Background Bruton's Tyrosine Kinase (BTK) is a cytoplasmic signaling molecule downstream from a group of cellular receptors important for disease initiation, propagation, and tissue destruction associated with a variety of autoimmune diseases including rheumatoid arthritis. There is strong pre-clinical validation for BTK as a therapeutic target for autoimmune diseases based on multiple animal models. Principia discovered a potent, selective inhibitor of BTK that targets cysteine through a reversible covalent interaction which results in prolonged residence time and inhibition of the target.

Objectives To characterize Principia's novel reversible covalent inhibitor of BTK and demonstrate efficacy in an animal model of rheumatoid arthritis through prolonged target residence time rather than continuous high exposure to the drug.

Methods Biochemical characterization of PRN1008 was performed utilizing Caliper-based kinase assays, TR-FRET-based off-rate assays, and mass-spectrometry-based reversibility assays. Binding of PRN1008 to BTK was assessed in Ramos B cells, human PBMC and rat splenocytes (for PK/PD studies) using a fluorescent probe-based occupancy assay. Functional effects in B cells were measured by effects of PRN1008 on B cell CD69 expression and proliferation induced by anti-IgM. Cellular selectivity for BTK was demonstrated by lack of potency against a range of off-target cell-based assays. The in vivo efficacy of PRN1008 was tested in a rat model of collagen-induced arthritis.

Results PRN1008 was found to be very potent against BTK (IC₅₀ =1.3±0.5 nM) and highly selective when tested against a panel of 251 other kinases. Cysteine targeting of BTK by PRN1008 results in a slow off-rate demonstrated by retention of 79±2% of binding to BTK in PBMC 18 hours after washing away the compound in vitro. The covalent cysteine binding was completely reversible after denaturation of the target. Anti-IgM induced human B cell proliferation (10% serum) and B cell CD69 expression (whole blood) were inhibited by PRN1008 with IC₅₀ of 5±2.4 nM and 123±38 nM, respectively. PRN1008 did not block EGFR signaling in epithelial cells or TCR and calcium flux stimulated T cell activation. PRN1008 also did not block IL-4 stimulation of B cells and did not exhibit cytotoxicity in an epithelial cell line HCT-116. In addition, PRN1008 did not block antibody dependent cellular cytotoxicity in combination with anti-CD20 antibodies allowing for potential combination therapies. In vivo PRN1008 demonstrated enduring pharmacodynamic effects after the compound had cleared from circulation, consistent with extended target residence time. PRN1008 also suppressed collagen-induced arthritis in rats in a dose dependent manner which allowed correlation of target occupancy and disease modification.

Conclusions Our characterization of PRN1008 demonstrated it to be a potent, selective and reversible covalent inhibitor of BTK with extended PD effects in vivo and efficacy in collagen-induced arthritis in rats. These data support continued development of PRN1008 as a therapeutic agent for rheumatoid arthritis.

Disclosure of Interest R. Hill Shareholder of: Principia Biopharma, Employee of: Principia Biopharma, J. Bradshaw Employee of: Principia Biopharma, A. Bisconte Employee of: Principia Biopharma, D. Tam Employee of: Principia Biopharma, T. Owens Shareholder of: Principia Biopharma, Employee of: Principia Biopharma, K. Brameld Shareholder of: Principia Biopharma, Employee of: Principia Biopharma, P. Smith Consultant for: Principia Biopharma, J. Funk Shareholder of: Principia Biopharma, Employee of: Principia Biopharma, D. Goldstein Shareholder of: Principia Biopharma, Employee of: Principia Biopharma, P. Nunn Employee of: Principia Biopharma