Discovery of Reversible Covalent Bruton's Tyrosine Kinase Inhibitors PRN473 and PRN1008 (Rilzabrutinib).

Bruton's tyrosine kinase (BTK), a Tec family tyrosine kinase, is critical in immune pathways as an essential intracellular signaling element, participating in both adaptive and immune responses. Currently approved BTK inhibitors are irreversible covalent inhibitors and limited to oncology indications. Herein, we describe the design of covalent reversible BTK inhibitors and the discoveries of PRN473 (11) and rilzabrutinib (PRN1008, 12). These compounds have exhibited potent and durable inhibition of BTK, in vivo efficacy in rodent arthritis models, and clinical efficacy in canine pemphigus foliaceus. Compound 11 has completed phase 1 trials as a topical agent, and 12 is in phase 3 trials for pemphigus vulgaris and immune thrombocytopenia.

Phase 1 clinical trial evaluating safety, exposure and pharmacodynamics of BTK inhibitor tolebrutinib (PRN2246, SAR442168).

Bruton's tyrosine kinase (BTK), expressed in B cells and cells of innate immunity, including microglia, is an essential signaling element downstream of the B-cell receptor and Fc-receptors. Tolebrutinib (PRN2246, SAR442168) is a potent BTK inhibitor that covalently binds the kinase, resulting in durable inhibition with the potential to target inflammation in the periphery and central nervous system (CNS). Tolebrutinib crosses the blood-brain barrier and potently inhibits BTK in microglial cells isolated from the CNS. A first-in-human randomized, double-blind, placebo-controlled study of tolebrutinib was conducted. The trial design consisted of five single ascending dose arms with oral administration of a single dose of 5, 15, 30, 60, and 120 mg (n = 6 per arm, n = 2 placebo), five multiple ascending dose arms with oral administration of 7.5, 15, 30, 60, and 90 mg (n = 8 per arm, n = 2 placebo) over 10 days, and one arm (n = 4) in which cerebral spinal fluid (CSF) exposure was measured 2 h after a single 120 mg dose. Tolebrutinib was well-tolerated in the study and all treatment-related treatment emergent adverse events were mild. Tolebrutinib was rapidly absorbed following oral administration with a rapid half-life of ~ 2 h. Peripheral BTK occupancy was assessed at various timepoints by an enzyme-linked immunosorbent assay-based readout using an irreversible probe. Assessments demonstrated extensive and prolonged peripheral BTK occupancy at steady-state with once daily doses as low as 7.5 mg. Further, CSF exposure was demonstrated 2 h after administration at 120 mg.

Preclinical Mechanisms of Topical PRN473, a Bruton Tyrosine Kinase Inhibitor, in Immune-Mediated Skin Disease Models.

The expression of Bruton tyrosine kinase (BTK) in B cells and innate immune cells provides essential downstream signaling for BCR, Fc receptors, and other innate immune cell pathways. The topical covalent BTK inhibitor PRN473 has shown durable, reversible BTK occupancy with rapid on-rate and slow off-rate binding kinetics and long residence time, resulting in prolonged, localized efficacy with low systemic exposure in vivo. Mechanisms of PRN473 include inhibition of IgE (FcεR)-mediated activation of mast cells and basophils, IgG (FcγR)-mediated activation of monocytes, and neutrophil migration. In vivo, oral PRN473 was efficacious and well tolerated in the treatment of canine pemphigus foliaceus. In this study, we evaluated in vitro selectivity and functionality, in vivo skin Ab inflammatory responses, and systemic pharmacology with topically administered PRN473. Significant dose-dependent inhibition of IgG-mediated passive Arthus reaction in rats was observed with topical PRN473 and was maintained when given 16 h prior to challenge, reinforcing extended activity with once-daily administration. Similarly, topical PRN473 resulted in significant dose-dependent inhibition of the mouse passive cutaneous anaphylaxis IgE-mediated reaction. Multiday treatment with topical PRN473 in rodents resulted in low-to-no systemic accumulation, suggesting that efficacy was mainly due to localized exposure. Reduced skin Ab inflammatory activity was also confirmed with oral PRN473. These preclinical studies provide a strong biologic basis for targeting innate immune cell responses locally in the skin, with rapid onset of action following once-daily topical PRN473 administration and minimal systemic exposure. Dose-dependent inhibition in these preclinical models of immune-mediated skin diseases support future clinical studies.

Preclinical Efficacy and Anti-Inflammatory Mechanisms of Action of the Bruton Tyrosine Kinase Inhibitor Rilzabrutinib for Immune-Mediated Disease.

Bruton tyrosine kinase (BTK) is expressed in B cells and innate immune cells, acting as an essential signaling element in multiple immune cell pathways. Selective BTK inhibition has the potential to target multiple immune-mediated disease pathways. Rilzabrutinib is an oral, reversible, covalent BTK inhibitor designed for immune-mediated diseases. We examined the pharmacodynamic profile of rilzabrutinib and its preclinical mechanisms of action. In addition to potent and selective BTK enzyme and cellular activity, rilzabrutinib inhibited activation and inflammatory activities of B cells and innate cells such as macrophages, basophils, mast cells, and neutrophils, without cell death (in human and rodent assay systems). Rilzabrutinib demonstrated dose-dependent improvement of clinical scores and joint pathology in a rat model of collagen-induced arthritis and demonstrated reductions in autoantibody-mediated FcγR signaling in vitro and in vivo, with blockade of rat Arthus reaction, kidney protection in mouse Ab-induced nephritis, and reduction in platelet loss in mouse immune thrombocytopenia. Additionally, rilzabrutinib inhibited IgE-mediated, FcεR-dependent immune mechanisms in human basophils and mast cell-dependent mouse models. In canines with naturally occurring pemphigus, rilzabrutinib treatment resulted in rapid clinical improvement demonstrated by anti-inflammatory effects visible within 2 wk and all animals proceeding to complete or substantial disease control. Rilzabrutinib is characterized by reversible covalent BTK binding, long BTK residence time with low systemic exposure, and multiple mechanistic and biological effects on immune cells. Rilzabrutinib's unique characteristics and promising efficacy and safety profile support clinical development of rilzabrutinib for a broad array of immune-mediated diseases.

Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity.

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible proteasome variant comprising the proteolytic subunits LMP2 (ß1i), MECL-1 (ß2i), and LMP7 (ß5i). Targeting the immunoproteasome in pre-clinical models of autoimmune diseases with the epoxyketone inhibitor ONX 0914 has proven to be effective. ONX 0914 was previously described as a selective LMP7 inhibitor. Here, we show that PRN1126, developed as an exclusively LMP7-specific inhibitor, has limited effects on IL-6 secretion, experimental colitis, and experimental autoimmune encephalomyelitis (EAE). We demonstrate that prolonged exposure of cells with ONX 0914 leads to inhibition of both LMP7 and LMP2. Co-inhibition of LMP7 and LMP2 with PRN1126 and LMP2 inhibitors LU-001i or ML604440 impairs MHC class I cell surface expression, IL-6 secretion, and differentiation of naïve T helper cells to T helper 17 cells, and strongly ameliorates disease in experimental colitis and EAE. Hence, co-inhibition of LMP2 and LMP7 appears to be synergistic and advantageous for the treatment of autoimmune diseases.

PRN473, an inhibitor of Bruton's tyrosine kinase, inhibits neutrophil recruitment via inhibition of macrophage antigen-1 signalling.

BACKGROUND AND PURPOSE: Following inflammatory stimuli, neutrophils are recruited to sites of inflammation and exert effector functions that often have deleterious effects on tissue integrity, which can lead to organ failure. Bruton's tyrosine kinase (Btk) is expressed in neutrophils and constitutes a promising pharmacological target for neutrophil-mediated tissue damage. Here, we evaluate a selective reversible inhibitor of Btk, PRN473, for its ability to dampen neutrophil influx via inhibition of adhesion receptor signalling pathways. EXPERIMENTAL APPROACH: In vitro assays were used to assess fMLP receptor 1 (Fpr-1)-mediated binding of ligands to the adhesion receptors macrophage antigen-1 (Mac-1) and lymphocyte function antigen-1. Intravital microscopy of the murine cremaster was used to evaluate post-adhesion strengthening and endoluminal crawling. Finally, neutrophil influx was visualized in a clinically relevant model of sterile liver injury in vivo. Btk knockout animals were used as points of reference for Btk functions. KEY RESULTS: Pharmacological inhibition of Btk by PRN473 reduced fMLP-induced phosphorylation of Btk and Mac-1 activation. Biochemical experiments demonstrated the specificity of the inhibitor. PRN473 (20 mg·kg-1 ) significantly reduced intravascular crawling and neutrophil recruitment into inflamed tissue in a model of sterile liver injury, down to levels seen in Btk-deficient animals. A higher dose did not provide additional reduction of intravascular crawling and neutrophil recruitment. CONCLUSIONS AND IMPLICATIONS: PRN473, a highly selective inhibitor of Btk, potently attenuates sterile liver injury by inhibiting the activation of the ß2 -integrin Mac-1 and subsequently neutrophil recruitment into inflamed tissue.

Cutting edge: Modulation of intestinal autoimmunity and IL-2 signaling by sphingosine kinase 2 independent of sphingosine 1-phosphate.

Sphingosine kinase (Sphk) phosphorylates sphingosine into sphingosine-1-phosphate (S1P), but its recently identified isoform Sphk2 has been suggested to have distinct subcellular localization and substrate specificity. We demonstrate here that, surprisingly, Sphk2(-/-) CD4(+) T cells exhibit a hyperactivated phenotype with significantly enhanced proliferation and cytokine secretion in response to IL-2 as well as reduced sensitivity to regulatory T cell-mediated suppression in vitro, apparently independent of effects upon S1P. Such findings appear to reflect a requirement for Sphk2 to suppress IL-2 signaling because, in Sphk2(-/-) CD4(+) T cells, IL-2 induced abnormally accentuated STAT5 phosphorylation and small interfering RNA knockdown of STAT5 abrogated their hyperactive phenotype. This pathway physiologically modulates autoinflammatory responses, because Sphk2(-/-) T cells induced more rapid and robust inflammatory bowel disease in scid recipients. Thus, Sphk2 regulates IL-2 pathways in T cells, and the modulation of Sphk2 activity may be of therapeutic utility in inflammatory and/or infectious diseases.

IL-25 regulates Th17 function in autoimmune inflammation.

Interleukin (IL)-25 is a member of the IL-17 family of cytokines. However, unlike the other members of this family, IL-25 promotes T helper (Th) 2 responses. We now show that IL-25 also regulates the development of autoimmune inflammation mediated by IL-17-producing T cells. We have generated IL-25-deficient (il25-/-) mice and found that they are highly susceptible to experimental autoimmune encephalomyelitis (EAE). The accelerated disease in the il25-/- mice is associated with an increase of IL-23 in the periphery and a subsequent increase in the number of inflammatory IL-17-, IFNgamma-, and TNF-producing T cells that invade the central nervous system. Neutralization of IL-17 but not IFNgamma in il25-/- mice prevented EAE, suggesting that IL-17 is a major disease-promoting factor. IL-25 treatment at several time points during a relapse-remitting model or chronic model of EAE completely suppressed disease. IL-25 treatment induced elevated production of IL-13, which is required for suppression of Th17 responses by direct inhibition of IL-23, IL-1beta, and IL-6 expression in activated dendritic cells. Thus, IL-25 and IL-17, being members of the same cytokine family, play opposing roles in the pathogenesis of organ-specific autoimmunity.

Anti-IL-23 therapy inhibits multiple inflammatory pathways and ameliorates autoimmune encephalomyelitis.

IL-23 is a member of the IL-12 cytokine family that drives a highly pathogenic T cell population involved in the initiation of autoimmune diseases. We have shown that IL-23-dependent, pathogenic T cells produced IL-17 A, IL-17 F, IL-6, and TNF but not IFN-gamma or IL-4. We now show that T-bet and STAT1 transcription factors are not required for the initial production of IL-17. However, optimal IL-17 production in response to IL-23 stimulation appears to require the presence of T-bet. To explore the clinical efficacy of targeting the IL-23 immune pathway, we generated anti-IL-23p19-specific antibodies and tested to determine whether blocking IL-23 function can inhibit EAE, a preclinical animal model of human multiple sclerosis. Anti-IL-23p19 treatment reduced the serum level of IL-17 as well as CNS expression of IFN-gamma, IP-10, IL-17, IL-6, and TNF mRNA. In addition, therapeutic treatment with anti-IL-23p19 during active disease inhibited proteolipid protein (PLP) epitope spreading and prevented subsequent disease relapse. Thus, therapeutic targeting of IL-23 effectively inhibited multiple inflammatory pathways that are critical for driving CNS autoimmune inflammation.

IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.

Interleukin (IL)-23 is a heterodimeric cytokine composed of a unique p19 subunit, and a common p40 subunit shared with IL-12. IL-12 is important for the development of T helper (Th)1 cells that are essential for host defense and tumor suppression. In contrast, IL-23 does not promote the development of interferon-gamma-producing Th1 cells, but is one of the essential factors required for the expansion of a pathogenic CD4(+) T cell population, which is characterized by the production of IL-17, IL-17F, IL-6, and tumor necrosis factor. Gene expression analysis of IL-23-driven autoreactive T cells identified a unique expression pattern of proinflammatory cytokines and other novel factors, distinguishing them from IL-12-driven T cells. Using passive transfer studies, we confirm that these IL-23-dependent CD4(+) T cells are highly pathogenic and essential for the establishment of organ-specific inflammation associated with central nervous system autoimmunity.

IL-12 and IL-23: master regulators of innate and adaptive immunity.

Initiation of an effective immune response requires close interactions between innate and adaptive immunity. Recent advances in the field of cytokine biology have led to an increased understanding of how myeloid cell-derived factors regulate the immune system to protect the host from infections and prevent tumor development. In this review, we focus on the function of interleukin (IL)-23, a new member of the IL-12 family of regulatory cytokines produced by activated macrophages and dendritic cells. We propose that IL-12 and IL-23 promote two distinct immunological pathways that have separate but complementary functions. IL-12 is required for antimicrobial responses to intracellular pathogens, whereas IL-23 is likely to be important for the recruitment and activation of a range of inflammatory cells that is required for the induction of chronic inflammation and granuloma formation. These two cytokines work in concert to regulate cellular immune responses critical for host defense and tumor suppression.

Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation.

Interleukin (IL) 23 is a heterodimeric cytokine composed of a p19 subunit and the p40 subunit of IL-12. IL-23 affects memory T cell and inflammatory macrophage function through engagement of a novel receptor (IL-23R) on these cells. Recent analysis of the contribution of IL-12 and IL-23 to central nervous system autoimmune inflammation demonstrated that IL-23 rather than IL-12 was the essential cytokine. Using gene-targeted mice lacking only IL-12 (p35-/-) or IL-23 (p19-/-), we show that the specific absence of IL-23 is protective, whereas loss of IL-12 exacerbates collagen-induced arthritis. IL-23 gene-targeted mice did not develop clinical signs of disease and were completely resistant to the development of joint and bone pathology. Resistance correlated with an absence of IL-17-producing CD4+ T cells despite normal induction of collagen-specific, interferon-gamma-producing T helper 1 cells. In contrast, IL-12-deficient p35-/- mice developed more IL-17-producing CD4+ T cells, as well as elevated mRNA expression of proinflammatory tumor necrosis factor, IL-1beta, IL-6, and IL-17 in affected tissues of diseased mice. The data presented here indicate that IL-23 is an essential promoter of end-stage joint autoimmune inflammation, whereas IL-12 paradoxically mediates protection from autoimmune inflammation.