Ricardo T. Paniagua

Selective tyrosine kinase inhibition by imatinib mesylate for the treatment of autoimmune arthritis

Tyrosine kinases play a central role in the activation of signal transduction pathways and cellular responses that mediate the pathogenesis of rheumatoid arthritis. Imatinib mesylate (imatinib) is a tyrosine kinase inhibitor developed to treat Bcr/Abl-expressing leukemias and subsequently found to treat c-Kit-expressing gastrointestinal stromal tumors. We demonstrate that imatinib potently prevents and treats murine collagen-induced arthritis (CIA). We further show that micromolar concentrations of imatinib abrogate multiple signal transduction pathways implicated in RA pathogenesis, including mast cell c-Kit signaling and TNF-alpha release, macrophage c-Fms activation and cytokine production, and fibroblast PDGFR signaling and proliferation. In our studies, imatinib attenuated PDGFR signaling in fibroblast-like synoviocytes (FLSs) and TNF-alpha production in synovial fluid mononuclear cells (SFMCs) derived from human RA patients. Imatinib-mediated inhibition of a spectrum of signal transduction pathways and the downstream pathogenic cellular responses may provide a powerful approach to treat RA and other inflammatory diseases.

Immunosuppression by the JAK3 inhibitor CP-690,550 delays rejection and significantly prolongs kidney allograft survival in nonhuman primates.

Background. Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common chain (&ggr;c). Because mutations in genes encoding &ggr;c or JAK3 result in immunodeficiency, we investigated the potential of a rationally designed inhibitor of JAK3, CP-690,550, to prevent renal allograft rejection in nonhuman primates. Methods. Life-supporting kidney transplantations were performed between mixed leukocyte reaction-mismatched, ABO blood group-matched cynomolgus monkeys. Animals were treated with CP-690,550 (n=18) or its vehicle (controls, n=3) and were euthanized at day 90 or earlier if there was allograft rejection. Results. Mean survival time (± standard error of mean) in animals treated with CP-690,550 (53±7 days) was significantly longer than in control animals (7±1 days, P=0.0003) and was positively correlated with exposure to the drug (r=0.79, P<0.01). Four treated animals were euthanized at 90 days with a normal renal function and low-grade rejection at final pathology. Occurrence of rejection was significantly delayed in treated animals (46±7 days from transplantation vs. 7±1 days in controls, P=0.0003). Persistent anemia, polyoma virus-like nephritis (n=2), and urinary calcium carbonate accretions (n=3) were seen in animals with high exposure. Natural killer cell and CD4+ and CD8+ T-cell numbers were significantly reduced in treated animals. Blood glucose, serum lipid levels, and arterial blood pressure were within normal range in treated animals, and no cancers were demonstrated. Conclusions. CP-690,550 is the first reported JAK3 inhibitor combining efficacy and good tolerability in a preclinical model of allotransplantation in nonhuman primates and thus has interesting potential for immunosuppression in humans.

Molecular Framework for Response to Imatinib Mesylate in Systemic Sclerosis

Systemic sclerosis (SSc) is an autoimmune disease in which the tyrosine kinases platelet-derived growth factor receptor (PDGFR) and Abl are hypothesized to contribute to the fibrosis and vasculopathy of the skin and internal organs. Herein we describe 2 patients with early diffuse cutaneous SSc (dcSSc) who experienced reductions in cutaneous sclerosis in response to therapy with the tyrosine kinase inhibitor imatinib mesylate. Immunohistochemical analyses of skin biopsy specimens demonstrated reductions of phosphorylated PDGFRbeta and Abl with imatinib therapy. By gene expression profiling, an imatinib-responsive signature specific to dcSSc was identified (P < 10(-8)). The response of these patients and the findings of the analyses suggest that PDGFRbeta and Abl play critical, synergistic roles in the pathogenesis of SSc, and that imatinib targets a gene expression program that is frequently dysregulated in dcSSc.

Effects of JAK3 inhibition with CP-690,550 on immune cell populations and their functions in nonhuman primate recipients of kidney allografts.

Background. Janus Kinase (JAK) 3 is a tyrosine kinase essential for proper signal transduction downstream of selected cytokine receptors and for robust T-cell and natural killer cells activation and function. JAK3 inhibition with CP-690,550 prevents acute allograft rejection. To provide further insight into the mechanisms of efficacy, we investigated the immunomodulatory effects of CP-690,550 in vitro and in vivo in nonhuman primates. Methods. Pharmacodynamic assessments of lymphocyte activation, function, proliferation and phenotype were performed in three settings: in vitro in whole blood isolated from untransplanted cynomolgus monkeys (cynos), in vivo in blood from untransplanted cynos dosed with CP-690,550 for 8 days, and in vivo in blood from transplanted cynos immunosuppressed with CP-690,550. Cell surface activation markers expression, IL-2- enhanced IFN-γ production, lymphocyte proliferation and immune cell phenotype analyzes were performed with multiparametric flow cytometry. Results. In vitro exposure to CP-690,550 resulted in significant reduction of IL-2-enhanced IFN-γ production by T-cells (maximum inhibition of 55-63%), T-cell surface expression of CD25 (50% inhibitory concentration (IC50); 0.18 &mgr;M) and CD71 (IC50; 1.6 &mgr;M), and T-cell proliferative capacities measured by proliferating cell nuclear antigen expression (IC50; 0.87 &mgr;M). Similar results were observed in animals dosed with CP-690,550. In addition, transplanted animals displayed significant reduction of NK cell (90% from baseline) and T-cell numbers whereas CD8+ effector memory T-cell populations were unaffected. Conclusions. Potent in vitro and in vivo immunomodulatory effects of the JAK3 inhibitor CP-690,550 likely contribute to its efficacy in the prevention of organ allograft rejection.

c-Fms-mediated differentiation and priming of monocyte lineage cells play a central role in autoimmune arthritis

IntroductionTyrosine kinases are key mediators of multiple signaling pathways implicated in rheumatoid arthritis (RA). We previously demonstrated that imatinib mesylate--a Food and Drug Administration (FDA)-approved, antineoplastic drug that potently inhibits the tyrosine kinases Abl, c-Kit, platelet-derived growth factor receptor (PDGFR), and c-Fms--ameliorates murine autoimmune arthritis. However, which of the imatinib-targeted kinases is the principal culprit in disease pathogenesis remains unknown. Here we examine the role of c-Fms in autoimmune arthritis.MethodsWe tested the therapeutic efficacy of orally administered imatinib or GW2580, a small molecule that specifically inhibits c-Fms, in three mouse models of RA: collagen-induced arthritis (CIA), anti-collagen antibody-induced arthritis (CAIA), and K/BxN serum transfer-induced arthritis (K/BxN). Efficacy was evaluated by visual scoring of arthritis severity, paw thickness measurements, and histological analysis. We assessed the in vivo effects of imatinib and GW2580 on macrophage infiltration of synovial joints in CIA, and their in vitro effects on macrophage and osteoclast differentiation, and on osteoclast-mediated bone resorption. Further, we determined the effects of imatinib and GW2580 on the ability of macrophage colony-stimulating factor (M-CSF; the ligand for c-Fms) to prime bone marrow-derived macrophages to produce tumor necrosis factor (TNF) upon subsequent Fc receptor ligation. Finally, we measured M-CSF levels in synovial fluid from patients with RA, osteoarthritis (OA), or psoriatic arthritis (PsA), and levels of total and phosphorylated c-Fms in synovial tissue from patients with RA.ResultsGW2580 was as efficacious as imatinib in reducing arthritis severity in CIA, CAIA, and K/BxN models of RA. Specific inhibition of c-Fms abrogated (i) infiltration of macrophages into synovial joints of arthritic mice; (ii) differentiation of monocytes into macrophages and osteoclasts; (iii) osteoclast-mediated bone resorption; and (iv) priming of macrophages to produce TNF upon Fc receptor stimulation, an important trigger of synovitis in RA. Expression and activation of c-Fms in RA synovium were high, and levels of M-CSF were higher in RA synovial fluid than in OA or PsA synovial fluid.ConclusionsThese results suggest that c-Fms plays a central role in the pathogenesis of RA by mediating the differentiation and priming of monocyte lineage cells. Therapeutic targeting of c-Fms could provide benefit in RA.

Tyrosine kinases as targets for the treatment of rheumatoid arthritis

As critical regulators of numerous cell signaling pathways, tyrosine kinases are implicated in the pathogenesis of several diseases, including rheumatoid arthritis (RA). In the absence of disease, synoviocytes produce factors that provide nutrition and lubrication for the surrounding cartilage tissue; few cellular infiltrates are seen in the synovium. In RA, however, macrophages, neutrophils, T cells and B cells infiltrate the synovium and produce cytokines, chemokines and degradative enzymes that promote inflammation and joint destruction. In addition, the synovial lining expands owing to the proliferation of synoviocytes and infiltration of inflammatory cells to form a pannus, which invades the surrounding bone and cartilage. Many of these cell responses are regulated by tyrosine kinases that operate in specific signaling pathways, and inhibition of a number of these kinases might be expected to provide benefit in RA.

Anti-non-Gal porcine endothelial cell antibodies in acute humoral xenograft rejection of hDAF-transgenic porcine hearts in cynomolgus monkeys.

Abstract:  Background:  Anti‐Galα1–3Gal (Gal) antibodies play a major role in hyperacute rejection and acute humoral xenograft rejection (AHXR) in porcine‐to‐nonhuman primate transplantation. The role of anti‐non‐Gal antibodies in AHXR is less well defined.

In vivo evaluation of the novel calcineurin inhibitor ISATX247 in Non-Human primates

BACKGROUND ISA(TX)247 is a novel calcineurin inhibitor that has shown more potency than cyclosporine in vitro. This is the first in vivo study of the effects of ISA(TX)247 on lymphocyte functions in non-human primates. METHODS Groups of cynomolgus monkeys were treated orally twice daily for 7 days, each dose consisting of 25 mg/kg cyclosporine (n = 5), 25 mg/kg ISA(TX)247 (n = 6) or 50 mg/kg ISA(TX)247 (n = 6). Levels of cyclosporine and ISA(TX)247 in whole blood were measured by liquid chromatography/mass spectrometry. After mitogen stimulation, lymphocyte proliferation was assessed by tritium-labeled thymidine incorporation and by flow cytometry (expression of proliferating cell nuclear antigen in cells in S/G(2)M phase). Flow cytometry was also used to assess production of intracellular cytokines by T cells (interleukin-2, interferon-gamma, tumor necrosis factor-alpha) and expression of T-cell surface activation antigens (CD25, CD71, CD11a, CD95, CD154). RESULTS Trough (C(14 hr)) and peak (C(3 hr)) drug levels, as well as area under the concentration-time curve, were significantly higher for cyclosporine than ISA(TX)247 (370 ng/ml vs 70 ng/ml, 877 ng/ml vs 303 ng/ml and 6,262 ng. h/ml vs 1,979 ng. h/ml, respectively). On Day 7 at C(14 hr), lymphocyte proliferation had been suppressed by approximately 50% in all groups compared with proliferation before treatment. Three hours after dosing, lymphocyte proliferation was inhibited significantly more by ISA(TX)247 (approximately 80%, with no differences between the two ISA(TX)247 dose levels) than by cyclosporine (65% inhibition). Similar differences between the immunosuppressive effects of ISA(TX)247 and cyclosporine were found for inhibition of expression of T-cell surface activation antigens. Despite lower ISA(TX)247 exposures compared with cyclosporine, the cyclosporine treatment only rarely suppressed cytokine production more than treatment with ISA(TX)247. CONCLUSIONS In non-human primates, ISA(TX)247 produces a greater or similar inhibition of lymphocyte proliferation, expression of T-cell activation surface antigens, and cytokine production when compared with cyclosporine, despite ISA(TX)247s lower blood levels and total exposure. We conclude that ISA(TX)247 suppresses diverse T-cell functions more potently than cyclosporine in non-human primates in vivo.

Autoimmunity against Fibrinogen Mediates Inflammatory Arthritis in Mice

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the presence of anticitrullinated protein Abs, although the exact targets and role of anticitrullinated protein autoimmunity in the pathogenesis of RA remain to be defined. Fibrinogen, which can be citrullinated, has recently emerged as a candidate autoantigen. To determine whether autoimmunity against fibrinogen can mediate inflammatory arthritis, we immunized a variety of common mouse strains with fibrinogen and found that DBA/1 and SJL mice developed an inflammatory and erosive arthritis. Mice with fibrinogen-induced arthritis (FIA) possess fibrinogen-reactive T cells that produce the proinflammatory cytokines IL-6, IL-17, TNF-α, and IFN-γ. FIA can be adoptively transferred with either plasma or fibrinogen-specific T cells from diseased mice. Mice with FIA possess rheumatoid factor, circulating immune complexes, and anticyclic citrullinated peptide Abs, all of which are characteristic of human RA. These observations demonstrate that fibrinogen is arthritogenic in mice and that the pathogenesis of FIA is mediated by both autoantibodies and fibrinogen-reactive T cells.

Hyperacute rejection of hDAF-transgenic pig organ xenografts in cynomolgus monkeys: Influence of pre-existing anti-pig antibodies and prevention by the αGal glycoconjugate GAS914

Abstract:  Background:  Our introductory pig‐to‐cynomolgus monkey heart or kidney transplantation using organs from pigs transgenic for human decay‐accelerating factor (hDAF), showed a high incidence of hyperacute rejection (HAR), which was ascribed to extraordinary high levels of anti‐pig antibodies. We evaluated the efficacy of GAS914, a Galα1–3Gal trisaccharide linked to a poly‐l‐lysine backbone, in inhibition of HAR.