Volume 7 Supplement 1

25th European Workshop for Rheumatology Research

Open Access

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

  • JW van der Heijden1,
  • AH Gerards2,
  • R Oerlemans1,
  • WF Lems1,
  • RJ Scheper3,
  • LA Aarden4,
  • BAC Dijkmans1 and
  • G Jansen1
Arthritis Research & Therapy20057(Suppl 1):P84

DOI: 10.1186/ar1605

Received: 11 January 2005

Published: 17 February 2005

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)

    

Mean IC-50 for TNF-α inhibition

Drug

Target

Reduced folate carrier affinity (cell uptake)

Folylpolyglutamate synthetase affinity (cell retention)

Rheumatoid arthritis patients (n = 11)

Controls (n = 6)

MTX

DHFR

++

+

78 (77) nM

57 (27) nM

PT523

DHFR

+++

-

5.8 (3.2) nM

6.4 (8.3) nM

ZD1694

TS

+++

+++

16 (26) nM

4.8 (3.0) nM

SSZ

NF-κB

-

-

278 (216) μM

191 (29) μM

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.

Declarations

Acknowledgement

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

Authors’ Affiliations

(1)
Department of Rheumatology, Vrije Universiteit Medical Center
(2)
Vlietland Hospital
(3)
Department of Pathology, Vrije Universiteit Medical Center
(4)
Department of Immunopathology, Sanquin-Research

References

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Copyright

© BioMed Central Ltd 2005

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