B-cell kinase inhibitors in rheumatoid arthritis

Introduction The pathogenesis of rheumatoid arthritis (RA) involves systemic dysregulation of T and B lymphocytes resulting in immune responses against self-antigens, and ultimately leading to downstream effects such as cartilage destruction, pannus formation, and bone erosion. Clinical evidence for the role of B cells in RA includes the effectiveness of rituximab, an antibody designed to deplete autoreactive B cells. A separate and newer approach to RA treatment involves interference with key intracellular signalling kinases present in B cells, notably spleen tyrosine kinase (SYK) and Bruton’s tyrosine kinase (BTK). Inhibition of SYK, a non-receptor tyrosine kinase predominantly expressed in hematopoietic cells that is involved in the activation of immune cells critical to RA, has proven effective in animal models of inflammation and inflammatory arthritis. BTK is a Tecfamily kinase prominently expressed in B cells, with well-established function in B-cell receptor–mediated activation and survival. Ibrutinib, a first-in-class BTK inhibitor recently approved in mantle cell lymphoma and currently in clinical trials for Bcell malignancies, potently and dosedependently reverses clinical arthritis, preventing cartilage and bone erosion in animal models of arthritis. Here we briefly review available data for SYK and BTK inhibitors in RA and other inflammatory diseases and outline their current clinical status. Conclusion With multiple small-molecule inhibitors being developed to treat chronic inflammation, the future appears promising for an increased variety of treatment options for patients with RA.


Introduction
Rheumatoid arthritis (RA) is a systemic disease characterized by circulating autoantibodies, synovial inflammation, pannus formation, and cartilage and bone destruction in affected joints.Initiation of the disease involves the systemic dysregulation of T and B lymphocytes, which results in immune responses directed against self-antigens.
During the chronic inflammatory phase of the disease, autoantibodies and immune complexes further activate a variety of cells such as neutrophils, monocytes/macrophages, dendritic cells, and mast cells that infiltrate the synovium and release proinflammatory cytokines and matrix metalloproteases, leading to cartilage destruction.Synovial hyperplasia leads to the formation of a pannus that invades the surrounding cartilage and bone, and inflammation enhances the activity of resident osteoclasts, leading to bone erosion 1,2 .
During the multi-step pathogenesis of RA, a host of intracellular signalling kinases are involved in initiation, inflammation, and bone resorption, including Janus kinase 1/3 (JAK1/3) in T cells and, in B cells, spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK).These mediate cell survival and the inflammatory response 2 .As such, RA has been a disease of interest for tyrosine kinase modulation, with small molecule JAK inhibitors leading the way in this approach to the clinical treatment of patients with RA.Modulation of the JAK/signal transducers and activators of transcription (STAT) pathway has proven to be effective in these patients, as evidenced by tofacitinib, which was approved in November 2012 for the treatment of patients with moderateto-severe RA with an inadequate response to or intolerance of methotrexate.Tofacitinib primarily inhibits JAK1 and JAK3, and, to a lesser extent, JAK2 3,4 .Multiple JAK inhibitors targeting different JAK homodimers are also being developed for patients with RA.The introduction of small molecule inhibition to RA treatment has led to clinical trials of other targeted therapies in various phases of development.This review focuses primarily on inhibitors of the B-cell kinases SYK and BTK that are currently in clinical development.

Discussion
The authors have referenced some of their own studies in this review.These referenced studies have been conducted in accordance with the Declaration of Helsinki (1964) and the protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.All human subjects, in these referenced studies, gave informed consent to participate in these studies.

Clinical Significance of B Cells in RA
Ample clinical data support the role of B cells in the pathogenesis of RA.Rituximab, a monoclonal antibody targeting CD20 expressed on B cells, was the first B-cell-targeted therapy to gain approval for treatment of patients with RA.It was developed with the intention of depleting autoreactive B cells in order to reduce the production of pathogenic autoantibodies such as rheumatoid factor 5 .The efficacy of rituximab was established in patients who still had active disease despite being treated with methotrexate 6,7 and in patients who were refractory or intolerant to anti-tumour necrosis B-cell modulation for treatment of patients with RA has been attempted via B-lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), which play important functional roles in B-cell maturation, proliferation, and survival 9 .Elevated BLyS and APRIL levels have been reported in mouse models of RA and in the serum and synovial fluid of patients with RA.
Atacicept, a fully human, recombinant fusion protein of the transmembrane activator and CAML interactor (TACI) receptor and of human immunoglobulin G (IgG), binds to BLyS and APRIL to prevent binding to their respective receptors on B cells.Early-phase clinical trials with atacicept in patients with RA demonstrated decreases in Ig and autoantibody production, whereas its effect on inflammatory markers and clinical outcomes was less profound 9 .Tabalumab, an anti-BLyS human monoclonal antibody 10 , was tested in phase 2 clinical trials for the treatment of patients with active RA and an inadequate response to methotrexate 11,12 .In one 16-week study, significantly more patients in each of the tabalumab dosing groups met American College of Rheumatology 20% improvement criteria (achieved an ACR20 response) than in the placebo group 11 .Observed ACR50 response rates in a longer study suggested that an initial response to treatment was not sustained to week 24 12 .In another phase 2 clinical trial, ACR50 response rates at week 16 were not significantly different between tabalumab-treated patients with active RA with an inadequate response to TNF inhibitors and the placebo group 13 .Subsequently, a phase 3 RA program was discontinued after an interim analysis of efficacy results.
The results of tabalumab and atacicept therapy for patients with active RA suggest that, while B cells may play a role in the perpetuation of the inflammatory response in RA as demonstrated by the studies with rituximab, only part of this response is related to the BLyS/APRIL pathway.Therefore, SYK is a central player in the activation of the immune cells that are critical to the pathogenesis of RA 15,16 .SYK is expressed differentially in RA synovia relative to osteoarthritis, and its inhibition suppresses both inflammation and bone erosions in animal models of RA 17 .SYK inhibitors such as R406/R788 (Table 1) 15,18,19,20,21,22,23 have been shown to be effective in inhibiting immune complex-mediated activations in animal models of inflammation (eg, reverse passive anaphylaxis [RPA] reactions, inflammatory arthritis, collagen-induced arthritis [CIA], and collagen antibody-induced arthritis [CAIA]) 24 .P505-15, a selective SYK inhibitor, was also shown to be effective in CIA and CAIA models 23 (Table 1).

Mechanisms of Action for Kinase
BTK is a Tec-family kinase that is specifically required for B-cell activation following engagement of the BCR 25 .BTK is prominently expressed in B cells, and has a well-established function in BCR-mediated cell activation and survival 26,27,28 .In the lymphoid lineage, expression of BTK is restricted to B cells and is not found in T cells.Functional null mutations of BTK in humans cause the inherited disease X-linked agammaglobulinemia (XLA), which is characterized by a lack of peripheral B cells and very low levels of serum Ig 29,30 .In the mouse, point mutation or deletion of the BTK gene causes X-linked immunodeficiency (xid), with approximately 50% fewer conventional B2 B cells, absent B1 B BTK is activated following FϲγR/FϲεR crosslinking by immune complexes in these cells 32,33,34,35 .Lacking BTK, xid mice have reduced FϲεR-dependent mast-cell degranulation 33 and impaired macrophage function 34,36 , including impaired TNF-α production 37 .Accordingly, xid mice have demonstrated resistance to disease manifestations in CIA models 38 , and BTK has been shown to be important for murine autoantibody production 39,40,41 .The involvement of BTK not only in B cells but also in these myeloid cells provides further support for the potential role of BTK inhibition in treating RA.

SYK Inhibition in RA
Fostamatinib (R788) was developed as an orally bioavailable small-molecule prodrug that is metabolized to R406, an active SYK inhibitor (Table 1).It has been tested in different populations of patients with RA with mixed results 17 .Three phase 2 studies have reported results with fostamatinib treatment in patients with RA.Two of the studies examined the effect of fostamatinib therapy in patients with active RA who had an inadequate response to methotrexate 42,43 .One of these was a 12-week, randomized, placebocontrolled study that enrolled 189 patients in a 3:1 ratio to receive  fostamatinib in an ascending-dose manner versus placebo 42 .The primary endpoint was the ACR20 response rate at week 12. Significantly more patients in two of the fostamatinib dosing groups (100 mg and 150 mg twice daily) achieved an ACR20 response (65% and 72%, respectively) than in the placebo group (38%; P < 0.01 for both doses vs placebo).Higher percentages of patients from these two dosing groups also achieved ACR50 and ACR70 responses and Disease Activity Score in 28 joints (DAS28) scores <2.6 than the placebo group.

Critical review
The second phase 2 study enrolled 457 patients with active RA who had an inadequate response to methotrexate.These patients were randomized to fostamatinib or placebo for 6 months, with the primary outcome of ACR20 response at month 6 43 .The fostamatinib doses of 100 mg twice daily and 150 mg twice daily were significantly superior to placebo with regard to the primary endpoint (ACR20 response rates of 67% and 57%, respectively, vs 35%; P < 0.001 for both doses vs placebo) as well as ACR50 (43% and 32% vs 19%; P < 0.001 for 100-mg dose vs placebo, P = 0.007 for 150-mg dose vs placebo) and ACR70 (28% and 14% vs 10%; P < 0.001 for 100-mg dose vs placebo, P = 0.34 for 150-mg dose vs placebo).This study confirmed that ACR20, ACR50, and ACR70 responses could be maintained for 6 months with fostamatinib therapy in this population of patients with RA.Subsequently, in OSKIRA-1, a phase 3 trial that compared fostamatinib with placebo in 923 patients with RA who had an inadequate response to methotrexate, the individual fostamatinib-treated groups had 24week ACR20 response rates that were significantly higher than those seen in the placebo group; however, the study failed to achieve the co-primary endpoint, which was to achieve a significantly different modified Total Sharp Score at 24 weeks between the fostamatinib and placebo groups 44 .
Fostamatinib has also been tested in patients with active RA who did not respond to biologic agents 45 .A 3month, randomized, placebocontrolled, phase 2 study in a total of 219 patients with RA reported no significant differences achieved in the ACR20, ACR50, or ACR70 response levels at 3 months between patients receiving fostamatinib and those given placebo.
Contrary to the previous phase 2 studies, the primary endpoint of ACR20 response at month 3 was not significantly different between the fostamatinib group and the placebo group.The authors noted that this endpoint may have been compromised by the study's difficultto-treat patient population, as well as by differences in baseline disease activity and distribution of patients with refractory disease between the fostamatinib and placebo groups.
Additional phase 3 trials with fostamatinib include OSKIRA-2, which studied patients with RA who had inadequate responses to diseasemodifying antirheumatic drugs (DMARDs) 46 , and OSKIRA-3, which studied patients with RA who had inadequate responses to methotrexate and a single TNF-α antagonist 47 .Both of these studies demonstrated ACR20 response rates at 24 weeks that were significantly higher for the fostamatinib-treated groups than they were with placebo, but no further phase 3 development with fostamatinib in RA has occurred since the results of these trials 48 .

BTK Inhibition in RA
Selective BTK inhibitors have been shown to block receptor signalling in human B cells 18,19,20,22,49 , although BTK inhibitors are just emerging in clinical trials of patients with RA.Ibrutinib, a first-in-class BTK inhibitor that is approved for the treatment of mantle cell lymphoma in patients who have received at least one prior therapy, is currently being developed clinically to treat patients with various B-cell malignancies 50 .It has also demonstrated therapeutic activity in animal models of RA 49 .Ibrutinib potently and dosedependently reversed clinical arthritis and prevented cartilage and bone erosion in an aggressive late-stage CIA model and a CAIA model (Table 1).In both models, anti-collagen antibodies produced by B cells formed immune complexes that engaged and activated the Fϲ macrophages, neutrophils, and mast cells, resulting in the infiltration or trafficking of these effector cells to antigen sites, such as the joints, where they released mediators that initiated synovitis, bone erosion, and pain 51,52,53 .
In an RPA assay in which neutrophils, macrophages, and mast cells acted as the key effector cells independently of the complement system, ibrutinib potently inhibited acute vasculitis mediated by immune complexes and FϲγR 54 .Collectively, these studies established the dose-dependent inhibition of ibrutinib on immune complex mediated inflammation in vivo.
BTK inhibition by ibrutinib has also been shown to inhibit cytokine and chemokine release upon FϲγR activation of monocytes and macrophages, as well as upon FϲεR activation of mast cells.In both CIA and CAIA models, ibrutinib inhibited clinical inflammation and pannus formation and protected against cartilage and bone damage.Mechanistically, ibrutinib potently inhibited the release of FϲγRinduced cytokines such as TNF-α, interleukin (IL)-6, IL-1β and monocyte chemoattractant protein 1 (MCP-1) in monocytes, albeit less potently in macrophages.Additional benefit in the CIA model might have been derived from the inhibition of RANKL-induced osteoclastic differentiation, as suggested by findings that BTK and Tec are key downstream elements in RANK signalling 55 .and MCP-1, and MCP-1 were also potently suppressed, consistent with in vitro results with primary monocytes and macrophages (Figure 1).
In combination with the in vitro studies with human primary cells and with preclinical data for other inhibitors of BTK (Table 1), these results suggest that BTK inhibition affects multiple cell types that contribute to the pathogenesis of RA, and that it is highly effective in models of several inflammatory diseases, including CIA, CAIA, and RPA.Inhibition of BTK by ibrutinib or other molecules thus constitutes a promising direction for therapeutic trials in diseases such as RA and other immune complex-mediated inflammatory diseases.

Conclusion
Biologic therapies such as rituximab that modulate the contribution of B cells to inflammatory pathways associated with RA are now used in clinical practice.Monoclonal antibodies and related biologics that are administered intravenously or subcutaneously have been the first targeted therapies for the treatment of patients with RA.
The regulatory approval of tofacitinib as a small-molecule inhibitor of the JAK-STAT pathway, however, has opened the path for the development of other targeted oral RA therapies.

Looking
ahead, intracellularly targeted small molecules will likely rival the therapeutic space now inhabited by biologic therapies.Particularly, pathways that have proven efficacy in B-cell hematologic malignancies are viable candidates for modulation of RA, such as BTK signalling.Challenges involved in inhibiting intracellular targets include designing a therapeutic drug that is effective and durable over time while maintaining a safety profile that will limit off-target toxicities.With multiple small-molecule inhibitors being developed to treat chronic inflammation, the future appears promising for an increased variety of treatment options for patients with RA.
) therapy8 .B-cell depletion and improved clinical responses were demonstrated.Rituximab is approved for use in combination with methotrexate in adult patients with moderately to severely active RA who have experienced an inadequate response to one or more TNF therapies.
Inhibitors SYK is a non-receptor tyrosine kinase predominantly expressed in hematopoietic cells.SYK binds to the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of B-cell antigen receptor (BCR) and Fϲγ receptor (FϲγR), and plays a critical role in the signal transduction of early B-cell development and activation.It is also involved in FϲγR and FϲεR signaling of IgG and IgE immune complexmediated activations and inflammations in macrophages and mast cells, respectively; Figure 1 14 .

Figure 1 :
Figure 1: Role of inhibitors of BTK and SYK in the pathogenesis of rheumatoid arthritis.
et al found a nearly complete inhibition of infiltrating cells (neutrophils and macrophages) in the synovial joints of mice.Cytokines and chemokines such IL-1β, IL-6, TNF-α, FOR CITATION PURPOSES: Chu AD, Chang BY.B-cell kinase inhibitors in rheumatoid arthritis.OA Arthritis 2013 Oct 27;1(2):17.