Association Of Selective Spleen Tyrosine Kinase Inhibitorp505-15 And Rheumatoid Arthritis

BackgroundRheumatoid arthritis (RA) is a chronic, progressive, systemic and autoimmune disease with the characteristics of symmetrical and multi arthritis. RA is a complex degenerative disease and involves many inflammatory and non-inflammatory cell types. RA is characterized by synovial hyperplasia, inflammation and irreversible joint destruction. This causes an irreversible damage to the joints and commonly leads to a significant disability, and it does great harm to human health.The pathology of RA is a complex process and involves various signaling cascades, and eventually activate various downstream signaling pathways. The autoimmune reaction is the central in the pathogenesis of rheumatoid arthritis. In the past few years, significant progress has been made in the pathogenesis of rheumatoid arthritis and related cellular and molecular pathophysiology, and we have also learned that the Spleen tyrosine kinase (Syk) plays a key role in the pathogenesis of RA.Syk is a master regulator in coupling activated immunoreceptors to the mobilization of downstream signal transduction cascades that affect diverse biological functions. And it has a central role in transmission of antigen receptor signals that are critical for autoantibody production and the various innate immune effector functions.Syk plays a very important role in the pathogenesis and progression of RA, which makes Syk inhibitors become a promising candidate for the treatment of RA. Many pharmaceutical and Biotech Corp have shown great interest in the inhibitors. At present, many Syk small molecule inhibitors are in different stages of development for RA therapy and other indications, and many animal experiments are being actively studied.P505-15 is a novel, highly selective inhibitor of spleen tyrosine kinase,and it has not been systematically studied.ObjectiveHere, we investigate the effect of p505-15 on the inhibition of Syk and the effective inhibition of CIA through molecular biology, cytology and animal experiments, in vivo/in vitro experiments to explore whether p505-15 impacted the development of collagen-induced arthritis (CIA) disease in vivo in mice and to evaluate the effect of p505-15 in rheumatoid arthritis.MethodsWestern blot analyses of whole-cell lysates of SUDHL4 cells were performed to evaluate Syk kinase activity and Lyn kinase activity to assess the specificity of p505-15. Human whole blood was stimulated with anti-BCR or PMA to induce signaling and B cell activation,and then was analyzed by flow cytometry to assess the specific inhibition of Syk suppresses BCR signaling and B cell activation. DBA/1 mice received a single oral dose of 15 mg/kg P505-15.Blood was for ex vivo stimulation with anti-mouse IgD antibody. Blood was processed for intracellular phospho-flow cytometry to evaluate BCR signaling to assess the high efficiency and the reversibility.CIA models were established to evaluate the AI, the paw thickness index, the histologic analysis by stained with H&E and Safranin O-fast green for light microscopy. The mouse joint sections were scored for changes in synovial inflammation, and bone erosion,and the synovial macrophages.The serum levels of anti-CII IgGl, IgG2a, TNF-a, IL-6 and IL-17 were measured by ELISA to assess the effect of p505-15 in CIA. And RA synovial cells were cultured and the supernatants were assayed for TNF-a, IL-6 and IL-17 to assess the effect of p505-15 in human RA.Results1. P505-15 inhibited BLNK Tyr84 phosphorylation in a concentrationdependent manner, whereas no inhibition of Lyn activity was observed. Densitometric analysis of Western blots was used to calculate P505-15 concentrations needed for half-maximal inhibition and the IC50 was during 0.16-0.4uM.2. P505-15 suppressed Syk-mediated pERK Tyr204 (mean IC50 S.E.M.,0.27 uM±0.02) and cellular activation of B cells (CD69; mean IC50 S.E.M.,0.28 uM±0.03) but did not inhibit Lyn-dependent signaling to pSyk Y352.3. After oral administration of 15 mg/kg p505-15, more than 50% suppression of Syk activity was observed between 2 and 6 h. Syk activity was reduced by 80% at 3 h, and Syk activity remained after 6 h postdosing, returning to levels of vehicle control treatment by 24 h. And the Lyn activity was not inhibited.4. The mice CIA model was successfully obtained, and the AI score increased sharply at the first 28-30 days, then tended to be flat, and the highest score was 9.2±0.7 on the 36 day after the experiment. In CIA mice, the thickness of the ankle joint was increased with time, and reached to the peak on 32 day after the first immunization, and the highest value was 42±2%. Compared with vehicle controls, Oral p505-15 can decreased the paw swelling degree and AI markedly, the maximum reduction of the foot swelling is 20%, the maximum reduction of AI was 3.4, and the P<0.01.5. In the vehicle-treated group, H&E staining showed that a marked chronic synovitis, with thickened joints and synovial lining and infiltration of leukocytes. In mice treated with 15 mg/kg P505-15, the damage to the joints was significantly reduced and seemed indistinguishable from normal mice. Blinded histopathology scores of bone erosion were reduced.(mean score 1±0.1; versus vehicle 2.9±0.4, P< 0.01.)6. Safranin-O staining indicated that the joints of CIA mice showed evident cartilage destruction, which was significantly reduced in the CIA mice treated with P505-15.and correlated with Photomicrographs of tarsal joints from mice treated with vehicle demonstrate intra-articular neutrophils and fibrin with primarily neutrophils in the surrounding stroma, loss of articular cartilage with an irregular hyaline cartilage surface, and higher magnification reveals loss of cartilage to tidemark and pyknotic chondrocytes. When treated with P505-15 tarsal joints were indistinguishable from those of naive mice. And the mean score was 1.3±0.1; versus vehicle 2.7±0.2, P< 0.01.7. Monoclonal anti-CD68 antibody was used to identify the synovial macrophages using immunohistochemistry. A significant reduction in CD68 expression was observed in the CIA mice treated with P505-15, significantly different from the control group, (mean score 0.8±0.1; versus vehicle 3.1±0.3, P< 0.01.)8. Serum levels of anti-CII IgG1 and IgG2a were measured by ELISA. p505-15 treatment significantly reduced all the anti-CII antibodies relative to vehicle-treated CIA controls. (p<0.01).9. Serum TNF-a, IL-6 and IL-17 levels were measured by ELISA. These results suggest that P505-15 may have a therapeutic effect in terms of CIA severity by inhibiting the production of inflammatory cytokines.(p<0.01).10. RA synovial cells were cultured, and the supernatants were assayed for TNF-a, IL-6 and IL-17. The data indicate that P505-15 treatment significantly reduced the levels of TNF-a, IL-6 and IL-17. These results show that p505-15 attenuates experimental arthritis via inhibiting production of proinflammatory response.ConclusionSyk has the potential to be an important therapeutic target in a variety of diseases of the immune system. P505-15 is a highly specific and potent small-molecule inhibitor of Syk that demonstrates potent immunomodulatory activity in cellular assays and in vivo. We conclude from these data that the specific inhibition of Syk may be a sufficient and safe strategy to control immune function in inflammatory disease. P505-15 is an alternative proposal for the treatment of inflammatory diseases.