Bruton's Tyrosine Kinase (BTK) Inhibitors and Autoimmune Diseases: Making Sense of BTK Inhibitor Specificity Profiles and Recent Clinical Trial Successes and Failures.

Clinical development of BTK kinase inhibitors for treating autoimmune diseases has lagged behind development of these drugs for treating cancers, due in part from concerns over the lack of selectivity and associated toxicity profiles of first generation drug candidates when used in the long term treatment of immune mediated diseases. Second generation BTK inhibitors have made great strides in limiting off-target activities for distantly related kinases, though they have had variable success at limiting cross-reactivity within the more closely related TEC family of kinases. We investigated the BTK specificity and toxicity profiles, drug properties, disease associated signaling pathways, clinical indications, and trial successes and failures for the 13 BTK inhibitor drug candidates tested in phase 2 or higher clinical trials representing 7 autoimmune and 2 inflammatory immune-mediated diseases. We focused on rheumatoid arthritis (RA), multiple sclerosis (MS), and systemic lupus erythematosus (SLE) where the majority of BTK nonclinical and clinical studies have been reported, with additional information for pemphigus vulgaris (PV), Sjogren's disease (SJ), chronic spontaneous urticaria (CSU), graft versus host disease (GVHD), and asthma included where available. While improved BTK selectivity versus kinases outside the TEC family improved clinical toxicity profiles, less profile distinction was evident within the TEC family. Analysis of genetic associations of RA, MS, and SLE biomarkers with TEC family members revealed that BTK and TEC family members may not be drivers of disease. They are, however, mediators of signaling pathways associated with the pathophysiology of autoimmune diseases. BTK in particular may be associated with B cell and myeloid differentiation as well as autoantibody development implicated in immune mediated diseases. Successes in the clinic for treating RA, MS, PV, ITP, and GVHD, but not for SLE and SJ support the concept that BTK plays an important role in mediating pathogenic processes amenable to therapeutic intervention, depending on the disease. Based on the data collected in this study, we propose that current compound characteristics of BTK inhibitor drug candidates for the treatment of autoimmune diseases have achieved the selectivity, safety, and coverage requirements necessary to deliver therapeutic benefit.

Discovery of the Selective Protein Kinase C-θ Kinase Inhibitor, CC-90005.

The PKC-θ isoform of protein kinase C is selectively expressed in T lymphocytes and plays an important role in the T cell antigen receptor (TCR)-triggered activation of mature T cells, T cell proliferation, and the subsequent release of cytokines such as interleukin-2 (IL-2). Herein, we report the synthesis and structure-activity relationship (SAR) of a novel series of PKC-θ inhibitors. Through a combination of structure-guided design and exploratory SAR, suitable replacements for the basic C4 amine of the original lead (3) were identified. Property-guided design enabled the identification of appropriately substituted C2 groups to afford potent analogs with metabolic stability and permeability to support in vivo testing. With exquisite general kinase selectivity, cellular inhibition of T cell activation as assessed by IL-2 expression, a favorable safety profile, and demonstrated in vivo efficacy in models of acute and chronic T cell activation with oral dosing, CC-90005 (57) was selected for clinical development.

Spebrutinib (CC-292) Affects Markers of B Cell Activation, Chemotaxis, and Osteoclasts in Patients with Rheumatoid Arthritis: Results from a Mechanistic Study.

INTRODUCTION: Spebrutinib (CC-292) is an orally administered, covalent, small-molecule inhibitor of Bruton's tyrosine kinase (BTK), part of the B-cell and Fc receptor signaling pathways. This study evaluated spebrutinib pharmacology and mechanism of action over a 4-week treatment period in patients with active rheumatoid arthritis (RA). METHODS: Primary human B cells, T cells, natural killer cells, macrophages, dendritic cells, basophils, and osteoclasts were treated with spebrutinib in vitro. Clinical pharmacodynamics were studied in 47 patients with active RA on background methotrexate therapy randomized to oral spebrutinib 375 mg/day or placebo. RESULTS: In vitro, spebrutinib inhibited B-cell proliferation more potently than T-cell proliferation and reduced both lymphoid and myeloid cytokine production and degranulation, as well as osteoclastogenesis. Clinical efficacy trended higher in spebrutinib-treated RA patients, with 41.7% (10/24) achieving ≥ 20% improvement in ACR response criteria (ACR20) versus 21.7% (5/23) of placebo patients at week 4 (P = 0.25). Treatment-emergent adverse events were comparable between treatment groups. In spebrutinib-treated patients, median BTK occupancy in peripheral blood was 83%, and significant increases in total CD19+ and mature-naive CD27-CD38-IgD+ B cells and decreases in transitional CD27-CD38+ B cells were observed. Spebrutinib significantly reduced serum chemokines chemokine ligand 13 (CXCL13), macrophage inflammatory protein-1ß (MIP-1ß), and the bone resorption biomarker carboxy-terminal collagen cross-linking telopeptide (CTX-I) (P < 0.05). Clinical response to spebrutinib was associated with lower increases in CD19+ B cells and greater decreases in CXCL13 and MIP-1ß from baseline to week 4. High CD19+ B cells and low CTX-I at baseline were associated with better spebrutinib clinical response. CONCLUSIONS: Spebrutinib inhibited various leukocyte responses in vitro, including those of B cells and osteoclasts. In this small study in RA patients, spebrutinib was well tolerated, showed a downward trend for symptoms, significantly modulated B-cell populations, and reduced markers of chemotaxis and osteoclast activity. TRIAL REGISTRATION: NCT01975610.

Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosus.

OBJECTIVES: IKZF1 and IKZF3 (encoding transcription factors Ikaros and Aiolos) are susceptibility loci for systemic lupus erythematosus (SLE). The pharmacology of iberdomide (CC-220), a cereblon (CRBN) modulator targeting Ikaros and Aiolos, was studied in SLE patient cells and in a phase 1 healthy volunteer study. METHODS: CRBN, IKZF1 and IKZF3 gene expression was measured in peripheral blood mononuclear cells (PBMC) from patients with SLE and healthy volunteers. Ikaros and Aiolos protein levels were measured by Western blot and flow cytometry. Anti-dsDNA and anti-phospholipid autoantibodies were measured in SLE PBMC cultures treated for 7 days with iberdomide. Fifty-six healthy volunteers were randomised to a single dose of iberdomide (0.03-6 mg, n=6 across seven cohorts) or placebo (n=2/cohort). CD19+ B cells, CD3+ T cells and intracellular Aiolos were measured by flow cytometry. Interleukin (IL)-2 and IL-1ß production was stimulated with anti-CD3 and lipopolysaccharide, respectively, in an ex vivo whole blood assay. RESULTS: SLE patient PBMCs expressed significantly higher CRBN (1.5-fold), IKZF1 (2.1-fold) and IKZF3 (4.1-fold) mRNA levels compared with healthy volunteers. Iberdomide significantly reduced Ikaros and Aiolos protein levels in B cells, T cells and monocytes. In SLE PBMC cultures, iberdomide inhibited anti-dsDNA and anti-phospholipid autoantibody production (IC50 ≈10 nM). Single doses of iberdomide (0.3-6 mg) in healthy volunteers decreased intracellular Aiolos (minimum mean per cent of baseline: ≈12%-28% (B cells); ≈0%-33% (T cells)), decreased absolute CD19+ B cells, increased IL-2 and decreased IL-1ß production ex vivo. CONCLUSIONS: These findings demonstrate pharmacodynamic activity of iberdomide and support its further clinical development for the treatment of SLE. TRIAL REGISTRATION NUMBER: NCT01733875; Results.

Aiolos Overexpression in Systemic Lupus Erythematosus B Cell Subtypes and BAFF-Induced Memory B Cell Differentiation Are Reduced by CC-220 Modulation of Cereblon Activity.

BAFF is a B cell survival and maturation factor implicated in the pathogenesis of systemic lupus erythematosus (SLE). In this in vitro study, we describe that soluble BAFF in combination with IL-2 and IL-21 is a T cell contact-independent inducer of human B cell proliferation, plasmablast differentiation, and IgG secretion from circulating CD27+ memory and memory-like CD27-IgD- double-negative (DN) B cells, but not CD27-IgD+ naive B cells. In contrast, soluble CD40L in combination with IL-2 and IL-21 induces these activities in both memory and naive B cells. Blood from healthy donors and SLE patients have similar circulating levels of IL-2, whereas SLE patients exhibit elevated BAFF and DN B cells and reduced IL-21. B cell differentiation transcription factors in memory, DN, and naive B cells in SLE show elevated levels of Aiolos, whereas Ikaros levels are unchanged. Treatment with CC-220, a modulator of the cullin ring ligase 4-cereblon E3 ubiquitin ligase complex, reduces Aiolos and Ikaros protein levels and BAFF- and CD40L-induced proliferation, plasmablast differentiation, and IgG secretion. The observation that the soluble factors BAFF, IL-2, and IL-21 induce memory and DN B cell activation and differentiation has implications for extrafollicular plasmablast development within inflamed tissue. Inhibition of B cell plasmablast differentiation by reduction of Aiolos and Ikaros may have utility in the treatment of SLE, where elevated levels of BAFF and Aiolos may prime CD27+ memory and DN memory-like B cells to become Ab-producing plasmablasts in the presence of BAFF and proinflammatory cytokines.

Teriflunomide attenuates immunopathological changes in the dark agouti rat model of experimental autoimmune encephalomyelitis.

Teriflunomide is an oral disease-modifying therapy recently approved in several locations for relapsing-remitting multiple sclerosis. To gain insight into the effects of teriflunomide, immunocyte population changes were measured during progression of experimental autoimmune encephalomyelitis in Dark Agouti rats. Treatment with teriflunomide attenuated levels of spinal cord-infiltrating T cells, natural killer cells, macrophages, and neutrophils. Teriflunomide also mitigated the disease-induced changes in immune cell populations in the blood and spleen suggesting an inhibitory effect on pathogenic immune responses.

Neurodegenerative disease and the neuroimmune axis (Alzheimer's and Parkinson's disease, and viral infections).

The appearance of activated immune cells and the accumulation of inflammation-associated proteins are common phenomena associated with neurodegenerative diseases. These inflammatory components of central nervous system (CNS) diseases have most often been described in the context of an immune response to damage and cell loss already occurring in the affected brain area. There has, however, been a renewed interest in how the neuroimmune axis might itself be involved in the etiology of these neurodegenerative diseases, particularly in cases involving slow, chronic, progressive neuropathology. This review addresses immune activation in Alzheimer's, Parkinson's, and brain viral infections that may be causative of, rather than responsive to, the observed neuronal loss in these pathologies.

P2X7 receptor modulation of beta-amyloid- and LPS-induced cytokine secretion from human macrophages and microglia.

To test whether extracellular ATP can play a role in the neuroimmunopathology of Alzheimer's disease (AD), we evaluated the capacity of the ATP-binding purinoreceptor, P2X7, to modulate cytokine secretion on cultured human macrophages and microglia pre-activated 24 h with the 42 amino acid beta-amyloid peptide (Abeta(1-42)) or lipopolysaccharide. Thirty minutes of exposure to the selective P2X7 agonist 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) resulted in the secretion of IL-1beta after either Abeta(1-42) or LPS stimulation of human macrophages that was dependent on the concentration of the stimulus used to pre-activate the cells. Further tests on human microglia treated with BzATP (300 microM) resulted in a 1.5- and 3.5-fold enhancement of IL-1alpha and IL-1beta secretion, respectively, from cells pre-activated by 10 microM Abeta(1-42) and a 1.6- and 3.9-fold enhancement of IL-1alpha and IL-1beta secretion, respectively, from cells pre-activated by 1 microg/ml LPS. BzATP induction of IL-1alpha and IL-1beta secretion from microglia was completely reversed by pre-incubation of the cells with the P2X7 antagonist, adenosine 5'-triphosphate 2',3'-acyclic dialcohol (oxidized ATP). In contrast to its effects on IL-1alpha and IL-1beta secretion, BzATP induced TNF-alpha after LPS stimulation, but not after stimulation with Abeta(1-42), induced IL-18 secretion regardless of whether microglia were pre-activated and attenuated IL-6 secretion after either LPS or Abeta(1-42) pre-activation. These results demonstrate that extracellular ATP can modulate Abeta-induced cytokine secretion from human macrophages and microglia and thus may play a role in the neuroimmunopathology of AD.

IL-10 and glucocorticoids inhibit Abeta(1-42)- and lipopolysaccharide-induced pro-inflammatory cytokine and chemokine induction in the central nervous system.

Alzheimer's disease (AD) is characterized by neuropil threads composed of structurally abnormal neurites, neurons containing paired helical filaments of tau protein, and extracellular deposits of amyloid-beta (Abeta) peptide, a protein fragment having neurotoxic and glial immune response activating potential. In the present study, we demonstrate that an acute intracerebroventricular (icv) injection of Abeta(1-42) in the mouse induces a time- and dose-dependent production of IL-1alpha, IL-1beta, IL-6 and MCP-1 in the hippocampus and cortex as measured by ELISA. Cytokine and chemokine levels were maximal at 9 h, with MCP-1 and IL-1alpha remaining elevated over a 24 h period and IL-1beta remaining elevated over a 48 h period. The temporal profile of Abeta-induced cytokine induction differed from that observed for LPS. Following an icv injection of LPS, maximal levels of IL-1alpha, IL-1beta, IL-6 and MCP-1 were attained by 9 h and, except for MCP-1, returned to levels indistinguishable from control at 24 h. MCP-1 remained elevated at 24 h and returned to basal levels at 48 h. In contrast, little production of TNF-alpha was observed under either Abeta or LPS acute stimulus conditions. Treatment with anti-inflammatory agents such as prednisolone, dexamethasone, or IL-10 inhibited both Abeta- and LPS-induced cytokine and chemokine production in the brain. In summary, icv administration of Abeta and LPS induced elevated brain levels of pro-inflammatory cytokines that could be inhibited by immune suppressing drugs and anti-inflammatory proteins, thus providing support for the utility of anti-inflammatory therapeutics in modulating the immunopathology observed in brain inflammatory diseases such as AD.