Julie DeMartino

RN486, a Selective Bruton's Tyrosine Kinase Inhibitor, Abrogates Immune Hypersensitivity Responses and Arthritis in Rodents

Genetic mutation and pharmacological inhibition of Brutons tyrosine kinase (Btk) both have been shown to prevent the development of collagen-induced arthritis (CIA) in mice, providing a rationale for the development of Btk inhibitors for treating rheumatoid arthritis (RA). In the present study, we characterized a novel Btk inhibitor, 6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one (RN486), in vitro and in rodent models of immune hypersensitivity and arthritis. We demonstrated that RN486 not only potently and selectively inhibited the Btk enzyme, but also displayed functional activities in human cell-based assays in multiple cell types, blocking Fcε receptor cross-linking-induced degranulation in mast cells (IC50 = 2.9 nM), Fcγ receptor engagement-mediated tumor necrosis factor α production in monocytes (IC50 = 7.0 nM), and B cell antigen receptor-induced expression of an activation marker, CD69, in B cells in whole blood (IC50 = 21.0 nM). RN486 displayed similar functional activities in rodent models, effectively preventing type I and type III hypersensitivity responses. More importantly, RN486 produced robust anti-inflammatory and bone-protective effects in mouse CIA and rat adjuvant-induced arthritis (AIA) models. In the AIA model, RN486 inhibited both joint and systemic inflammation either alone or in combination with methotrexate, reducing both paw swelling and inflammatory markers in the blood. Together, our findings not only demonstrate that Btk plays an essential and conserved role in regulating immunoreceptor-mediated immune responses in both humans and rodents, but also provide evidence and mechanistic insights to support the development of selective Btk inhibitors as small-molecule disease-modifying drugs for RA and potentially other autoimmune diseases.

Suppression of Glomerulonephritis in Lupus-Prone NZB × NZW Mice by RN486, a Selective Inhibitor of Bruton's Tyrosine Kinase

OBJECTIVE Brutons tyrosine kinase (BTK) plays a critical role in B cell development and function. We recently described a selective BTK inhibitor, RN486, that blocks B cell receptor (BCR) and Fcγ receptor signaling and is efficacious in animal models of arthritis. The aim of this study was to examine the potential efficacy of BTK in systemic lupus erythematosus (SLE), using an NZB × NZW mouse model of spontaneous SLE. METHODS Mice received RN486 or its vehicle (administered in chow) at a final concentration of 30 mg/kg for 8 weeks, starting at 32 weeks of age. RESULTS The administration of RN486 completely stopped disease progression, as determined by histologic and functional analyses of glomerular nephritis. The efficacy was associated with striking inhibition of B cell activation, as demonstrated by a significant reduction in CD69 expression in response to BCR crosslinking. RN486 markedly reduced the secretion of IgG anti-double-stranded DNA (anti-dsDNA) secretion, as determined by enzyme-linked immunosorbent and enzyme-linked immunospot assays. Flow cytometric analysis demonstrated depletion of CD138(high) B220(low) plasma cells in the spleen. RN486 inhibited secretion of IgG anti-dsDNA but not IgM anti-dsDNA, suggesting that pharmacologic blockade of BTK resembles the reported transgenic expression of low levels of endogenous BTK in B cells. In addition, RN486 may also impact the effector function of autoantibodies, as evidenced by a significant reduction in immune complex-mediated activation of human monocytes in vitro and down-regulation of the expression of macrophage-related and interferon-inducible genes in both the kidneys and spleens of treated mice. CONCLUSION Collectively, our data suggest that BTK inhibitors may simultaneously target autoantibody-producing and effector cells in SLE, thus constituting a promising therapeutic alternative for this disease.

Targeting the SYK–BTK axis for the treatment of immunological and hematological disorders: Recent progress and therapeutic perspectives

Spleen Tyrosine Kinase (SYK) and Brutons Tyrosine Kinase (BTK) are non-receptor cytoplasmic tyrosine kinases that are primarily expressed in cells of hematopoietic lineage. Both are key mediators in coupling activated immunoreceptors to downstream signaling events that affect diverse biological functions, from cellular proliferation, differentiation and adhesion to innate and adaptive immune responses. As such, pharmacological inhibitors of SYK or BTK are being actively pursued as potential immunomodulatory agents for the treatment of autoimmune and inflammatory disorders. Deregulation of SYK or BTK activity has also been implicated in certain hematological malignancies. To date, from a clinical perspective, pharmacological inhibition of SYK activity has demonstrated encouraging efficacy in patients with rheumatoid arthritis (RA), while patients with relapsed or refractory chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) have benefited from covalent inhibitors of BTK in early clinical studies. Here, we review and discuss recent insights into the emerging role of the SYK-BTK axis in innate immune cell function as well as in the maintenance of survival and homing signals for tumor cell progression. The current progress on the clinical development of SYK and BTK inhibitors is also highlighted.

Characterization of a novel CRAC inhibitor that potently blocks human T cell activation and effector functions

Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC current inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4(+) T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (mixed lymphocyte reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and another calcineurin inhibitor FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC current inhibitor that potently inhibits human T cell functions.

BET Bromodomain Proteins Mediate Downstream Signaling Events following Growth Factor Stimulation in Human Lung Fibroblasts and Are Involved in Bleomycin-Induced Pulmonary Fibrosis

Epigenetic alterations, such as histone acetylation, regulate the signaling outcomes and phenotypic responses of fibroblasts after growth factor stimulation. The bromodomain and extra-terminal domain–containing proteins (Brd) bind to acetylated histone residues, resulting in recruitment of components of the transcriptional machinery and subsequent gene transcription. Given the central importance of fibroblasts in tissue fibrosis, this study sought to determine the role of Brd proteins in human lung fibroblasts (LFs) after growth factor stimulation and in the murine bleomycin model of lung fibrosis. Using small interfering RNA against human Brd2 and Brd4 and pharmacologic Brd inhibitors, this study found that Brd2 and Brd4 are essential in mediating the phenotypic responses of LFs downstream of multiple growth factor pathways. Growth factor stimulation of LFs causes increased histone acetylation, association of Brd4 with growth factor–responsive genes, and enhanced transcription of these genes that could be attenuated with pharmacologic Brd inhibitors. Of note, lung fibrosis induced after intratracheal bleomycin challenge in mice could be prevented by pretreatment of animals with pharmacologic inhibitors of Brd proteins. This study is the first demonstration of a role for Brd2 and Brd4 proteins in mediating the responses of LFs after growth factor stimulation and in driving the induction of lung fibrosis in mice in response to bleomycin challenge.

Selective inhibition of spleen tyrosine kinase (SYK) with a novel orally bioavailable small molecule inhibitor, RO9021, impinges on various innate and adaptive immune responses: implications for SYK inhibitors in autoimmune disease therapy

IntroductionSpleen tyrosine kinase (SYK) is a key integrator of intracellular signals triggered by activated immunoreceptors, including Bcell receptors (BCR) and Fc receptors, which are important for the development and function of lymphoid cells. Given the clinical efficacy of Bcell depletion in the treatment of rheumatoid arthritis and multiple sclerosis, pharmacological modulation of Bcells using orally active small molecules that selectively target SYK presents an attractive alternative therapeutic strategy.MethodsA SYK inhibitor was developed and assayed in various in vitro systems and in the mouse model of collagen-induced arthritis (mCIA).ResultsA novel ATP-competitive inhibitor of SYK, 6-[(1R,2S)-2-Amino-cyclohexylamino]-4-(5,6-dimethyl-pyridin-2-ylamino)-pyridazine-3-carboxylic acid amide, designated RO9021, with an adequate kinase selectivity profile and oral bioavailability, was developed. In addition to suppression of BCR signaling in human peripheral blood mononuclear cells (PBMC) and whole blood, FcγR signaling in human monocytes, and FcϵR signaling in human mast cells, RO9021 blocked osteoclastogenesis from mouse bone marrow macrophages in vitro. Interestingly, Toll-like Receptor (TLR) 9 signaling in human Bcells was inhibited by RO9021, resulting in decreased levels of plasmablasts, immunoglobulin (Ig) M and IgG upon B-cell differentiation. RO9021 also potently inhibited type I interferon production by human plasmacytoid dendritic cells (pDC) upon TLR9 activation. This effect is specific to TLR9 as RO9021 did not inhibit TLR4- or JAK-STAT-mediated signaling. Finally, oral administration of RO9021 inhibited arthritis progression in the mCIA model, with observable pharmacokinetics (PK)-pharmacodynamic (PD) correlation.ConclusionsInhibition of SYK kinase activity impinges on various innate and adaptive immune responses. RO9021 could serve as a starting point for the development of selective SYK inhibitors for the treatment of inflammation-related and autoimmune-related disorders.

Bruton's Tyrosine Kinase mediates platelet receptor-induced generation of microparticles: A potential mechanism for amplification of inflammatory responses in rheumatoid arthritis synovial joints

Platelet microparticles (pMPs) are small membrane-coated vesicles that are released from the plasma membrane upon platelet activation. In the joint fluid of patients with rheumatoid arthritis, pMP can interact with and activate fibroblast-like synoviocytes (FLS), which are important effector cells that mediate both immune activation and joint destruction. The signaling process by which engagement of glycoprotein VI (GPVI), a surface glycoprotein receptor for collagen which is expressed on platelets, triggers pMP generation is poorly understood, but has been suggested to involve Spleen Tyrosine Kinase (SYK), best known as an upstream activator of Brutons Tyrosine Kinase (BTK) in B cells. In this study, we showed that activation of human platelets triggered by convulxin or collagen, specific ligands for GPVI receptor, or alternatively by antibody-mediated cross-linking of another platelet receptor, C type lectin-like receptor 2 (CLEC2), resulted in phosphorylation of BTK and downstream effector, phospholipase Cγ2 (PLCγ2). A potent and selective BTK inhibitor, RN486, inhibited GPVI- or CLEC2-mediated PLCγ2 phosphorylation and pMP production in a dose-dependent manner. BTK is also an essential effector of B cell receptor (BCR)-induced B cell signaling. Consistent with the biology, the IC50s of BTK inhibitors with varying potencies in a BCR-dependent B cell activation marker assay correlated with those in the GPVI-mediated PLCγ2 phosphorylation. In a co-culture system consisting of human primary synovial FLS and activated human platelets, convulxin stimulation resulted in elevated production of pro-inflammatory cytokines, IL-6 and IL-8, an effect which was dose-dependently blocked by RN486. The effects are specific as RN486 abrogated platelet aggregation induced by GPVI ligands but not by other platelet surface receptor agonists. Taken together, our data further support the potential therapeutic utility of BTK inhibitors in RA therapy, by inhibiting GPVI-mediated platelet activation and thus subsequent amplification of inflammation driven by pMP-induced FLS cytokines production.

Natural regulatory T cells are resistant to calcium release-activated calcium (CRAC/ORAI) channel inhibition

Organ transplant patients are often treated with immunosuppressants, such as the calcineurin phosphatase inhibitor, cyclosporin A, to block T cell-mediated graft rejection. The calcium release-activated calcium (CRAC/ORAI) channels, which act upstream of calcineurin, are essential for calcium entry and CD4(+) T-cell activation. Although cyclosporine A has also been shown to inhibit FoxP3(+) Tregs both in vitro and in vivo, the role of ORAI channel inhibition in natural Tregs (nTregs) or inducible Tregs (iTregs) has not been investigated. We found that, despite inhibition of calcium influx through the ORAI channels, ORAI channel inhibitors were unable to repress FoxP3 expression in mouse and human nTregs, whereas FoxP3 expression was inhibited in iTregs. In contrast, cyclosporin A inhibited FoxP3 expression in both nTregs and iTregs. We also generated mice with a T cell-specific, conditional knockout of ORAI1 and found that the mice have normal nTreg development and suppressive activity. Moreover, iTregs derived from ORAI1 conditional knockout mice develop normally and are still susceptible to ORAI channel inhibition. Our data indicate that unlike CD4(+) T cells and iTregs, nTregs are resistant to ORAI-mediated inhibition. Targeting ORAI channels potentially offers a novel way to inhibit pathologic T cells, while sparing nTreg-mediated tolerance.

Btk inhibition suppresses agonist-induced human macrophage activation and inflammatory gene expression in RA synovial tissue explants

Objectives Brutons tyrosine kinase (Btk) is required for B lymphocyte and myeloid cell contributions to pathology in murine models of arthritis. Here, we examined the potential contributions of synovial Btk expression and activation to inflammation in rheumatoid arthritis (RA). Materials and methods Btk was detected by immunohistochemistry and digital image analysis in synovial tissue from biologically naive RA (n=16) and psoriatic arthritis (PsA) (n=12) patients. Cell populations expressing Btk were identified by immunofluorescent double labelling confocal microscopy, quantitative (q-) PCR and immunoblotting. The effects of a Btk-specific inhibitor, RN486, on gene expression in human macrophages and RA synovial tissue explants (n=8) were assessed by qPCR, ELISA and single-plex assays. Results Btk was expressed at equivalent levels in RA and PsA synovial tissue, restricted to B lymphocytes, monocytes, macrophages and mast cells. RN486 significantly inhibited macrophage IL-6 production induced by Fc receptor and CD40 ligation. RN486 also reduced mRNA expression of overlapping gene sets induced by IgG, CD40 ligand (CD40L) and RA synovial fluid, and significantly suppressed macrophage production of CD40L-induced IL-8, TNF, MMP-1 and MMP-10, LPS-induced MMP-1, MMP-7 and MMP-10 production, and spontaneous production of IL-6, PDGF, CXCL-9 and MMP-1 by RA synovial explants. Conclusions Btk is expressed equivalently in RA and PsA synovial tissue, primarily in macrophages. Btk activity is needed to drive macrophage activation in response to multiple agonists relevant to inflammatory arthritis, and promotes RA synovial tissue cytokine and MMP production. Pharmacological targeting of Btk may be of therapeutic benefit in the treatment of RA and other inflammatory diseases.

Development of a Pharmacodynamic Assay Based on PLCγ2 Phosphorylation for Quantifying Spleen Tyrosine Kinase (SYK)–Bruton’s Tyrosine Kinase (BTK) Signaling

Spleen tyrosine kinase (SYK) and Bruton’s tyrosine kinase (BTK) are key mediators in coupling cell surface receptors, such as the B-cell receptor (BCR), to downstream signaling events affecting diverse biological functions. There is therefore tremendous interest in the development of pharmacological inhibitors targeting the SYK-BTK axis for the treatment of inflammatory disorders and hematological malignancies. A good pharmacodynamic (PD) assay, ideally a blood-based assay that measures proximal events, is warranted for evaluation of such inhibitors. In platelets, collagen-induced activation of membrane glycoprotein GPVI is dependent on the SYK-BTK axis. Here, we report the development of a novel immunoassay that uses the dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) to measure GPVI-mediated phosphorylation of phospholipase C γ2 (PLCγ2), a direct substrate of SYK and BTK, in platelets. The assay was validated using SYK or BTK inhibitors and generated IC50 correlated with those from the BCR-induced B-cell activation assay. Furthermore, this assay showed good stability and uniformity over a period of 24 h in different donors. Interestingly, compound IC50 values using blood from patients with rheumatoid arthritis were slightly higher compared with those produced using samples from healthy donors. This novel platelet PLCγ2 phosphorylation-based immunoassay should serve as a promising PD assay for preclinical and clinical development of inhibitors targeting the SYK-BTK axis.