The Bruton tyrosine kinase inhibitor PCI-32765 ameliorates autoimmune arthritis by inhibition of multiple effector cells

Abstract

Introduction: The aim was to determine the effect of the Bruton tyrosine kinase (Btk)-selective inhibitor PCI-32765, currently in Phase I/II studies in lymphoma trials, in arthritis and immune-complex (IC) based animal models and describe the underlying cellular mechanisms.

Methods: PCI-32765 was administered in a series of murine IC disease models including collagen-induced arthritis (CIA), collagen antibody-induced arthritis (CAIA), reversed passive anaphylactic reaction (RPA), and passive cutaneous anaphylaxis (PCA). Clinical and pathologic features characteristic of each model were examined following treatment. PCI-32765 was then examined in assays using immune cells relevant to the pathogenesis of arthritis, and where Btk is thought to play a functional role. These included proliferation and calcium mobilization in B cells, cytokine and chemokine production in monocytes/macrophages, degranulation of mast cells and its subsequent cytokine/chemokine production.

Results: PCI-32765 dose-dependently and potently reversed arthritic inflammation in a therapeutic CIA model with an ED(50) of 2.6 mg/kg/day. PCI-32765 also prevented clinical arthritis in CAIA models. In both models, infiltration of monocytes and macrophages into the synovium was completely inhibited and importantly, the bone and cartilage integrity of the joints were preserved. PCI-32765 reduced inflammation in the Arthus and PCA assays. In vitro, PCI-32765 inhibited BCR-activated primary B cell proliferation (IC(50) = 8 nM). Following FcγR stimulation, PCI-32765 inhibited TNFα, IL-1β and IL-6 production in primary monocytes (IC(50) = 2.6, 0.5, 3.9 nM, respectively). Following FcεRI stimulation of cultured human mast cells, PCI-32765 inhibited release of histamine, PGD(2), TNF-α, IL-8 and MCP-1.

Conclusions: PCI-32765 is efficacious in CIA, and in IC models that do not depend upon autoantibody production from B cells. Thus PCI-32765 targets not only B lymphocytes but also monocytes, macrophages and mast cells, which are important Btk-expressing effector cells in arthritis.

Figure 1

PCI-32765 dose-dependently inhibits inflammation, bone erosion, cellular infiltration and synovial fluid cytokines/chemokines in CIA models. (a) Mice (n = 12) were treated with PCI-32765 orally once daily. Daily average clinical scores are plotted over 18 days of treatment; *** P < 0.001 compared with vehicle (Mann-Whitney U-test). (b) Using the fluorescent probe PCI-33380 [19], the percentage of Btk occupied by PCI-32765 in collagen-induced arthritis (CIA) mice was measured in splenocytes collected at three hours (left panel, n = 6) and 24 hours (right panel, n = 6) following last dose (D18) of drug treatment. (c) Average J score was determined following micro-CT scanned reconstructed images of hind legs from CIA study (n = 6). (d) Average cellularity (n = 12) in synovial fluid from the knee of CIA mice following PCI-32765 treatment. Giemsa-Wright stained slides were enumerated for granulocytes, macrophages, and lymphocytes under 100×, and five random fields were scored and averaged. (e) Synovial fluid cytokine and chemokine levels from the 12.5 mg/kg treatment group (n = 12) are shown; * P < 0.05, ** P < 0.01; *** P < 0.001 compared with vehicle, analysis of variance.

Figure 2

Histopathology images and scores from CIA mice. (a) Histopathology images from collagen-induced arthritis (CIA) mice: 1, 2, 5, and 6 were treated with vehicle; 3, 4, 7, and 8 were treated with 12.5 mg/kg PCI-32765; 1, 2, 3, and 4 were from carpus, 5, 6, 7, and 8 were from tarsus. Images 1, and 3 are magnified 40 ×; 2, 4, 5, 6, 7, and 8 are magnified 100× under the microscope. Bone resorption is depicted with arrowheads; asterisks show carpal bones in panels 1, and 3. R, radius; U, ulna; MC, metacarpal. In panel 5, and 6 asterisks show severe inflammation, and arrows show loss and necrosis of cartilage. In panels 7, and 8 arrowheads show minimum synovial inflammation, and arrow points to minor loss of articular chondrocytes. (b) Histopathology Scores from four joints (carpus and tarsus, both sides) of the CIA mice were averaged and scored by degree of inflammation, pannus, cartilage damage, and bone damage. * P < 0.05, ** P < 0.01; *** P < 0.001 compared with vehicle, analysis of variance.

Figure 3

PCI-32765 potently inhibits anti-IgM stimulation of pBtk and pERK, calcium mobilization, early activation marker and anti-IgM induced cell proliferation in human primary B lymphocytes. (a) Inhibition of intracellular pBtk (Y551) and pERK1/2 staining in B cells following anti-IgM stimulation with flow cytometry methods. (b) Inhibition of calcium mobilization following B-cell antigen receptor (BCR) stimulation in primary B cells. (c) Inhibition of the early activation marker CD69 following BCR stimulation in primary B cells. (d) PCI-32765 inhibits B lymphocyte proliferation stimulated by anti-IgM but not phorbol myristate acetate. Anti-CD3/28 failed to stimulate purified human B lymphocytes (not shown).

Figure 4

PCI-32765 inhibits FcγR signaling in monocytes. (a) Inhibition of Btk signaling in THP-1 cells. THP-1 cells were stimulated with IFN-γ and pretreated with PCI-32765 (1 μM or 0.1 μM) or vehicle and stimulated by cross-linking with human IgG and goat anti-human IgG in the presence of H₂O₂ and IFNγ. Cell lysates were subjected to western blot analysis and probed with the indicated antibodies. (b) Inhibition of calcium mobilization in THP-1 cells (upper panel) or human monocytes (lower panel) stimulated with cross-linked IgG as described in Materials and Methods.

Figure 5

PCI-32765 dose-dependently inhibits disease in the CAIA, Arthus, and PCA models. (a) Mice (n = 10) were treated with PCI-32765 orally once daily. Daily average clinical scores are plotted over 14 days of treatment; ***P < 0.001 (Student t-test). (b) Tissue sections from six joints (carpus, tarsus, and knee) of treated mice stained with H&E and Safranin-O were evaluated through histopathology for inflammation, pannus, cartilage damage, and bone erosion (n = 10). PCI-32765 treatments significantly inhibited inflammation, pannus, cartilage, and bone damages. (c) Representative reconstructed micro-CT images of forelimb of mice treated with vehicle, PCI-32765 (12. 5 mg/kg) or dexamethasone (0.2 mg/kg). (d) Mean J scores of bone destruction calculated from micro-CT images (n = 3). (e) PCI-32765 treatments were evaluated in Arthus reactions induced by OVA and anti-OVA injections. Evans blue dye extravasation area measurements (left) and tissue biopsies evaluated for change of OD (right) are shown (n = 6). (f) PCI-32765 inhibits anti-DNP (dinitrophenol)-IgE and DNP-BSA mediated skin PCA. Extravasation area (left panel) and change of Evans blue OD (optical density) measurements (n = 10) (right panel) are displayed. * P < 0.05, ** P < 0.01; *** P < 0.001 compared with vehicle, analysis of variance. CAIA, collagen antibody-induced arthritis; OVA, ovalbumin; PCA, passive cutaneous anaphylaxis.