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.

Suppression of glomerulonephritis in lupus-prone NZB × NZW mice by RN486, a selective inhibitor of Bruton's tyrosine kinase.

OBJECTIVE: Bruton's 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.

Selective Btk inhibition by PRN1008/PRN473 blocks human CLEC-2, and PRN473 reduces venous thrombosis formation in mice.

ABSTRACT: Platelet C-type lectin-like receptor 2 (CLEC-2) is a hem-immunoreceptor tyrosine-based activation motif-containing receptor that has a critical role in venous thrombosis but minimal involvement in hemostasis. CLEC-2 can be blocked by Btk inhibitors. Treatment with ibrutinib is associated with increased bleeding due to off-target inhibition of Src family kinases (SFKs). Patients with X-linked agammaglobulinemia (XLA) who lack Btk, however, do not bleed, suggesting selective Btk inhibition as a viable antithrombotic strategy. We assessed the effects of selective Btk inhibitors PRN1008 (rilzabrutinib) and PRN473 on platelet signaling and function mediated by CLEC-2 and glycoprotein-VI. We used healthy donors and XLA platelets to determine off-target inhibitor effects. Inferior vena cava (IVC) stenosis and Salmonella infection mouse models were used to assess antithrombotic effects of PRN473 in vivo. PRN1008 and PRN473 potently inhibited CLEC-2-mediated platelet activation to rhodocytin. No off-target inhibition of SFKs was seen. PRN1008 treatment of Btk-deficient platelets resulted in minor additional inhibition of aggregation and tyrosine phosphorylation, likely reflecting inhibition of Tec. No effect on G protein-coupled receptor-mediated platelet function was observed. PRN473 significantly reduced the number of thrombi in podoplanin-positive vessels after Salmonella infection and the presence of IVC thrombosis after vein stenosis. The potent inhibition of human platelet CLEC-2 and reduced thrombosis in in vivo models, together with the lack of off-target SFK inhibition and absence of bleeding reported in rilzabrutinib-treated patients with immune thrombocytopenia, suggest Btk inhibition as a promising antithrombotic strategy.

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.

Discovery of the Irreversible Covalent FGFR Inhibitor 8-(3-(4-Acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (PRN1371) for the Treatment of Solid Tumors.

Aberrant signaling of the FGF/FGFR pathway occurs frequently in cancers and is an oncogenic driver in many solid tumors. Clinical validation of FGFR as a therapeutic target has been demonstrated in bladder, liver, lung, breast, and gastric cancers. Our goal was to develop an irreversible covalent inhibitor of FGFR1-4 for use in oncology indications. An irreversible covalent binding mechanism imparts many desirable pharmacological benefits including high potency, selectivity, and prolonged target inhibition. Herein we report the structure-based design, medicinal chemistry optimization, and unique ADME assays of our irreversible covalent drug discovery program which culminated in the discovery of compound 34 (PRN1371), a highly selective and potent FGFR1-4 inhibitor.

The Irreversible Covalent Fibroblast Growth Factor Receptor Inhibitor PRN1371 Exhibits Sustained Inhibition of FGFR after Drug Clearance.

An increasing number of cancers are known to harbor mutations, translocations, or amplifications in the fibroblast growth factor receptor (FGFR) family of kinases. The FGFR inhibitors evaluated in clinical trials to date have shown promise at treating these cancers. Here, we describe PRN1371, an irreversible covalent inhibitor of FGFR1-4 targeting a cysteine within the kinase active site. PRN1371 demonstrated strong FGFR potency and excellent kinome-wide selectivity in a number of biochemical and cellular assays, including in various cancer cell lines exhibiting FGFR alterations. Furthermore, PRN1371 maintained FGFR inhibition in vivo, not only when circulating drug levels were high but also after the drug had been cleared from circulation, indicating the possibility of sustained FGFR inhibition in the clinic without the need for continuous drug exposure. Durable tumor regression was also obtained in multiple tumor xenografts and patient-derived tumor xenograft models and was sustained even using an intermittent dosing strategy that provided drug holidays. PRN1371 is currently under clinical investigation for treatment of patients with solid tumors. Mol Cancer Ther; 16(12); 2668-76. ©2017 AACR.