title: Methotrexate enhances the anti-inflammatory effect of CF101 via up-regulation of the A3 adenosine receptor expression.
abstract: 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.
authors.affiliation: Can-Fite Biopharma Ltd, 10 Bareket Street, Kiryat-Matalon, Petah-Tikva, 49170, Israel.
authors.forename: Avivit
authors.initials: A
authors.lastname: Ochaion
authors.forename: Sara
authors.initials: S
authors.lastname: Bar-Yehuda
authors.forename: Shira
authors.initials: S
authors.lastname: Cohn
authors.forename: Luis
authors.initials: L
authors.lastname: Del Valle
authors.forename: Georginia
authors.initials: G
authors.lastname: Perez-Liz
authors.forename: Lea
authors.initials: L
authors.lastname: Madi
authors.forename: Faina
authors.initials: F
authors.lastname: Barer
authors.forename: Motti
authors.initials: M
authors.lastname: Farbstein
authors.forename: Sari
authors.initials: S
authors.lastname: Fishman-Furman
authors.forename: Tatiana
authors.initials: T
authors.lastname: Reitblat
authors.forename: Alexander
authors.initials: A
authors.lastname: Reitblat
authors.forename: Howard
authors.initials: H
authors.lastname: Amital
authors.forename: Yair
authors.initials: Y
authors.lastname: Levi
authors.forename: Yair
authors.initials: Y
authors.lastname: Molad
authors.forename: Reuven
authors.initials: R
authors.lastname: Mader
authors.forename: Moshe
authors.initials: M
authors.lastname: Tishler
authors.forename: Pnina
authors.initials: P
authors.lastname: Langevitz
authors.forename: Alexander
authors.initials: A
authors.lastname: Zabutti
authors.forename: Pnina
authors.initials: P
authors.lastname: Fishman
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D001168:Arthritis / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D015153:Blotting, Western; D004359:Drug Therapy, Combination; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology*; D008875:Middle Aged; D051381:Rats; D011917:Rats, Inbred Lew; D043684:Receptor, Adenosine A3 / Q000096:biosynthesis / Q000187:drug effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
chemical_list: D000893:Anti-Inflammatory Agents; C478920:CF101; D043684:Receptor, Adenosine A3; D000241:Adenosine; D008727:Methotrexate