Background. The A₃ adenosine receptor (A₃AR) is overexpressed in the tumor and in the peripheral blood mononuclear cells of patients with hepatocellular carcinoma (HCC). The orally active drug candidate CF102, an A₃AR agonist, induces apoptosis of HCC cells via deregulation of the Wnt signaling pathway. In this open label phase I/II trial, the safety and clinical effects of CF102 were assessed in patients with advanced unresectable HCC. Methods. The primary objectives of this trial were to examine the safety and pharmacokinetic (PK) behavior of CF102 given orally (1, 5, and 25 mg BID) in 28-day cycles. Evaluation of anti-tumor effects and the utilization of A₃AR as a biological predictive marker of response to CF102 were the secondary objectives. Results. Eighteen patients received CF102—six at each dose level. No serious drug-related adverse events or dose-limiting toxicities were observed. CF102 demonstrated good oral bioavailability and linear PK behavior. Median overall survival in the study population, 67% of whom had received prior sorafenib, was 7.8 months, and for Child Pugh B patients (28%) it was 8.1 months. Stable disease by RECIST was observed in four patients for at least 4 months. CF102 maintained liver function over a 6-month period. A correlation between receptor overexpression levels at baseline and patients' overall survival was found. One of the patients who presented with skin nodules that were biopsy-proven to be HCC metastases prior to the trial showed complete metastasis regression during three months of treatment with CF102. Conclusions. CF102 is safe and well-tolerated, showing favorable PK characteristics in Child Pugh A and B HCC patients, justifying further clinical development.

The anti-inflammatory effect of adenosine was previously found to be mediated via activation of the A₃adenosine 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.

Adenosine is a purine nucleoside that acts as a regulatory molecule by binding to specific G-protein-coupled A1, A_2A, A_2B, and A3 cell surface receptors. We have recently demonstrated that adenosine inhibits tumour cell growth and concomitantly stimulates bone marrow cell proliferation via activation of the A3 adenosine receptor (A3AR). In the present study, we show that a synthetic agonist to the A3AR, CF101, at the low nanomolar concentration range, inhibits HCT-116 human colon carcinoma cell growth. This effect was reversed by the selective A3AR antagonist MRS1523, demonstrating the specificity of the response. CF101 (given orally) was efficacious in inhibiting the development of primary tumours in xenograft and syngeneic models in which mice were inoculated subcutaneously with human HCT-116 or murine CT-26 colon carcinoma cells, respectively. Moreover, CF101 suppressed (50%, P<0.01) colon cancer liver metastases in syngeneic mice inoculated to the spleen with CT-26 cells. The mechanism of action entailed upregulation of interleukin-12 production in the CF101-treated groups and potentiation of NK cell activity. In the HCT-116 xenograft model in which a combined therapy of CF101 and 5-fluorouracyl (5-FU) was examined, an additive antitumour effect was demonstrated. Moreover, CF101 prevented the 5-FU-induced myelotoxicity, resulting in normal values of white blood cell and neutrophil counts. We conclude that the A3AR agonist CF101, a small orally bioavailable molecule, exerts systemic anticancer, antimetastatic, and myeloprotective effects in colon carcinoma-bearing mice, and may serve as an adjuvant treatment to enhance the chemotherapeutic index and prevent myelotoxicity.