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Inhibition of tumour necrosis factor alpha production by activated T cells of rheumatoid arthritis patients by novel anti-folate drugs: an ex vivopilot study

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Introduction

The folate antagonist methotrexate (MTX) is the 'anchor-drug' in the treatment of patients with rheumatoid arthritis (RA) [1]. The main target of MTX in intracellular folate metabolism is dihydrofolate reductase (DHFR) but several other targets have been described (e.g. thymidylate synthase [TS] and 5-amino-imidazole-4-carboxamide ribonucleotide [AICAR]). At present the exact mechanism of action of MTX in RA still remains elusive [1]. Despite the potent anti-rheumatic capacity of MTX many patients (at least 50%) become resistant to MTX during long-lasting therapy. However, little is known about the mechanisms of resistance against MTX in RA patients [2].

From the field of oncology, where MTX is used against haematological malignancies, new anti-folate drugs were developed to circumvent MTX resistance [3]. These new folate antagonists have the following characteristics: are better transported through the reduced folate carrier, are retained intracellular more efficiently by polyglutamylation via folylpolyglutamate synthetase, and/or have other targets in the folate pathway besides DHFR (e.g. TS).

Objective

To investigate whether two new-generation anti-folate drugs, PT523 (DHFR-inhibitor) and ZD1694 (TS-inhibitor), have equal or better anti-inflammatory capacity compared with MTX based on their capacity to inhibit tumour necrosis factor alpha (TNF-α) production by activated T cells.

Methods

Whole blood from 11 RA patients and six healthy volunteers was incubated ex vivo with MTX, PT523, ZD1694 and, as a control, the DMARD sulphasalazine (SSZ) after T-cell stimulation with α-CD3/CD28. Inhibition of TNF-α production was measured after 72 hours by ELISA [4].

The IC-50 values (defined as the drug concentration exerting 50% inhibition of TNF-α production) are used as a value for the anti-inflammatory capacity of the drug (Table 1).

Table 1 Characteristics of drugs and mean IC-50 values (± standard deviation)

Results

Both PT523 and D1694 turned out to inhibit TNF-α production by activated T cells much more efficiently than MTX (5–15 times). For comparison, the DMARD SSZ is effective at much higher concentration (μM range). The inhibition of TNF-α production by the anti-folate drugs does not seem to be a result of (apoptotic) cell death of T cells whereas SSZ induces apoptosis of T cells (data not shown).

Conclusion

In an ex vivo setting, two novel anti-folate drugs designed to circumvent MTX resistance proved to be very effective in inhibiting TNF-α production by activated T cells from RA patients and healthy volunteers. Future experiments are designed to evaluate ex vivo anti-folate sensitivity profiles for 'MTX-responders' and 'MTX-non-responders' to investigate whether these novel generation of antifolate drugs can be useful in cases of clinical failure on MTX.

References

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    Kremer JM: Toward a better understanding of methotrexate. Arthritis Rheum. 2004, 50: 1370-1382. 10.1002/art.20278.

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    Jansen G, Scheper RJ, Dijkmans BAC: Multidrug resistance proteins in rheumatoid arthritis, role in disease modifying antirheumatic drug efficacy and inflammatory processes: an overview. Scand J Rheumatol. 2003, 32: 325-339. 10.1080/03009740310004333.

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    Rots MG, Pieters R, Peters GJ, van Zantwijk CH, Mauritz R, Noordhuis P, Willey JC, Hahlen K, Creutzig U, Janka-Schaub G, et al: Circumvention of methotrexate resistance in childhood leukemia subtypes by rationally designed antifolates. Blood. 1999, 94: 3121-3128.

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    Gerards AH, de Lathouder S, de Groot ER, Dijkmans BAC, Aarden LA: Inhibition of cytokine production by methotrexaat. Studies in healthy volunteers and patients with rheumatoid arthritis. Rheumatology. 2003, 42: 1189-1196. 10.1093/rheumatology/keg323.

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Acknowledgement

This study is supported by the Dutch Arthritis Association (Grant NRF-03-I-40).

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Keywords

  • Rheumatoid Arthritis Patient
  • Inhibit Tumour Necrosis Factor
  • AICAR
  • Sulphasalazine
  • Reduce Folate Carrier