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469Baharav, et al: A3 adenosine receptor agonists

Antiinflammatory Effect of A3 Adenosine ReceptorAgonists in Murine Autoimmune Arthritis ModelsEHUD BAHARAV, SARA BAR-YEHUDA, LEA MADI, DANIEL SILBERMAN, LEA RATH-WOLFSON, MARISA HALPREN, AVIVIT OCHAION, ABRAHAM WEINBERGER, and PNINA FISHMAN

ABSTRACT. Objective. CF101, an A3 adenosine receptor (A3AR) agonist, is a small orally bioavailable moleculeknown to suppress in vitro the production of tumor necrosis factor-α (TNF-α). We evaluated its ther-apeutic potential and antiinflammatory effects in 3 murine models of adjuvant induced arthritis(AIA).Methods. The antiinflammatory effect of CF101 was examined in rat AIA, in mouse collageninduced arthritis, and in tropomyosin induced arthritis. The clinical effect of another A3AR agonist,Cl-IB-MECA, was examined in rat AIA. The effect of low dose (10 or 100 mg/kg/day) A3ARagonists administered orally once daily on arthritis severity was assessed clinically and histologi-cally. The effect of CF101 on the protein expression level of TNF-α in the synovial tissue, draininglymph nodes, and spleen cells was determined by Western blot.Results. CF101 and Cl-IB-MECA markedly ameliorated the clinical and histological features ofarthritis in the 3 models when administered orally at a low dose of 10 mg/kg body weight in the 3autoimmune arthritis models. The lower dose of 10 mg/kg of either CF101 or Cl-IB-MECA had bet-ter antiinflammatory effect than the higher 100 mg/kg dose. Decreased expression of TNF-α wasnoted in protein extracts of synovia, draining lymph nodes, and spleen tissues.Conclusion. The results provide evidence that A3AR agonists exert significant antirheumatic effectsin different autoimmune arthritis models by suppression of TNF-α production. The beneficial activ-ity of the drugs at the low dose demonstrates that the effect is A3AR mediated. (J Rheumatol2005;32:469–76)

Key Indexing Terms:A3 ADENOSINE RECEPTOR AGONIST CF101 ADJUVANT ARTHRITISCOLLAGEN INDUCED ARTHRITIS TROPOMYOSIN INDUCED ARTHRITIS

TUMOR NECROSIS FACTOR-α

From Can-Fite BioPharma Ltd., Kiryat-Matalon, Petach-Tikva;Department of Medicine B, Beilinson Campus, Laboratory of JointsPhysiopathology, Felsenstein Medical Research Center; Laboratory ofClinical and Tumor Immunology, Felsenstein Medical Research Center,Tel-Aviv University Sackler Faculty of Medicine, Rabin Medical Center;Department of Pathology, Golda Campus, Rabin Medical Center andSackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

E. Baharav, MD, Can-Fite BioPharma Ltd. and Laboratory of JointsPhysiopathology, Felsenstein Medical Research Center; S. Bar-Yehuda,PhD, Can-Fite BioPharma Ltd. and Laboratory of Clinical and TumorImmunology, Felsenstein Medical Research Center; L. Madi, PhD; D. Silberman, VetD, Can-Fite BioPharma Ltd.; L. Rath-Wolfson, MD; M. Halpren, MD, Department of Pathology, Rabin Medical Center andSackler Faculty of Medicine, Tel Aviv University; A. Weinberger, MD,Laboratory of Joints Physiopathology, Felsenstein Medical ResearchCenter; P. Fishman, PhD, Can-Fite BioPharma Ltd. and Laboratory ofClinical and Tumor Immunology, Felsenstein Medical Research Center.

Address reprint requests to Prof. P. Fishman, Can-Fite Bio Pharma Ltd.,10 Bareket Street, Kiryat-Matalon, Petach-Tikva, Israel 49170. E-mail: [email protected]

Submitted September 3, 2004; revision accepted October 13, 2004.

Rheumatoid arthritis (RA) is a chronic autoimmune disorderwith predominant involvement of the joints. The etiology ofRA is unknown but it is evident that tumor necrosis factor-α (TNF-α) plays a key role in the development and persist-ence of the disease1. Transgenic mice hyperexpressing thehuman TNF-α gene develop spontaneous arthritis resem-

bling RA2. Recently, anti-TNF-α treatments have beenfound to be highly effective. These drugs modify the diseasecourse and in the majority of patients prevent periarticularbone damage3. TNF-α production is regulated mainly bynuclear factor-κB (NF-κB), a cytoplasmic protein, whichupon translocation to the nucleus induces transcription ofTNF-α gene4.

Adenosine (9-ß-d-ribofuranosyladenine), the purinenucleoside, mediates various physiological cellular activi-ties, such as cell growth, differentiation, and cell death5,6.Adenosine is released into the extracellular environmentfrom activated or metabolically stimulated cells and binds toselective G-protein-associated membrane receptors, desig-nated A1, A2A, A2B, and A37. The cumulative effects of acti-vated adenosine A2 and A3 receptors (A2AR, A3AR) onmononuclear cells of the immune system are consideredantiinflammatory. Modification of monocyte/macrophagefunction by adenosine is expressed by decreased productionof proinflammatory cytokines, such as TNF-α, interleukin 6(IL-6), and IL-8, and the increase of the antiinflammatorycytokine IL-108-10. Moreover, the cytokine milieu alters theexpression and function of adenosine membrane receptorson inflammatory cells11. In addition, adenosine was found toact as an antirheumatic agent in autoimmune arthritis mod-

Personal non-commercial use only. The Journal of Rheumatology Copyright © 2005. All rights reserved.

els12. A3AR was found to mediate part of the antiinflamma-tory effect of adenosine13. A3AR activation in macrophagesand lymphocytes was shown to decrease the intracellularlevel of NF-κB, leading to a decrease in the transcription ofTNF-α14, and IB-MECA has been shown to decrease IL-12production15.

We assessed the antiinflammatory effect of two A3ARsynthetic agonists, CF101 and Cl-IB-MECA, on autoim-mune murine models of arthritis. Adjuvant induced arthritis(AIA) in rats and collagen induced arthritis (CIA) in miceare considered classic murine models for RA. The thirdexperimental model is an autoimmune condition resemblingBehçet’s disease and is characterized by T cell dependentjoint inflammation. Indeed, the data show that the 2 agonistsmarkedly inhibit the manifestations of autoimmune arthritis,suggesting that A3AR may serve as a target to downregulatethe inflammatory process.

MATERIALS AND METHODSDrugs. The A3AR agonist CF101, a GMP grade of the compound knowngenerically as 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purine-9-yl]-N-methyl-D-ribofuranuronamide (IB-MECA), was synthesized forCan-Fite BioPharma by Albany Molecular Research Inc. (Albany, NY,USA). The additional A3AR agonist, 2-chloro-N6-(3-iodobenzyl)-adeno-sine-5’- N-methyl-uronamide (CI-IB-MECA) and MRS 1220, a highlyselective A3AR antagonist, were purchased from RBI/Sigma (Natick, MA,USA). For both agonists and antagonists a stock solution of 10 mM wasprepared in DMSO and further dilutions in culture medium or phosphatebuffered saline (PBS) were performed to reach the desired concentration.Methotrexate (MTX) was purchased from Abic (Petach-Tikva, Israel).

Animal models. AIA. Female Lewis rats aged 8–12 weeks obtained fromHarlan Laboratories (Jerusalem, Israel) were injected subcutaneously (SC)at the tail base with 100 µl of suspension composed of incomplete Freund’sadjuvant (IFA) with 10 mg/ml heat killed Mycobacterium tuberculosisH37Ra (Difco, Detroit, MI, USA).

CIA. Male DBA mice aged 10 weeks were injected SC with 100 µl of TypeII collagen 200 µg in complete Freund’s adjuvant (CFA). On Day 21 abooster injection of the same emulsion was administered. Mice wereinspected daily for symptoms of clinical arthritis.

Tropomyosin induced arthritis (TIA). TIA was induced by vaccinating thefemale Lewis rats at tail base SC with 100 µl emulsion composed of 100µg bovine α-tropomyosin dissolved in PBS, emulsified in an equal volumeof IFA containing 4 mg/ml heat killed M. tuberculosis H37Ra.Intraperitoneal injections of pertussis toxin 200 ng/100 µl PBS were admin-istered on Day 0 (induction) and on Day 216.

Animals received standardized pelleted diet and tap water ad libitum.Experiments were performed in accordance with the guidelines estab-

lished by the Institutional Animal Care and Use Committee at Can-FiteBioPharma.

Each group contained 10 animals and each experiment was conductedat least 3 times.

Treatment protocols. Drugs were orally administered by gavage, oncedaily. The positive control received vehicle only (DMSO in a dilution cor-responding to that of the drug), while the treatment groups received 10µg/kg or 100 µg/kg of CF101 or Cl-IB-MECA. In the AIA and TIA mod-els treatment was initiated on Day 7 after vaccination, while in the CIAmodel, mice were treated at onset of clinical arthritis. To compare the effectof CF101 to the standard therapy, we treated an additional group of animalswith MTX. The drug (3 mg/kg) was given intraperitoneally twice weekly,starting on Day 3 after immunization.

Clinical disease activity score. In the AIA and TIA models, the animalswere inspected every second day for clinical arthritis. The scoring systemranged from 0 to 4 for each limb as follows: 0: no arthritis; 1: redness orswelling of one toe/finger joint; 2: redness and swelling of more than onetoe/finger joint; 3: ankle and tarsal-metatarsal joint involvement; and 4:entire paw redness or swelling. The arthritic score was calculated by addingthe 4 individual leg scores to a maximum of 1617,18.

Inflammatory intensity in the CIA model was determined in accordwith the increase in hindpaw diameter, measured by calipers. The meanscore in each experimental group was designated as the clinical score.

Histology score. Animals were sacrificed and the legs were removed up tothe knee level, fixed in 10% formaldehyde, decalcified, dehydrated,embedded in paraffin, cut into 4 µm sections, and stained by hematoxylin-eosin.

Assessment of all pathologic findings was performed blind (by LRWand MH) using semiquantitative grading scales of 0 to 4 for the followingmeasures: (1) extent of inflammatory cell infiltration to the joint tissues; (2)synovial lining cell hyperplasia; (3) pannus formation; (4) joint cartilagelayer destruction; and (5) bone damage and erosion score, graded 0 to 5 asfollows: 0: normal, 1: minimal loss of cortical bone at a few sites, 2: mildloss of cortical trabecular bone, 3: moderate loss of bone at many sites, 4:marked loss of bone at many sites, 5: marked loss of bone at many siteswith fragmenting and full thickness penetration of inflammatory process orpannus into the cortical bone18,19. The mean of all the histological measurescores was designated the histology score.

Measurement of TNF-α expression. TNF-α was measured in synovia,draining lymph nodes, and spleen tissues derived from control and CF101treated rats with AIA. This experiment was repeated 3 times, and in eachexperiment protein extracts were prepared by pooling the relative organsample from 5 different animals.

To detect the level of expression of TNF-α, Western blot analysis wasperformed. Spleen and draining lymph node mononuclear cells and syn-ovial tissue were rinsed with ice-cold PBS and transferred to ice-cold lysisbuffer (TNN buffer, 50 mM Tris buffer, pH 7.5, 150 mM NaCl, NP 400.5%) for 20 min. Cell debris was removed by centrifugation for 10 min at7500 g. The supernatant was utilized for Western blot analysis. Protein con-centrations were determined using the Bio-Rad protein assay dye reagent.Equal amounts of the sample (50 µg) were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis using 12% polyacrylamide gels.Resolved proteins were then electroblotted onto nitrocellulose membranes(Schleicher & Schuell, Keene, NH, USA). Membranes were blocked with1% bovine serum albumin and incubated with polyclonal goat anti-ratTNF-α antibodies (dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz,CA, USA) for 24 h at 4°C. Blots were then washed and incubated with rab-bit anti-goat polyclonal antibodies for 1 h at room temperature. Bands wererecorded using BCIP/NBT color development kit (Promega, Madison, WI,USA). Densitometry of protein expression was normalized against ß-actinand expressed as percentage of control.

Statistical analysis. Results were evaluated using the Student t test.Comparison between the mean values of different experiments was carriedout. The criterion for statistical significance was p < 0.05.

RESULTSEffects of A3AR agonists on development of arthritis.Adjuvant induced arthritis. In the control group, arthritisappeared on Day 15, disease incidence was 70%, and themean of the maximal clinical arthritis score (6.3 ± 1.56) wasobserved on Day 21. In the 10 µg/kg and 100 µg/kg CF101treated groups, the incidence of disease was only 20%, andthe maximal clinical arthritis score was delayed to Day 26and had lower values of 1.0 ± 0.7 and 2.7 ± 0.9, respective-ly (Figure 1A). The effect of CF101 (10 µg/kg) was similar

470 The Journal of Rheumatology 2005; 32:3



Figure 1. Effect of CF101 on development of adjuvant arthritis. Rats wereinjected with CFA and treatment with CF101 (10 or 100 µg/kg) was initiat-ed 7 days later; an additional group was treated with MTX (3 mg/kg) twicedaily starting on Day 3 after immunization. A. Effect of CF101 on diseaseclinical score. B. Hind paws of representative rat from group treated with10 µg/kg CF101 and control, on Day 26. C. Effect of CF101 on histologyfeatures of joint destruction: representative section was obtained after ratswere sacrificed on Day 26 (left panel 40× magnification, right 20×). D.Mean histology score.


to that of MTX. Interestingly, when treatment with CF101(100 µg/kg) started on Day 14, the effect was even betterthan in the group in which treatment was initiated on Day 7(maximal clinical score 0.9 ± 0.12).

Figure 1B depicts the paw of the vehicle treated rat (leftpanel) that is markedly swollen and red, while the leg ofCF101 treated rat (10 µg/kg) appears completely normal(right panel).

Figure 1C shows representative hind foot joint histology.In the control, prominent infiltration of predominantmononuclear inflammatory cells is visible. The joint cleftwas obliterated by these cells as well as cell debris. In addi-tion, there is destruction of the cartilage and bone degrada-tion. In contrast, the treatment with CF101 preserved thenormal histology of rat hind limb joints. No damage to car-tilage and bone was detected in the treated groups. Figure1D shows the mean histology score, which is significantlyreduced in the 2 CF101 treated groups.

In another set of experiments, the effect of an additionalA3AR agonist, Cl-IB-MECA (10 and 100 µg/kg/day), onthe development of AIA was examined. In the control groupthe incidence of disease was 80%, the arthritis started onDay 14, and the maximal clinical arthritis score (6.3 ± 1.56)was observed on Day 22. In both Cl-IB-MECA treatedgroups the incidence of disease was 60%, and the maximalarthritis score was delayed to Day 26 and had a statisticallysignificant lower value [1.4 ± 0.66 (p < 0.01) and 3.4 ± 1.23(p < 0.05), respectively; Figure 2].

Since similar or even better results were obtained withthe lower dose (10 µg/kg/day) than with the higher dose(100 µg/kg/day), the subsequent experiments were conduct-ed utilizing 10 µg/kg/day CF 101 only.

Collagen induced arthritis. In the following experiments,the effect of CF101 (10 µg/kg/day) on the development of

CIA was evaluated. DBA mice developed the disease sever-al days after the second immunization with Type II colla-gen/CFA emulsion. In each individual mouse, CF101 wasadministered at the onset of disease. In the control groupmaximal hindpaw swelling was observed 6 days after dis-ease onset, whereas in the CF101 treated group it was notedon Day 14 (Figure 3A). The intensity of the arthritis wasreduced throughout the experiment period, reaching a max-imum on Day 21 (p < 0.02). In the CF101 treated group his-tology sections showed minimal inflammatory changescompared to the extensive changes in the control group(Figure 3B). In addition, the histology score was markedlylower in the CF101 treated group compared to the controlgroup (3.87 ± 1.2 vs 1.0 ± 0.6; p < 0.01; Figure 3C). Theseclinical measurements combined with the histology evi-dence reinforce the previous conclusion that the activationof A3AR has an antirheumatic effect, demonstrated in dif-ferent experimental models of RA.

Tropomyosin induced arthritis. CF101 tested in TIA induceda remarkable antiinflammatory effect, expressed by thereduced maximal clinical arthritis score (9.3 ± 1.62 vs 4.1 ±0.72; p < 0.001; Figure 4A). In pathological sections of con-trols the inflammation and the joint structure destructionwere prominent, whereas in CF101 treated mice the jointstructure and synovial tissue were preserved (Figure 4B).The histology score supports the clinical observation thatmild inflammation of joints was visualized in the treated rats(Figure 4C). Overall, these results demonstrate that the anti-inflammatory effect of CF101 is not limited to a specificmurine model of arthritis.

Measurement of TNF-α. We measured TNF-α expression byWestern blot analysis of protein extracts from draininglymph nodes, splenocytes, and synovial tissue. Figure 5shows representative data of one experiment (this experi-


Figure 2. Disease clinical scores show the effect of Cl-IB-MECA on development of adjuvantarthritis. Rats were injected with CFA and treatment with Cl-IB-MECA (10 or 100 µg/kg) wasstarted 7 days later.


ment was repeated 3 times). In each experiment proteinextracts were prepared by pooling the relative organ samplesfrom 5 different animals. A decreased level of the cytokinewas found in the different tissues.

DISCUSSIONOur series of experiments revealed a remarkable antiinflam-matory effect mediated by administering A3AR agonists todifferent mouse/rat experimental models of inflammatoryarthritis. The incidence of animals affected with arthritis wasreduced significantly and the maximal and mean jointinflammation intensity was decreased.

CF101 and Cl-IB-MECA reduced the inflammatory cellinfiltration and synovial involvement in the inflammatoryprocess. Moreover, minimal damage of the joint, cartilage,and bone was detected in the majority of the treated animalswith AIA. These protective effects are typical of diseasemodifying antirheumatic drugs, which in addition to theirability to negate the inflammatory manifestations of arthritiscan reduce the accumulative structural damage to the joints’hard tissues.

These findings indicate that CF101 exhibits a generalantiinflammatory effect in autoimmune models, and theeffect was observed in rat as well as in mouse models.

Interestingly, CF101 and Cl-IB-MECA had significantantiinflammatory effects at the low doses of 10 µg/kg and100 µg/kg, respectively, in the AIA model, suggesting thatthe antiinflammatory effect of CF101 is A3AR mediated.We have reported similar data in various murine tumor mod-els in which the animals were treated with IB-MECA or Cl-IB-MECA at the same doses, resulting in tumor suppres-sion20-22. It was hypothesized that efficacy at the low dosewas attributed to the high selectivity of CF101 and Cl-IB-MECA to A3AR (Ki 1.1 nM and 0.33 nM, respectively).Since the concentration of the agonists was very low (10µg/kg) in the present study, it could target the A3AR exclu-sively. It is reasonable that at the low agonist concentrationA3AR is predominantly activated, whereas at higher dosesother adenosine receptors may be involved, resulting in alower antiinflammatory response.

Supporting the role of A3AR in mediating antiinflamma-tory effects are the studies showing that in A3AR knockout


Figure 3. Effect of CF101 on development of collagen induced arthritis (CIA). Mice were immunized with type II collagen in CFA, and treatment with CF101(10 µg/kg) was started upon onset of disease. A. Effect of CF101 on joint swelling. B. Representative histology sections show effects of CF101 on jointdestruction in CIA, after mice were sacrificed on Day 21 (all 20× magnification). C. Mean histology score.


mice, selective agonists to the receptor did not inhibitlipopolysaccharide induced TNF-α production8. Moreover,Montesinos, et al showed that MTX treatment in A3ARknockout mice did not prevent the accumulation of inflam-matory cells in an air-pouch model in comparison to itseffect in wild-type mice23.

Szabo, et al24 showed the efficacy of IB-MECA in ame-liorating the manifestations of CIA in mice, utilizing highdose agonist (500 µg/kg). They concluded that the antiin-flammatory effect was attributed to activation of both A2ARand A3AR. In our study the effect is probably A3AR medi-ated due to the low dose of agonist.

Macrophage and T cell derived cytokines such as TNF-αappear to play a critical role in the induction and perpetua-tion of chronic inflammatory processes in rheumatoid jointsas well as in the systemic manifestations of this disease25,26.TNF-α is overproduced in joints of RA patients, and isknown to trigger synoviocyte proliferation and a cascade ofsecondary mediators involved in the recruitment of inflam-

matory cells and activation of osteoclast that mediates jointdestruction27. Accumulated data indicate that in RA patientsas well as in animal models, there is increased activity ofNF-κB, the transcription factor of TNF-α, and additionalproinflammatory mediators28,29. Moreover, in mouse strainsthat are genetically prone to develop spontaneous auto-immune diseases, higher levels of nuclear active NF-κBhave been detected, leading to greater expression of theproinflammatory cytokine IL-1230.

We found diminished TNF-α in the synovial tissue,splenocytes, and draining lymph node cells derived fromCF101 treated rats. TNF-α inhibition upon A3AR activationhas previously been observed in monocytes andmacrophages11,31-33. In these studies, both TNF-α mRNAand protein expression levels were downregulated, support-ing our data and the suggested mechanism. The antiinflam-matory effect of CF101 is probably related to its ability toregulate TNF-α levels.

MTX, the widely used RA disease modifying agent34,


Figure 4. Effect of CF101 on development of tropomyosin induced arthritis. Rats were immunized with bovine α-tropomyosin and treatment with CF101 (10µg/kg) was started 7 days later. A. Effect of CF101 on disease clinical score. B. Effect of CF101 on histology features: representative sections were obtainedafter rats were sacrificed on Day 28 (both 40× magnification). C. Mean histology score.


acts by increasing the extracellular adenosine concentra-tion35. In this pathway MTX inhibits the enzyme 5-aminoimidazole-4, carboxamide ribonuclease (AICAR)transformylase, and as a consequence intracellular AICARaccumulates, resulting in increased adenosine concentra-tion36. This means that at least part of the mechanism ofMTX is mediated by the metabolite adenosine. Furthermore,Montesinos, et al23 showed that in A3AR knockout mice,MTX lost its antiinflammatory effect, indicating that A3ARactivation is an essential step in mediating the antirheumat-ic properties of MTX. Chronic treatment with MTX hastoxic effects in the liver, lungs, hematopoietic system, skin,and mucosal membranes, and its use is forbidden duringpregnancy and lactation37,38. Thus, utilizing CF101, an oralnontoxic drug, may be advantageous.

Lately, a new class of antirheumatic biologic drugs, themonoclonal antibodies against the cytokines TNF-α and IL-1, has been developed. These drugs have shown beneficialeffects in about 80% of patients treated for severe disease ormultiple antirheumatic drug failure39-41. The anti-TNF-αantibodies are very costly, require parenteral administration,and have been associated with both acute adverse reactionsand longer-term sequelae42, including serious infectiouscomplications such as tuberculosis, histoplasmosis,increased incidence of squamous cell carcinoma of the skin,and hematological neoplasias43-45. Thus, despite recent

advances in the treatment of RA, there is still a need forconvenient, safe, and cost-effective medications.

The beneficial clinical and histological effects of CF101in experimental autoimmune arthritis models, its suppres-sive effect on TNF-α production, and its high oral bioavail-ability suggest CF101 as an attractive adjuvant therapy forautoimmune arthritis.

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