When, at the end of the last decade, the new and potent tumor necrosis factor α (TNFα) inhibitors were approved for the treatment of rheumatoid arthritis (RA), the era of methotrexate (MTX) as the gold standard for treatment of this disease seemed to have come to an inevitable end. On the one hand, there were the new, intelligently designed and highly specific cytokine inhibitors, a result of the fast-growing knowledge about immunopathologic mechanisms of inflammation. On the other hand, there was MTX, a byproduct of oncology research from decades past. The exact mode of antiarthritic action of this antifolate drug in the complex system of inflammation could only be hypothesized.

Now, several years later, MTX plays a more important role than ever as the basis of RA treatment. Due to its effectiveness and tolerability, MTX remains the drug of first choice in the treatment of RA. Moreover, in order to make the new biologic drugs as effective as possible, almost all of the new investigational drugs are tested in combination with MTX. The designs of these clinical trials mainly resemble the design of the original Anti–TNF Trial in RA with Concomitant Therapy (ATTRACT) study (1), in which MTX was given in combination with infliximab, at that time still with the purpose of inhibiting the formation of antibodies against the drug. Meanwhile, not only TNFα inhibitors (2, 3), but also many other new biologic drugs are given in combination with MTX because this strongly increases the magnitude and duration of the therapeutic response. Although there is now a growing number of new drugs available for the treatment of RA, it is widely accepted that the question is not whether a patient with RA should receive MTX or not, but rather, whether the patient should receive it with or without a biologic drug.

Thus, the principle of folate antagonism seems to remain the foundation of immunomodulatory treatment of RA, both now and in the future.

It should be emphasized that MTX is experiencing a comeback in RA clinical trials, although this drug has several pharmacologic disadvantages that one might think should be limiting its use. First, if given orally, MTX shows a wide variability of resorption, ranging in some studies from 28% to 88% (4). Second, although it is given only once a week, it has a short half-life in the circulation, consisting of only 5–8 hours (4). Therefore, it is thought that its antiarthritic effect might partly depend on the rate of polyglutamation, an intracellular transformation of MTX that results in intracellular persistence of the drug. The rate of this modification, which is possibly crucial for its therapeutic effect, has a marked interindividual variability (5) and is influenced by independent factors, such as estrogen, insulin, or dexamethasone (6). Finally, the intracellular uptake of the molecule and its pharmacologic actions on intracellular targets are characterized by a broad range of mechanisms (7). This makes differentiation between the mechanisms of the unwanted toxic effects of MTX and the wanted antiinflammatory effects difficult.

Different approaches have been tried in an attempt to overcome the disadvantageous properties of MTX. Braun et al (8) recently reported the results of a clinical trial showing that MTX is more effective when given subcutaneously instead of orally. With this mode of application, the unpredictability of resorption can be avoided. My coworkers and I (9,10) have reported that MTX accumulates in inflamed joints if it is covalently coupled to albumin. This coupling can be performed either by ex vivo synthesis of a compound of MTX and human serum albumin (9) or by synthesis of a conjugate of MTX and an albumin-binding peptide spacer, which targets and tightly binds to endogenous albumin after injection (10). Since albumin is metabolized in large quantities by activated inflammatory cells in arthritic joints, this new drug–protein complex serves as a drug carrier and targets MTX to sites of inflammation. In the collagen-induced arthritis model of RA in mice, the therapeutic efficiency of MTX can be increased by this novel therapeutic approach, and clinical studies are in preparation. First steps have been made toward predicting the response of RA patients to MTX by measuring the concentration of polyglutamated MTX in red blood cells and then calculating a pharmacogenetic index by analyzing genetic polymorphisms of folate metabolism (5, 11).

In order to improve and further develop RA therapy with folate antagonists, van der Heijden and coworkers present an interesting new project elsewhere in this issue of Arthritis & Rheumatism (12). They characterized new folate antagonist drugs according to their capacity to use a transport system of intracellular uptake of folate that has a high specificity for the rheumatoid synovium: folate receptor β (FRβ).

What is the scientific background of this approach? There are several transport mechanisms by which folate or its inhibitors enter the cell (Table 1). MTX has a particularly high affinity for the reduced folate carrier (RFC), a transmembrane folate transport mechanism that has a ubiquitous distribution in the body. In contrast, there are the folate receptors α, β, and γ, which, when not activated, have only a very low affinity for MTX (6). FRβ is a differentiation marker of the myelomonocytic lineage of hematopoietic cells, FRα is mainly expressed on epithelial cells and malignant tumors, and FRγ is the secreted form of the receptor. It has been shown that activated synovial macrophages from RA patients selectively express FRβ and that MTX is transported through that receptor into those cells (13). It was speculated that in activated states, glycosylation of FRβ on macrophages might occur and that this might result in an increased MTX binding capacity of the receptors. Therefore, and relatively specifically, the up-regulated and glycosylated FRβ on synovial macrophages might anchor folate antagonists such as MTX to the site of joint inflammation.

Table 1. Folate transport mechanisms and their cellular distribution and specificity for folate and methotrexate
Folate transport mechanismMajor cellular expressionUtilization by folate and methotrexate
 Reduced folate carrierUbiquitous distributionMethotrexate; leucovorin (5-formyl-tetrahydrofolate); folate
 Folate receptor αEpithelial cells (luminal surface), malignant cellsFolate
 Folate receptor βDifferentiated myelomonocytic cells, in particular, activated monocyte/macrophages, synoviocytes, neutrophilsFolate and methotrexate (possibly due to glycosylation on activated cells)
 Folate receptor γSecreted receptor form, constitutively expressed in hematopoietic tissueFolate

However, the affinity of MTX for RFC, with the result of widespread uptake, limits the magnitude of an inflammation-specific effect of FRβ utilization by MTX in RA. Thus, antifolate drugs that, unlike MTX, are FRβ-specific would have a stronger effect on synovial macrophages and a weaker effect on other types of cells that take up MTX by the ubiquitously expressed RFC. A higher therapeutic effect and a lower rate of side effects of FRβ-specific antifolates as compared with MTX could possibly be the result.

In their study, van der Heijden et al (12) first performed immunohistochemistry of synovial tissue samples from RA patients and noninflammatory synovial tissues from orthopedic patients and confirmed that FRβ expression is a phenomenon that is highly specific to macrophages in the RA synovial membrane. T lymphocyte areas in the synovium showed no FRβ expression. Polymerase chain reaction analysis showed that the highest concentration of messenger RNA (mRNA) for FRβ was detectable in RA synovial tissue and that mRNA for FRβ was also detectable, although in much lower concentrations, in ex vivo–activated monocyte-derived macrophages. In contrast, peripheral blood lymphocytes, monocytes, or ex vivo–activated T lymphocytes showed no or only marginal FRβ expression.

The investigators then evaluated the potential of FR-targeted folate antagonists. In the past, a number of second-generation antifolate drugs had been developed for the treatment of patients with malignant diseases, mostly in order to avoid common mechanisms of resistance to MTX, including impaired transport via the RFC. The new antifolates have a broad range of properties concerning their use of the RFC transport mechanism, their binding capacity for FRs, as well as their targeting of different key enzymes of folate metabolism. Van der Heijden et al examined these novel drugs in order to determine which ones will most likely be beneficial for the treatment of synovial inflammation.

Use of the RFC system—a property that is not preferred because of its nonspecific expression—was examined by measuring the growth inhibition of the non–FR-expressing human monocytic macrophage cell line THP-1 by the tested drugs. As expected, MTX showed a good effect. Moreover, 8 of 10 second-generation antifolates inhibited growth by the RFC pathway, and only 2 drugs (BGC 945 and CB300635), both of which are inhibitors of the folate-dependent enzyme thymidylate synthase, were not active. When the relative binding affinities of the antifolates to FRα and FRβ were tested using the FRα-expressing cell line KB and FRβ-transfected Chinese hamster ovary cells (CHO/FRβ), the latter 2 drugs showed strong binding to both receptors, comparable to that of folate. In contrast, the binding of MTX was ∼50-fold lower. A specificity for one of the FR subtypes, α or β, was not detectable in either of the drugs. This latter finding is in contrast to the other drugs they tested, such as raltitrexed, a novel antifolate agent approved for the treatment of colon cancer, which relatively specifically targets FRα.

Finally, they examined growth inhibition of CHO/FRβ cells by BGC 945 and CB300635 in contrast to wild-type CHO cells. It appeared that only BGC 945 was able to inhibit the growth of CHO/FRβ cells at low concentrations, and this growth was completely inhibited by the addition of folate, which demonstrates the receptor specificity of the process.

This stepwise approach resulted in the identification of the novel folate antagonist BGC 945, which is active through FR but has only a little affinity for the RFC. Due to the abundant expression of FRβ on activated macrophages in synovial tissue, this new drug might be useful for a novel approach of selective therapeutic intervention that targets FRβ. BGC 945 is a promising drug candidate for further preclinical and clinical studies of the treatment of RA. These results have possible implications for the future of folate antagonist therapy in RA.

Obviously, we must await further studies to learn whether the process of targeting FRβ will keep its promises. FRβ is highly expressed on synovial macrophages and probably also on neutrophils. Other cell types that are thought to play an important role in the pathology of RA are not targeted with this approach. On the other hand, MTX has been shown to increase the rate of apoptosis of T lymphocytes in RA (14) and to reduce cartilage invasion and degradation in the SCID mouse model of human RA (15), which is exclusively mediated through activated synovial fibroblasts. Thus, in contrast to MTX, an FRβ-specific folate antagonist might miss these cell types. Whether this might reduce its therapeutic effectiveness must await further study. Finally, an FR-specific approach with folate antagonist drugs uses the same transport system as folate itself, and thus, the drug competes with folate. Its therapeutic action might therefore be influenced by folate intake, and the efficiency of this treatment might possibly be restricted by, for example, an uncontrolled intake of folate-containing nutritional additives or even dietary variations. Despite these questions, which will not be answered until clinical development of the drug, the FR-specific intervention of folate antagonist treatment described by van der Heijeden et al represents a promising novel therapeutic approach and further progress in improving one of the most successful principles of antiarthritic drug treatment.


  1. Top of page
  • 1
    Maini R, St.Clair EW, Breedveld F, Furst D, Kalden J, Weisman M, et al, for the ATTRACT Study Group. Infliximab (chimeric anti-tumour necrosis factor α monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase III trial. Lancet 1999; 354: 19329.
  • 2
    Breedveld FC, Weisman MH, Kavanaugh AF, Cohen SB, Pavelka K, van Vollenhoven R, et al, for the PREMIER Investigators. 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 2006; 54: 2637.
  • 3
    Klareskog L, van der Heijde D, de Jager JP, Gough A, Kalden J, Malaise M, et al, for the TEMPO Study Investigators. 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 2004; 363: 67581.
  • 4
    Bannwarth B, Pehourcq F, Schaeverbeke T, Dehais J. Clinical pharmacokinetics of low-dose pulse methotrexate in rheumatoid arthritis. Clin Pharmacokinet 1996; 30: 194210.
  • 5
    Dervieux T, Furst D, Lein DO, Capps R, Smith K, Walsh M, et al. Polyglutamation of methotrexate with common polymorphisms in reduced folate carrier, aminoimidazole carboxamide ribonucleotide transformylase, and thymidylate synthase are associated with methotrexate effects in rheumatoid arthritis. Arthritis Rheum 2004; 50: 276674.
  • 6
    Kremer JM. Toward a better understanding of methotrexate [review]. Arthritis Rheum 2004; 50: 137082.
  • 7
    Wessels JA, Huizinga TW, Guchelaar HJ. Recent insights in the pharmacological actions of methotrexate in the treatment of rheumatoid arthritis. Rheumatology (Oxford) 2008; 47: 24955.
  • 8
    Braun J, Kastner P, Flaxenberg P, Wahrisch J, Hanke P, Demary W, et al, for the MC-MTX.6/RH Study Group. Comparison of the clinical efficacy and safety of subcutaneous versus oral administration of methotrexate in patients with active rheumatoid arthritis: results of a six-month, multicenter, randomized, double-blind, controlled, phase IV trial. Arthritis Rheum 2008; 58: 7381.
  • 9
    Wunder A, Muller-Ladner U, Stelzer EH, Funk J, Neumann E, Stehle G, et al. Albumin-based drug delivery as novel therapeutic approach for rheumatoid arthritis. J Immunol 2003; 170: 4793803.
  • 10
    Fiehn C, Kratz F, Sass G, Muller-Ladner U, Neumann E. Targeted drug delivery by in vivo coupling to endogenous albumin: an albumin-binding prodrug of methotrexate (MTX) is better than MTX in the treatment of murine collagen-induced arthritis. Ann Rheum Dis 2008; 67: 118891.
  • 11
    Dervieux T, Furst D, Lein DO, Capps R, Smith K, Caldwell J, 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 2005; 64: 11805.
  • 12
    Van der Heijden JW, Oerlemans R, Dijkmans BA, Qi H, van der Laken CJ, Lems WF, et al. Folate receptor β as a potential delivery route for novel folate antagonists to macrophages in the synovial tissue of rheumatoid arthritis patients. Arthritis Rheum 2009; 60: 1221.
  • 13
    Nakashima-Matsushita N, Homma T, Yu S, Matsuda T, Sunahara N, Nakamura T, et al. Selective expression of folate receptor β and its possible role in methotrexate transport in synovial macrophages from patients with rheumatoid arthritis. Arthritis Rheum 1999; 42: 160916.
  • 14
    Herman S, Zurgil N, Langevitz P, Ehrenfeld M, Deutsch M. The immunosuppressive effect of methotrexate in active rheumatoid arthritis patients vs. its stimulatory effect in nonactive patients, as indicated by cytometric measurements of CD4+ T cell subpopulations. Immunol Invest 2004; 33: 35162.
  • 15
    Fiehn C, Neumann E, Wunder A, Krienke S, Gay S, Muller-Ladner U. Methotrexate (MTX) and albumin coupled with MTX (MTX-HSA) suppress synovial fibroblast invasion and cartilage degradation in vivo. Ann Rheum Dis 2004; 63: 8846.