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Methotrexate enhances the anti-inflammatory effect of CF101 via up-regulation of the A3 adenosine receptor expression

Avivit Ochaion12, Sara Bar-Yehuda1, Shira Cohn12, Luis Del Valle3, Georginia Perez-Liz3, Lea Madi1, Faina Barer1, Motti Farbstein1, Sari Fishman-Furman1, Tatiana Reitblat4, Alexander Reitblat4, Howard Amital5, Yair Levi5, Yair Molad6, Reuven Mader7, Moshe Tishler8, Pnina Langevitz9, Alexander Zabutti1 and Pnina Fishman1*

Author Affiliations

1 Can-Fite Biopharma Ltd., 10 Bareket Street, Kiryat-Matalon, Petah-Tikva, 49170, Israel

2 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Heren Hyesod Street, Ramat-Gan, 5200, Israel

3 Department of Neuroscience, Neuropathology Core & Center for NeuroVirology, Temple University School of Medicine, North 12th Street, Philadelphia, PA 19122, USA

4 Internal Department D, The Barzilai Medical Center, Hahistadrut Street, Ashkelon, 78278, Israel

5 Internal Department D/E, Meir Medical Center, Tshernihovsky Street, Kfar Saba, 44281, Israel

6 Rheumatology Department, Rabin Medica Center, Zabutinsky Street, Petah-Tikva, 49100, Israel

7 Medical Clinic of Rheumatology, Ha'Emek Medical Center, Afula, 18101 Israel

8 Rheumatology Department, Assaf Harofeh Medical Center, Zerifin, Beer Yakov, 70300, Israel

9 Internal Department F, The Chaim Sheba Medical Center, Tel-Hashomer, 52621 Israel

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Arthritis Research & Therapy 2006, 8:R169  doi:10.1186/ar2078

Published: 13 November 2006


Methotrexate (MTX) exerts an anti-inflammatory effect via its metabolite adenosine, which activates adenosine receptors. The A3 adenosine receptor (A3AR) was found to be highly expressed in inflammatory tissues and peripheral blood mononuclear cells (PBMCs) of rats with adjuvant-induced arthritis (AIA). CF101 (IB-MECA), an A3AR agonist, was previously found to inhibit the clinical and pathological manifestations of AIA. The aim of the present study was to examine the effect of MTX on A3AR expression level and the efficacy of combined treatment with CF101 and MTX in AIA rats. AIA rats were treated with MTX, CF101, or both agents combined. A3AR mRNA, protein expression and exhibition were tested in paw and PBMC extracts from AIA rats utilizing immunohistochemistry staining, RT-PCR and Western blot analysis. A3AR level was tested in PBMC extracts from patients chronically treated with MTX and healthy individuals. The effect of CF101, MTX and combined treatment on A3AR expression level was also tested in PHA-stimulated PBMCs from healthy individuals and from MTX-treated patients with rheumatoid arthritis (RA). Combined treatment with CF101 and MTX resulted in an additive anti-inflammatory effect in AIA rats. MTX induced A2AAR and A3AR over-expression in paw cells from treated animals. Moreover, increased A3AR expression level was detected in PBMCs from MTX-treated RA patients compared with cells from healthy individuals. MTX also increased the protein expression level of PHA-stimulated PBMCs from healthy individuals. The increase in A3AR level was counteracted in vitro by adenosine deaminase and mimicked in vivo by dipyridamole, demonstrating that receptor over-expression was mediated by adenosine. In conclusion, the data presented here indicate that MTX induces increased A3AR expression and exhibition, thereby potentiating the inhibitory effect of CF101 and supporting combined use of these drugs to treat RA.