P. Fishman

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.

Involvement of Wnt signaling pathway in murine medulloblastoma induced by human neurotropic JC virus

By using the early genome of the human neurotropic polyomavirus, JCV, we have created transgenic animals that develop cerebellar primitive neuroectodermal tumors which model human medulloblastoma. Expression of T-antigen was found in some, but not all, tumor cells, and examination of the clonal cell lines derived from the tumor population showed enhanced tumorigenicity of cells expressing T-antigen in comparison to T-antigen negative cells. Considering the earlier notion on the potential involvement of β-catenin with human medulloblastoma, we investigated various components of the Wnt signaling pathway including β-catenin, its partner transcription factor, LEF-1, and their downstream target gene c-myc in these two cell populations. Immunohistochemical staining of the cells revealed enhanced nuclear appearance of β-catenin in T-antigen positive cells. Results from Western blot showed higher levels of β-catenin and LEF-1 in T-antigen positive cells in comparison to those in T-antigen negative cells. The enhanced level of LEF-1 expression correlated with the increase in DNA binding activity of this protein in nuclear extracts of T-antigen positive cells. Results from Northern and Western blot analyses revealed that the level of c-myc expression is augmented both at the RNA and protein levels in T-antigen positive cells. These observations corroborated results from transfection studies indicating the ability of JCV T-antigen to stimulate c-myc promoter activity. Further, co-transfection experiments revealed that the amount of c-myc and T-antigen protein in tumor cells may dictate the activity of JCV early promoter in these cells. These observations are interesting in light of recent discoveries on the association of JCV with human medulloblastoma and suggest that communication between JCV and the Wnt pathway may be an important event in the genesis of these tumors.

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.

384 Phase 1/2 trial of CF102, a selective A3 adenosine receptor (A3AR) agonist, in patients with hepatocellular carcinoma (HCC)

Methods: The objectives of this trial are to evaluate the safety and pharmacokinetic (PK) behavior of CF102 in HCC patients. Utilizing a “3+3” design, successive cohorts of patients with advanced HCC were enrolled at CF 102 doses of 1, 5, or 25 mg twice daily, given orally in continuous cycles of 28 days each. Progression to a higher-dose cohort was based on first cycle toxicity. Standard safety and PK assessments were performed; αfetoprotein (AFP) levels were obtained each cycle, and tumor imaging was obtained every other cycle.

CF101 enhances the apoptotic effect of chemotherapy on colon and pancreatic carcinoma cell lines: Molecular mechanisms involved

3173 Background: The A3 adenosine Gi protein-coupled receptor is highly expressed in tumor as compared to normal cells. Targeting the A3 adenosine receptor (A3AR) with the highly selective agonist CF101 induces growth inhibition of colon carcinoma and other tumor cell types via down-regulation of PKB/Akt and NF-kB. High expression level of these two signaling proteins is associated with the development of chemo-resistance in various tumor cell types, thus it prompted us to explore whether CF101 would enhance the anti-tumor effect of chemotherapy. METHODS HCT-116 human colon and Bx-PC3 human pancreatic carcinoma cell lines were used. Cells were cultured for 48h in the presence of 5-FU (0.625 μM), oxaliplatin (0.5 μM) or gemcitabine (0.02 mM) and CF101 (10nM) was introduced for an additional 24h. Proliferation assay (MTT) and protein expression profile were conducted at 72h. In vivo studies included nude mice that were inoculated with the tumor cells. Treatment with chemotherapy alone or in combination with CF101 was initiated when tumor reached a size of 150 mm3. Tumor size was monitored each 4 days and study was terminated after 35 days. RESULTS In HCT-116 human colon and Bx-PC3 human pancreatic carcinoma cells, CF101 enhanced the cytotoxic effect of 5-FU, oxaliplatin and gemcitabine in vitro by 20% 25% and 32% respectively (P<0.01). The molecular mechanism involved a marked decrease in the level of PKB/Akt and NF-kB followed by an increase in caspase-3 level resulting in apoptosis, as observed by DNA ladder analysis. In xenograft models of HCT-116 colon carcinoma, CF101 doubled the time to tumor progression (p<0.002) when given in combination with 5-FU. CONCLUSIONS CF101 enhanced the chemotherapeutic effect in vitro and doubled the time to tumor progression in xenograft models. It is thus suggested that CF101 may be further developed as an add-on treatment to chemotherapy for colon and pancreatic carcinoma. [Table: see text].