Background/Purpose: Tumor Necrosis Factor-α (TNF-α) is an essential component of the inflammatory stimuli leading to rheumatoid arthritis (RA).  Therapeutics targeting TNF-α are the standard of care but are not always capable of inhibiting disease progression.  Thus, there is an unmet need for novel targets for treatment of RA.  Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase that plays an important role in phagocyte (macrophage and neutrophil) activation.  Phagocytes are known to be important in the pathology of RA; the purpose of this study was to evaluate the impact of Syk inhibition in modulating phagocyte activation. 

Methods: Macrophages were generated by culturing peripheral blood monocytes from healthy human volunteers with M-CSF (5ng/mL) for five days. Neutrophils were isolated from human peripheral blood on a Ficoll gradient and separated from erythrocytes via dextran sedimentation.  As a surrogate for pathogenic autoantibodies in the arthritic synovial joint, immune complex was used for activation of macrophages and neutrophils.  Immune complex was prepared by preincubating chicken egg ovalbumin with goat anti-chicken egg ovalbumin IgG  at 37⁰C.  To mimic integrin-mediated activation, plate-bound poly-RGD (20 µg/mL) was used as a stimulus.  TNF-α release from macrophages was measured via ELISA (R and D Systems).  The SOD-inhibitable oxidative burst response in neutrophils was measured through cytochrome c reduction in a plate-based absorbance assay. Specific Syk inhibitor, PRT062607 (P505-15) was added in vitro to the experimental system containing human macrophages or neutrophils.

Results: Syk inhibition by PRT062607 suppressed inflammatory cytokine release. Cytokine levels, as measured by TNF-α release from macrophages stimulated with immune complex, was potently inhibited by PRT062607 (IC₅₀=0.070µM). Furthermore, PRT062607 treatment was a potent inhibitor of neutrophil-mediated superoxide production in response to various physiologically relevant stimuli (integrin ligation IC₅₀=0.037µM and immune complex stimulation IC₅₀=0.195µM). Interestingly TNF-α alone was able to facilitate neutrophil-mediated superoxide production, and this activity was potently suppressed by PRT062607 (IC₅₀=0.065µM). 

Conclusion: Together these data indicate a novel role for Syk inhibition in controlling TNF-α-mediated inflammation.  These results suggest that in addition to decreasing immune cell activation and inflammation in the joint, Syk inhibition could also partly mimic the effects of an anti- TNF-α therapy.  Thus, Syk inhibition by a small molecule kinase inhibitor has the potential to be a novel mode of therapy in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-specific-inhibitor-of-spleen-tyrosine-kinase-prt062607-is-a-potent-modulator-of-innate-immune-cell-function/