Methotrexate analogues display enhanced inhibition of TNF-α production in whole blood from RA patients

Abstract

Objectives: Although methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis (RA), patients experience clinical resistance to MTX upon prolonged treatment. We explored whether new-generation antifolates elicit superior anti-inflammatory properties when compared to MTX, based on their capacity to inhibit tumour necrosis factor (TNF)-α production.

Method: T cells in whole blood from 18 RA patients (including MTX-naïve, MTX- responsive, and MTX non-responsive patients) and seven healthy volunteers were stimulated with αCD3/αCD28 antibodies and incubated ex vivo for 72 h with MTX and eight novel antifolate drugs with potentially favourable biochemical and pharmacological properties. Drug concentrations exerting 50% inhibition (IC-50) of TNF-α production (by enzyme-linked immunosorbent assay, ELISA) were determined as an estimate for their anti-inflammatory capacity. In addition, induction of T-cell apoptosis was evaluated by flow cytometry.

Results: The new-generation antifolates PT523, PT644, raltitrexed, and GW1843 proved to be potent inhibitors of TNF-α production in activated T cells from all three groups of RA patients and from healthy volunteers. Based on IC-50 values, these antifolates were up to 10.3 times more potent than MTX. The anti-inflammatory effects were observed at drug concentrations that provoked suppression of T-cell activation and induction of apoptosis in 20–40% of activated T cells.

Conclusion: In an ex-vivo setting, novel antifolates elicited marked inhibition of TNF-α production in activated T cells from RA patients. Further clinical evaluation is warranted to investigate whether a low dosage of these antifolates can elicit immunosuppressive effects equivalent to MTX, and whether they are superior to MTX in patients who fail to respond to MTX.

Acknowledgements

We thank Professor LA Aarden for helpful advice and critical reading of the manuscript, and for assistance in designing the whole-blood assay. JW van der Heijden is recipient of the 2006 Rheumatology Grant from the Dutch Society for Rheumatology. YG Assaraf is recipient of a visiting professor fellowship to the VUmc provided by the Royal Netherlands Academy of Arts and Sciences, the Netherlands Organization of Scientific Research, and the Dutch Arthritis Foundation. This study was supported by grants from the Netherlands Organization for Health Research and Development (ZonMW; 920-03-362 to JW van der Heijden) and the Dutch Arthritis Foundation (NRF-03-I-40).