Background/Purpose: Janus Kinase (JAK) and Bruton’s tyrosine kinase (BTK) play critical roles in activation and function of T cells and B cells. Dysregulation of this process has been known to cause various immune-related diseases. Tofacitinib, a JAK inhibitor, was originally developed as a JAK3 inhibitor but it showed limited selectivity against JAK1 and JAK2. Although Tofacitinib has proved therapeutic effects in RA, serious side effects such as anemia and neutropenia have been frequently reported. Meanwhile, existing BTK inhibitors have insufficient clinical efficacy, which creates higher demands for treatment with improved efficacy. DWP212525 is a novel and selective JAK3 and BTK inhibitor which may have synergistic effects for the treatment of rheumatoid arthritis and other inflammatory diseases such as pemphigus vulgaris (PV).

Methods: Inhibition of JAK3 and BTK enzyme activity and selectivity against Cys-family kinase group were evaluated by a series of biochemical assays. Cellular activity for target phosphorylation in human T and B cells were measured by Phospho-STAT5 or phospho-BTK cellular kit. BCR dependent CD69 expressions were determined in hPBMC. To measure BTK occupancy, mouse spleen was extracted at several time points up to 24 hours after receiving an oral administration of DWP212525. Occupancy was quantified by the number of unbound BTK in ELISA-based assay using biotinylated-DWP212525, which binds to free active site of target after oral administration of DWP212525. Furthermore, the efficacy of DWP212525 was investigated in the mouse collagen-induced arthritis (CIA) model and mouse PV model, in comparison with selective BTK inhibitor.

Results: We developed a novel and potent dual target inhibitor, DWP212525, with JAK3 IC₅₀ value of 0.2 nM and BTK IC₅₀ value of 1.5 nM. More importantly, DWP212525 is highly selective against JAK3 and BTK, yet has low affinity toward JAK1, JAK2 and EGFR. DWP212525 shows successful inhibition of BCR-dependent CD69 expression in B cells (IC₅₀ value of 132nM). In vivo, oral administration of low-dose DWP212525 (1mg/kg) showed more than 80% BTK occupancy in mouse splenocytes. Interestingly, the plasma concentration of DWP212525 rapidly decreased, but high rate of BTK occupancy was maintained for up to 24 hours. In mouse CIA model, we observed a dose-dependent improvement of arthritis symptoms in the group treated with DWP212525. The ED₅₀ value of DWP212525 is 0.8 mg/kg. In mouse PV model, DWP212525 alleviated the severity of disease index score prevented body weight loss and confirmed that the survival rate was higher than the positive control group treated with a BTK inhibitor and the vehicle group at the end of the 32-day oral administration after the induction of the disease.

Conclusion: We developed a novel, highly potent, and selective covalent inhibitor of JAK3 and BTK, DWP212525. We demonstrated that DWP212525 has potent in vitro and in vivo pharmacological activities compared to Tofacitinib and existing selective BTK inhibitor. These results suggest that

DWP212525 can be more effective due to the addition of JAK3 inhibition than selective BTK inhibitor for the treatment of various autoimmune diseases, including RA and PV.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/discovery-of-dwp212525-a-potent-jak3-and-btk-dual-target-inhibitor-for-the-treatment-of-autoimmune-diseases/