Efficacy and safety of remibrutinib, a selective potent oral BTK inhibitor, in Sjögren's syndrome: results from a randomised, double-blind, placebo-controlled phase 2 trial.

OBJECTIVES: To evaluate the safety and efficacy of remibrutinib in patients with moderate-to-severe Sjögren's syndrome (SjS) in a phase 2 randomised, double-blind trial (NCT04035668; LOUiSSE (LOU064 in Sjögren's Syndrome) study). METHODS: Eligible patients fulfilling 2016 American College of Rheumatology/European League Against Rheumatism (EULAR) criteria for SjS, positive for anti-Ro/Sjögren's syndrome-related antigen A antibodies, with moderate-to-severe disease activity (EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) (based on weighted score) ≥ 5, EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) ≥ 5) received remibrutinib (100 mg) either one or two times a day, or placebo for the 24-week study treatment period. The primary endpoint was change from baseline in ESSDAI at week 24. Key secondary endpoints included change from baseline in ESSDAI over time, change from baseline in ESSPRI over time and safety of remibrutinib in SjS. Key exploratory endpoints included changes to the salivary flow rate, soluble biomarkers, blood transcriptomic and serum proteomic profiles. RESULTS: Remibrutinib significantly improved ESSDAI score in patients with SjS over 24 weeks compared with placebo (ΔESSDAI -2.86, p=0.003). No treatment effect was observed in ESSPRI score (ΔESSPRI 0.17, p=0.663). There was a trend towards improvement of unstimulated salivary flow with remibrutinib compared with placebo over 24 weeks. Remibrutinib had a favourable safety profile in patients with SjS over 24 weeks. Remibrutinib induced significant changes in gene expression in blood, and serum protein abundance compared with placebo. CONCLUSIONS: These data show preliminary efficacy and favourable safety of remibrutinib in a phase 2 trial for SjS.

Remibrutinib (LOU064) inhibits neuroinflammation driven by B cells and myeloid cells in preclinical models of multiple sclerosis.

BACKGROUND: Bruton's tyrosine kinase (BTK) is a key signaling node in B cell receptor (BCR) and Fc receptor (FcR) signaling. BTK inhibitors (BTKi) are an emerging oral treatment option for patients suffering from multiple sclerosis (MS). Remibrutinib (LOU064) is a potent, highly selective covalent BTKi with a promising preclinical and clinical profile for MS and other autoimmune or autoallergic indications. METHODS: The efficacy and mechanism of action of remibrutinib was assessed in two different experimental autoimmune encephalomyelitis (EAE) mouse models for MS. The impact of remibrutinib on B cell-driven EAE pathology was determined after immunization with human myelin oligodendrocyte glycoprotein (HuMOG). The efficacy on myeloid cell and microglia driven neuroinflammation was determined in the RatMOG EAE. In addition, we assessed the relationship of efficacy to BTK occupancy in tissue, ex vivo T cell response, as well as single cell RNA-sequencing (scRNA-seq) in brain and spinal cord tissue. RESULTS: Remibrutinib inhibited B cell-dependent HuMOG EAE in dose-dependent manner and strongly reduced neurological symptoms. At the efficacious oral dose of 30 mg/kg, remibrutinib showed strong BTK occupancy in the peripheral immune organs and in the brain of EAE mice. Ex vivo MOG-specific T cell recall response was reduced, but not polyclonal T cell response, indicating absence of non-specific T cell inhibition. Remibrutinib also inhibited RatMOG EAE, suggesting that myeloid cell and microglia inhibition contribute to its efficacy in EAE. Remibrutinib did not reduce B cells, total Ig levels nor MOG-specific antibody response. In brain and spinal cord tissue a clear anti-inflammatory effect in microglia was detected by scRNA-seq. Finally, remibrutinib showed potent inhibition of in vitro immune complex-driven inflammatory response in human microglia. CONCLUSION: Remibrutinib inhibited EAE models by a two-pronged mechanism based on inhibition of pathogenic B cell autoreactivity, as well as direct anti-inflammatory effects in microglia. Remibrutinib showed efficacy in both models in absence of direct B cell depletion, broad T cell inhibition or reduction of total Ig levels. These findings support the view that remibrutinib may represent a novel treatment option for patients with MS.

Remibrutinib inhibits hives effector cells stimulated by serum from chronic urticaria patients independently of FcεR1 expression level and omalizumab clinical response.

BACKGROUND: Despite advances in the treatment of chronic urticaria, in a significant percentage of the patients symptoms are not fully controlled with conventional approaches. New strategies under development include blocking intracellular mediators of mast cell and basophil activation. OBJECTIVE: We aim to investigate the effects of the Bruton's tyrosine kinase (BTK) inhibitor remibrutinib on human blood basophils and CD34+ -derived mast cells activation induced by serum obtained from chronic urticaria patients. METHODS: Twenty-two patients with chronic spontaneous urticaria (mean age 52 years, 27% women) and 22 patients with chronic inducible urticaria (46 years, 27% women) were included in the study together with a sex-matched control group. Patients were classified as responders or non-responders to anti-IgE therapy on the basis of their clinical data, FcεR1a expression on blood basophils and total IgE levels. Changes on CD63 expression-as an activation marker-, were used to evaluate in vitro the response of basophils and mast cells to serum exposure and the inhibitory effects of remibrutinib. RESULTS: Remibrutinib inhibits degranulation induced by IgE cross-linking in mast cells and basophils and also the activation triggered by factors present in the sera of spontaneous and inducible chronic urticaria patients. Patient's serum induces a greater degranulation of effector cells than controls. Activation of mast cells and basophils by patient sera and remibrutinib effects were not related to omalizumab responsiveness. CONCLUSION: Remibrutinib inhibits activation of human basophils and mast cells induced in vitro by exposure to the serum of chronic urticaria patients independently of their response to omalizumab.

Remibrutinib (LOU064): A selective potent oral BTK inhibitor with promising clinical safety and pharmacodynamics in a randomized phase I trial.

Safe and effective new oral therapies for autoimmune, allergic, and inflammatory conditions remain a significant therapeutic need. Here, we investigate the human pharmacokinetics, pharmacodynamics (PDs), and safety of the selective, covalent Bruton's tyrosine kinase (BTK) inhibitor, remibrutinib. Study objectives were explored in randomized single and multiple ascending dose (SAD and MAD, respectively) cohorts with daily doses up to 600 mg, and a crossover food effect (FE) cohort, in adult healthy subjects without (SAD [n =80]/FE [n =12]) or with asymptomatic atopic diathesis (MAD [n =64]). A single oral dose of remibrutinib (0.5-600 mg) was rapidly absorbed (time to maximum concentration = 0.5 h-1.25 h) with an apparent blood clearance of 280-560 L/h and apparent volume of distribution of 400-15,000 L. With multiple doses (q.d. and b.i.d.), no pronounced accumulation of remibrutinib was detected (mean residence time was <3 h). Food intake showed no clinically relevant effect on remibrutinib exposure suggesting no need for dose adaptation. With remibrutinib doses greater than or equal to 30 mg, blood BTK occupancy was greater than 95% for at least 24 h (SAD). With MAD, remibrutinib reached near complete blood BTK occupancy at day 12 predose with greater than or equal to 10 mg q.d. Near complete basophil or skin prick test (SPT) inhibition at day 12 predose was achieved at greater than or equal to 50 mg q.d. for CD63 and at greater than or equal to 100 mg q.d. for SPT. Remibrutinib was well-tolerated at all doses without any dose-limiting toxicity. Remibrutinib showed encouraging blood and skin PDs with a favorable safety profile, supporting further development for diseases driven by mast cells, basophils, and B-cells, such as chronic spontaneous urticaria, allergic asthma, or Sjögren's syndrome.

Discovery of LOU064 (Remibrutinib), a Potent and Highly Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncology indications based on their suboptimal kinase selectivity. We describe the discovery and preclinical profile of LOU064 (remibrutinib, 25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC90 of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in phase 2 clinical studies for chronic spontaneous urticaria and Sjoegren's syndrome.

Design of Potent and Selective Covalent Inhibitors of Bruton's Tyrosine Kinase Targeting an Inactive Conformation.

Bruton's tyrosine kinase (BTK) is a member of the TEC kinase family and is selectively expressed in a subset of immune cells. It is a key regulator of antigen receptor signaling in B cells and of Fc receptor signaling in mast cells and macrophages. A BTK inhibitor will likely have a positive impact on autoimmune diseases which are caused by autoreactive B cells and immune-complex driven inflammation. We report the design, optimization, and characterization of potent and selective covalent BTK inhibitors. Starting from the selective reversible inhibitor 3 binding to an inactive conformation of BTK, we designed covalent irreversible compounds by attaching an electrophilic warhead to reach Cys481. The first prototype 4 covalently modified BTK and showed an excellent kinase selectivity including several Cys-containing kinases, validating the design concept. In addition, this compound blocked FcγR-mediated hypersensitivity in vivo. Optimization of whole blood potency and metabolic stability resulted in compounds such as 8, which maintained the excellent kinase selectivity and showed improved BTK occupancy in vivo.

Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling.

Cardiac hypertrophy accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease, and it is a strong predictor of increased cardiovascular morbidity and mortality. Deletion of bone marrow kinase in chromosome X (Bmx), an arterial nonreceptor tyrosine kinase, has been shown to inhibit cardiac hypertrophy in mice. This finding raised the possibility of therapeutic use of Bmx tyrosine kinase inhibitors, which we have addressed here by analyzing cardiac hypertrophy in gene-targeted mice deficient in Bmx tyrosine kinase activity. We found that angiotensin II (Ang II)-induced cardiac hypertrophy is significantly reduced in mice deficient in Bmx and in mice with inactivated Bmx tyrosine kinase compared with WT mice. Genome-wide transcriptomic profiling showed that Bmx inactivation suppresses myocardial expression of genes related to Ang II-induced inflammatory and extracellular matrix responses whereas expression of RNAs encoding mitochondrial proteins after Ang II administration was maintained in Bmx-inactivated hearts. Very little or no Bmx mRNA was expressed in human cardiomyocytes whereas human cardiac endothelial cells expressed abundant amounts. Ang II stimulation of endothelial cells increased Bmx phosphorylation, and Bmx gene silencing inhibited downstream STAT3 signaling, which has been implicated in cardiac hypertrophy. Furthermore, activation of the mechanistic target of rapamycin complex 1 pathway by Ang II treatment was decreased in the Bmx-deficient hearts. Our results demonstrate that inhibition of the cross-talk between endothelial cells and cardiomyocytes by Bmx inactivation suppresses Ang II-induced signals for cardiac hypertrophy. These results suggest that the endothelial Bmx tyrosine kinase could provide a target to attenuate the development of cardiac hypertrophy.

BMX and its role in inflammation, cardiovascular disease, and cancer.

Bone marrow kinase on chromosome X (BMX) is a cytosolic tyrosine kinase and a member of the TEC kinase family. BMX is expressed in hematopoietic cells of the myeloid lineage where it participates in the immune response. It is also involved in the response to ischemia and pressure overload in the endocardium and the cardiac endothelium. Moreover, BMX is expressed in several types of cancers and very recently has been shown to mediate the survival and tumorigenicity of glioblastoma cancer stem cells. In the inflammatory response BMX regulates the secretion of proinflammatory cytokines induced by TNFα, IL-1ß, and TLR agonists. It is required for the activation of the MAP kinase and NFκB pathways and acts at the level of the essential TAK1/TAB complex. Cellular regulation of the IL-8 promoter by BMX is dependent on membrane localization mediated by its pleckstrin homology domain, as well as on BMX kinase activity. BMX deficiency confers protection from arthritis in a mouse model known to be dependent on macrophages and IL-1ß. Genetic replacement of BMX with a kinase-inactive allele surprisingly restored susceptibility to arthritis, suggesting that in vivo BMX kinase activity can be dispensable. This review summarizes recent advances in the knowledge of BMX biology and their relevance for translational medicine.

The tyrosine kinase BMX is an essential mediator of inflammatory arthritis in a kinase-independent manner.

Inflammatory cytokines like TNF play a central role in autoimmune disorders such as rheumatoid arthritis. We identified the tyrosine kinase bone marrow kinase on chromosome X (BMX) as an essential component of a shared inflammatory signaling pathway. Transient depletion of BMX strongly reduced secretion of IL-8 in cell lines and primary human cells stimulated by TNF, IL-1ß, or TLR agonists. BMX was required for phosphorylation of p38 MAPK and JNK, as well as activation of NF-κB. The following epistasis analysis indicated that BMX acts downstream of or at the same level as the complex TGF-ß activated kinase 1 (TAK1)-TAK1 binding protein. At the cellular level, regulation of the IL-8 promoter required the pleckstrin homology domain of BMX, which could be replaced by an ectopic myristylation signal, indicating a requirement for BMX membrane association. In addition, activation of the IL-8 promoter by in vitro BMX overexpression required its catalytic activity. Genetic ablation of BMX conferred protection in the mouse arthritis model of passive K/BxN serum transfer, confirming that BMX is an essential mediator of inflammation in vivo. However, genetic replacement with a catalytically inactive BMX allele was not protective in the same arthritis animal model. We conclude that BMX is an essential component of inflammatory cytokine signaling and that catalytic, as well as noncatalytic functions of BMX are involved.

CARM1 mediates the ligand-independent and tamoxifen-resistant activation of the estrogen receptor alpha by cAMP.

The estrogen receptor alpha (ERalpha) is activated as a transcription factor by both estrogen and a large variety of other extracellular signals. The mechanisms of this ligand-independent activation, notably by cAMP signaling, are still largely unknown. We now close the gap in the signaling pathway between cAMP and ERalpha. Whereas the direct phosphorylation of ERalpha by the cAMP-activated protein kinase A (PKA) is dispensable, the phosphorylation of the coactivator-associated arginine methyltransferase 1 (CARM1) by PKA at a single serine is necessary and sufficient for direct binding to the unliganded hormone-binding domain (HBD) of ERalpha, and the interaction is necessary for cAMP activation of ERalpha. Sustained PKA activity promoting a constitutive interaction may contribute to tamoxifen resistance of breast tumors. Binding and activation involve a novel regulatory groove of the ERalpha HBD. As a result, depending on the activating signal, ERalpha recruits different coactivator complexes to regulate alternate sets of target genes.

The lysosomal transmembrane protein 9B regulates the activity of inflammatory signaling pathways.

The intracellular signaling pathway by which tumor necrosis factor (TNF) induces its pleiotropic actions is well characterized and includes unique components as well as modules shared with other signaling pathways. In addition to the currently known key effectors, further molecules may however modulate the biological response to TNF. In our attempt to characterize novel regulators of the TNF signaling cascade, we have identified transmembrane protein 9B (TMEM9B, c11orf15) as an important component of TNF signaling and a module shared with the interleukin 1beta (IL-1beta) and Toll-like receptor (TLR) pathways. TMEM9B is a glycosylated protein localized in membranes of the lysosome and partially in early endosomes. The expression of TMEM9B is required for the production of proinflammatory cytokines induced by TNF, IL-1beta, and TLR ligands but not for apoptotic cell death triggered by TNF or Fas ligand. TMEM9B is essential in TNF activation of both the NF-kappaB and MAPK pathways. It acts downstream of RIP1 and upstream of the MAPK and IkappaB kinases at the level of the TAK1 complex. These findings indicate that TMEM9B is a key component of inflammatory signaling pathways and suggest that endosomal or lysosomal compartments regulate these pathways.

Identification of polymorphisms in the human 11beta-hydroxysteroid dehydrogenase type 2 gene promoter: functional characterization and relevance for salt sensitivity.

Reduced activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a role in essential hypertension and the sensitivity of blood pressure to dietary salt. Nonconservative mutations in the coding region are extremely rare and do not explain the variable 11beta-HSD2 activity. We focused therefore on the 5'-regulatory region and identified and characterized the first promoter polymorphisms. Transfections of variants G-209A and G-126A into SW620 cells reduced promoter activity and affinity for activators nuclear factor 1 (NF1) and Sp1. Chromatin immunoprecipitation revealed Sp1, NF1, and glucocorticoid receptor (GR) binding to the HSD11B2 promoter. Dexamethasone induced expression of mRNA and activity of HSD11B2. GR and/or NF1 overexpression increased endogenous HSD11B2 mRNA and activity. GR complexes cooperated with NF1 to activate HSD11B2, an effect diminished in the presence of the G-209A variant. When compared to salt-resistant subjects (96), salt-sensitive volunteers (54) more frequently had the G-209A variant, higher occurrence of alleles A4/A7 of polymorphic microsatellite marker, and higher urinary ratios of cortisol to cortisone metabolites. First, we conclude that the mechanism of glucocorticoid-induced HSD11B2 expression is mainly mediated by cooperation between GR and NF1 on the HSD11B2 promoter and, second, that the newly identified promoter variants reduce activity and cooperation of cognate transcription factors, resulting in diminished HSD11B2 transcription, an effect favoring salt sensitivity.

Evaluation of protease-activated receptor 2 in murine models of arthritis.

OBJECTIVE: Protease-activated receptor 2 (PAR-2) activation has been linked to pro- and antiinflammatory cellular responses. We undertook this study to explore the importance of PAR-2 activation in 4 murine models of arthritis and to analyze the expression of PAR-2 in human arthritic synovium. METHODS: Zymosan-induced arthritis (ZIA), K/BxN serum-induced arthritis, and Freund's complete adjuvant (CFA)-induced arthritis were generated in naive PAR-2(-/-) mice and PAR-2(+/+) littermates. Antigen-induced arthritis (AIA) was generated in immunized mice using methylated bovine serum albumin (mBSA). The severity of arthritis was assessed by clinical scoring, technetium uptake measurement, and histologic analysis. Immune responses to mBSA were also evaluated from AIA. The expression of PAR-2 in synovial tissues from rheumatoid arthritis (RA) and osteoarthritis (OA) patients was compared. RESULTS: In AIA, arthritis was significantly decreased in PAR-2-deficient mice and was associated with decreased levels of anti-mBSA IgG antibodies and lymph node cell proliferation. No difference in arthritis severity was seen in mice with ZIA, K/BxN serum-induced arthritis, and CFA-induced arthritis. Synovial biopsy specimens from RA patients demonstrated significantly increased expression of PAR-2 compared with those from OA patients. CONCLUSION: PAR-2 deficiency was found to modulate articular inflammation in murine models of arthritis that require prior immunization and was associated with reduced levels of anti-mBSA IgG and lymph node cell proliferation in AIA. Expression of PAR-2 in RA synovium was significantly higher than that in OA synovium, and this suggests that PAR-2 is implicated in the pathogenesis of immune-mediated forms of arthritis.

Disparate expression of the PTEN gene: a novel finding in B-cell chronic lymphocytic leukaemia (B-CLL).

One fifth of B-cell chronic lymphocytic leukaemia (B-CLL) patients exhibit loss of heterozygosity (LOH) at 10q23.3, the site of the tumour suppressor PTEN. Microsatellite markers mapped complete LOH to 10q23.3 in 2/41 B-CLL (5%) and allelic imbalances in 6/41 (15%). No PTEN gene mutations were found. PTEN protein expression was not detected in 11 B-CLL (28%), and was reduced in eight patients (20%). LOH or allelic imbalances at 10q23.3 were fairly frequent in B-CLL, but did not encompass the PTEN gene. Nevertheless, PTEN protein may be absent in B-CLL with a normal PTEN genotype, suggesting a role of this phosphatase in the molecular pathology of B-CLL.