/10.1038/nrrheum.2016.175

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