Shira Cohen

The PI3K–NF-κB signal transduction pathway is involved in mediating the anti-inflammatory effect of IB-MECA in adjuvant-induced arthritis

The anti-inflammatory effect of adenosine was previously found to be mediated via activation of the A3 adenosine receptor (A3AR). The aim of the present study was to decipher the molecular mechanism involved with the inhibitory effect of IB-MECA, an A3AR agonist, on adjuvant-induced arthritis.The adjuvant-induced arthritis rats responded to IB-MECA treatment with a decrease in the clinical score and the pathological score of the disease. The response to IB-MECA was neutralized by the antagonist MRS 1220, confirming that the efficacy of the synthetic agonist was A3AR mediated.The A3AR protein expression level was highly expressed in the synovia, in the peripheral blood mononuclear cells and in the drain lymph node (DLN) tissues of adjuvant-induced arthritis rats in comparison with naïve animals. Downregulation of A3AR expression was noted upon treatment with IB-MECA. Analysis of synovia and DLN protein extracts revealed a decreased expression level of PI3K, PKB/Akt, IKK, NF-κB and tumor necrosis factor alpha, known to affect survival and apoptosis of inflammatory cells, whereas the caspase-3 level was upregulated.Taken together, high A3AR expression is found in the synovia, in the immune cells in the DLN and in peripheral blood mononuclear cells. IB-MECA, an orally bioavailable molecule, activates the A3AR, inducing receptor downregulation and the initiation of a molecular mechanism that involves de-regulation of the PI3K–NF-κB signaling pathway. As a result, a potent anti-inflammatory effect manifested in the improvement of the disease clinical score and pathological score occurs. The finding that the A3AR expression level in the peripheral blood mononuclear cells and in the DLN reflects the receptor status in the remote inflammatory site suggests use of the A3AR as a follow-up biomarker.

Induction of an antiinflammatory effect and prevention of cartilage damage in rat knee osteoarthritis by CF101 treatment.

OBJECTIVE Studies have suggested that rheumatoid arthritis (RA) and osteoarthritis (OA) share common characteristics. The highly selective A(3) adenosine receptor agonist CF101 was recently defined as a potent antiinflammatory agent for the treatment of RA. The purpose of this study was to examine the effects of CF101 on the clinical and pathologic manifestations of OA in an experimental animal model. METHODS OA was induced in rats by monosodium iodoacetate, and upon disease onset, oral treatment with CF101 (100 microg/kg given twice daily) was initiated. The A(3) adenosine receptor antagonist MRS1220 (100 microg/kg given twice daily) was administered orally, 30 minutes before CF101 treatment. The OA clinical score was monitored by knee diameter measurements and by radiographic analyses. Histologic analyses were performed following staining with hematoxylin and eosin, Safranin O-fast green, or toluidine blue, and histologic changes were scored according to a modified Mankin system. Signaling proteins were assayed by Western blotting; apoptosis was detected via immunohistochemistry and TUNEL analyses. RESULTS CF101 induced a marked decrease in knee diameter and improved the changes noted on radiographs. Administration of MRS1220 counteracted the effects of CF101. CF101 prevented cartilage damage, osteoclast/osteophyte formation, and bone destruction. In addition, CF101 markedly reduced pannus formation and lymphocyte infiltration. Mechanistically, CF101 induced deregulation of the NF-kappaB signaling pathway, resulting in down-regulation of tumor necrosis factor alpha. Consequently, CF101 induced apoptosis of inflammatory cells that had infiltrated the knee joints; however, it prevented apoptosis of chondrocytes. CONCLUSION CF101 deregulated the NF-kappaB signaling pathway involved in the pathogenesis of OA. CF101 induced apoptosis of inflammatory cells and acted as a cartilage protective agent, which suggests that it would be a suitable candidate drug for the treatment of OA.

The anti-inflammatory effect of A3 adenosine receptor agonists: a novel targeted therapy for rheumatoid arthritis

Targeting the A3 adenosine receptor (A3AR) to combat inflammation is a new concept based on two findings. First, A3AR is highly expressed in inflammatory cells, whereas low expression is found in normal tissues. This receptor was also found to be overexpressed in peripheral blood mononuclear cells, reflecting receptor status in the remote inflammatory process. Second, A3AR activation with a specific agonist induces de-regulation of the NF-κB signaling pathway in inflammatory cells, as well as initiation of immunomodulatory effects. The A3AR agonist CF-101 (known generically as IB-MECA) induces anti-inflammatory effects in experimental animal models of collagen- and adjuvant-induced arthritis. Combined therapy with CF-101 and methotrexate in adjuvant-induced arthritis rats yielded an additive anti-inflammatory effect. Methotrexate induced upregulation of A3AR, rendering the inflammatory cells more susceptible to CF-101. In Phase I and in Phase IIa human studies, CF-101 was safe, well tolerated and showed strong evidence of an anti-inflammatory effect in rheumatoid arthritis patients. In peripheral blood mononuclear cells withdrawn from the patients at base line, a statistically significant correlation between A3AR expression level and response to the drug was noted. It is suggested that A3AR may serve as a biologic marker to predict patient response to the drug. Taken together, this information suggests that A3AR agonists may be a new family of orally bioavailable drugs to be developed as potent inhibitors of autoimmune–inflammatory diseases.

The A3 adenosine receptor agonist CF502 inhibits the PI3K, PKB/Akt and NF-κB signaling pathway in synoviocytes from rheumatoid arthritis patients and in adjuvant-induced arthritis rats

The A(3) adenosine receptor (A(3)AR) is over-expressed in inflammatory cells and was defined as a target to combat inflammation. Synthetic agonists to this receptor, such as IB-MECA and Cl-IB-MECA, exert an anti-inflammatory effect in experimental animal models of adjuvant- and collagen-induced arthritis. In this study we present a novel A(3)AR agonist, CF502, with high affinity and selectivity at the human A(3)AR. CF502 induced a dose dependent inhibitory effect on the proliferation of fibroblast-like synoviocytes (FLS) via de-regulation of the nuclear factor-kappa B (NF-kappaB) signaling pathway. Furthermore, CF502 markedly suppressed the clinical and pathological manifestations of adjuvant-induced arthritis (AIA) in a rat experimental model when given orally at a low dose (100 microg/kg). As is typical of other G-protein coupled receptors, the A(3)AR expression level was down-regulated shortly after treatment with agonist CF502 in paw and in peripheral blood mononuclear cells (PBMCs) derived from treated AIA animals. Subsequently, a decrease in the expression levels of protein kinase B/Akt (PKB/Akt), IkappaB kinase (IKK), I kappa B (IkappaB), NF-kappaB and tumor necrosis factor-alpha (TNF-alpha) took place. In addition, the expression levels of glycogen synthase kinase-3 beta (GSK-3beta), beta-catenin, and poly(ADP-ribose)polymerase (PARP), known to control the level and activity of NF-kappaB, were down-regulated upon treatment with CF502. Taken together, CF502 inhibits FLS growth and the inflammatory manifestations of arthritis, supporting the development of A(3)AR agonists for the treatment of rheumatoid arthritis.

Inhibition of experimental auto-immune uveitis by the A3 adenosine receptor agonist CF101.

Uveitis is an inflammation of the middle layer of the eye with a high risk of blindness. The Gi protein associated A3 adenosine receptor (A3AR) is highly expressed in inflammatory cells whereas low expression is found in normal cells. CF101 is a highly specific agonist at the A3AR known to induce a robust anti-inflammatory effect in different experimental animal models. The CF101 mechanism of action entails down-regulation of the NF-κB-TNF-α signaling pathway, resulting in inhibition of pro-inflammatory cytokine production and apoptosis of inflammatory cells. In this study the effect of CF101 on the development of retinal antigen interphotoreceptor retinoid-binding protein (IRBP)-induced experimental autoimmune uveitis (EAU) was assessed. Oral treatment with CF101 (10 µg/kg, twice daily), initiated upon disease onset, improved uveitis clinical score measured by fundoscopy and ameliorated the pathological manifestations of the disease. Shortly after treatment with CF101 A3AR expression levels were down-regulated in the lymph node and spleen cells pointing towards receptor activation. Downstream events included a decrease in PI3K and STAT-1 and proliferation inhibition of IRPB auto-reactive T cells ex vivo. Inhibition of interleukin-2, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production and up-regulation of interleukin-10 was found in cultured splenocytes derived from CF101-treated animals. Overall, the present study data point towards a marked anti-inflammatory effect of CF101 in EAU and support further exploration of this small molecule drug for the treatment of uveitis.

The A3 adenosine receptor (A3AR): therapeutic target and predictive biological marker in rheumatoid arthritis

The Gi protein-associated A3 adenosine receptor (A3AR) is over-expressed in inflammatory cells, and this high expression is also reflected in the peripheral blood mononuclear cells of patients with autoimmune inflammatory diseases such as rheumatoid arthritis, psoriasis, and Crohn’s disease. CF101, a selective agonist with high affinity to the A3AR, is known to induce robust anti-inflammatory effect in experimental animal models of adjuvant-, collagen-, and tropomyosin-induced arthritis. The effect is mediated via a definitive molecular mechanism entailing deregulation of the nuclear factor-κB (NF-κB) and the Wnt signal transduction pathways resulting in apoptosis of inflammatory cells. CF101 was found to be safe and well tolerated in all preclinical, phase I, and phase II human clinical studies. In two phase II clinical studies where CF101 was administered to rheumatoid arthritis (RA) patients as a stand-alone drug, a significant anti-rheumatic effect and a direct significant correlation were found between receptor expression at baseline and patients’ response to the drug, suggesting that A3AR may be utilized as a predictive biomarker. The A3AR is a promising therapeutic target in rheumatoid arthritis and can be used also as a biological marker to predict patients’ response to CF101. This is a unique type of a personalized medicine approach which may pave the way for a safe and efficacious treatment for this patient population.

Targeting the A3 adenosine receptor for glaucoma treatment (Review)

Glaucoma is a worldwide disease and the second leading cause of blindness. Current treatments are associated with a number of side-effects and poor compliance, due to the requirement for treatment administration several times a day. These treatments typically aim to lower intraocular pressure (IOP); however, they are unable to protect retinal ganglion cells (RGCs) from undergoing apoptosis, which is the main cause of vision loss. A3 adenosine receptor (A3AR) agonists have been found to protect normal cells from undergoing apoptosis via the downregulation of death signals. Furthermore, A3AR agonists have been reported to have several ophthalmological effects, including the prevention of ganglion cell apoptosis in vitro and in vivo and anti‑inflammatory effects in experimental models of autoimmune uveitis. CF101, an orally bioavailable A3AR agonist, has been analyzed in dry eye syndrome phase II clinical trials and was identified to be safe and well tolerated. The anti‑inflammatory effect of CF101 was shown to significantly improve corneal staining, tear meniscus and tear break‑up time in dry eye patients. In addition, CF101 was found to decrease IOP in patients. The safety and efficacy of CF101, together with its suitability for oral administration, indicates that it has potential as a candidate drug for the treatment of glaucoma.

A3 Adenosine Receptor Allosteric Modulator Induces an Anti-Inflammatory Effect: In Vivo Studies and Molecular Mechanism of Action

The A3 adenosine receptor (A3AR) is overexpressed in inflammatory cells and in the peripheral blood mononuclear cells of individuals with inflammatory conditions. Agonists to the A3AR are known to induce specific anti-inflammatory effects upon chronic treatment. LUF6000 is an allosteric compound known to modulate the A3AR and render the endogenous ligand adenosine to bind to the receptor with higher affinity. The advantage of allosteric modulators is their capability to target specifically areas where adenosine levels are increased such as inflammatory and tumor sites, whereas normal body cells and tissues are refractory to the allosteric modulators due to low adenosine levels. LUF6000 administration induced anti-inflammatory effect in 3 experimental animal models of rat adjuvant induced arthritis, monoiodoacetate induced osteoarthritis, and concanavalin A induced liver inflammation in mice. The molecular mechanism of action points to deregulation of signaling proteins including PI3K, IKK, IκB, Jak-2, and STAT-1, resulting in decreased levels of NF-κB, known to mediate inflammatory effects. Moreover, LUF6000 induced a slight stimulatory effect on the number of normal white blood cells and neutrophils. The anti-inflammatory effect of LUF6000, mechanism of action, and the differential effects on inflammatory and normal cells position this allosteric modulator as an attractive and unique drug candidate.

Therapeutic Effect of Oral CF101 in Patients with Rheumatoid Arthritis: A Randomized, Double-blind, Placebo-controlled Phase II Study

Background: CF101, an orally bioavailable A3 adenosine receptor (A3AR) agonist, demonstrated very good safety and anti-inflammatory effect in Phase II clinical studies in patients with rheumatoid arthritis (RA) and psoriasis. A3AR expression level in peripheral blood mononuclear cells has been defined as a biological predictive marker, based on a significant correlation found in a former RA Phase II study between its over expression at baseline and positive patients’ response to CF101 treatment. Methods: 79 patients were enrolled to a phase II, multicenter, randomized, double-blind, Placebo controlled, parallel-group study designed to assess the efficacy and safety of CF101 1mg vs. placebo, administered orally twice daily to patients with active RA for 12 weeks. Primary efficacy endpoint was ACR20 response at week 12 and secondary efficacy included ACR 50/70. Patients were enrolled based on A3AR mRNA expression level, utilized as an inclusion criterion. (NCT # NCT01034306) Results: CF101 was found to be safe and well tolerated. CF101 achieved ACR20 of 48.6%, statistically significantly higher than that of the placebo group (25.0%) at week 12 (P=0.0352). CF101 showed superiority in ACR50 and ACR70 values vs. placebo. Interestingly, ACR20, ACR50 and ACR70 response rate at week 12 in a subpopulation with no prior systemic therapy was impressively higher (ACR20 75%) compared to the response of the whole patient population treated with CF101. Conclusions: CF101 reached the primary endpoint in the current study demonstrating clear evidence of efficacy and safety when given orally as monotherapy for 12 weeks in patients with active RA.

Adenosine Receptors and Current Opportunities to Treat Cancer

Adenosine is an endogenous modulator exerting its physiological effects by activating four A1, A2A, A2B, and A3 adenosine receptors. This nucleoside increases in hypoxia that characterizes solid tumors, thus affecting vasculature, immunoescaping, and cancer growth. This chapter offers an updated overview on the current opportunities to treat tumors coming from the adenosinergic field. Several years of research has led to the conclusion that A2A and A3 subtypes are the most promising for drug development. As for A3 receptors, consequent to the efficacy of their agonists in numerous animal models of cancer, the lead compound, Namodenoson, has entered in clinical trials for hepatocellular carcinoma. Phase I results proved its optimal safety profile and efficacy, so that phase II studies are in progress. Specifically, A2A receptor is responsible for immunosuppressive effects, reducing antitumor immunity and promoting immunoescaping of cancer. Therefore, A2A receptor antagonists have been proposed to fight cancer by enhancing immunotherapy, supported also by their safety already demonstrated in clinical trials for Parkinson’s disease. Overall, from these positive results, it may be expected that A3 agonists and A2A antagonists may become future anticancer drugs with the ability to save and improve human health also for diseases with very limited treatment options.