Narrow Spectrum Kinase Inhibitors Demonstrate Promise for the Treatment of Dry Eye Disease and Other Ocular Inflammatory Disorders.

Purpose: The purpose of this study is to determine the potential of narrow spectrum kinase inhibitors (NSKIs) to treat inflammatory eye disorders. Methods: Human conjunctival epithelial (HCE) cells were retrieved from subjects via impression cytology. Real-time quantitative PCR (qPCR) was performed on HCE cells to determine gene expression of NSKI kinase targets and proinflammatory cytokines in dry eye disease (DED) patients versus healthy controls. qPCR also assessed p38α expression in hyperosmolar-treated Chang conjunctival epithelial cells. Interaction of NSKI TOP1362 with the kinases was evaluated in ATP-dependent Z-LYTE and competition binding assays. Anti-inflammatory activity was assessed in human peripheral blood mononuclear cells and primary macrophages. In an endotoxin-induced uveitis (EIU) study, lipopolysaccharide (LPS) was administered intravitreally to Lewis rats. TOP1362, dexamethasone, or vehicle was administered topically, and inflammatory cytokine levels were measured 6 hours after LPS injection. Results: HCE cells from DED patients showed significantly increased expression of p38α, spleen tyrosine kinase (Syk), Src, lymphocyte-specific protein tyrosine kinase (Lck), interleukin one beta (IL-1ß), interleukin eight (IL-8), monocyte chemotactic protein-1 (MCP-1), and matrix metalloproteinase-9 (MMP-9). TOP1362 strongly inhibited the kinase targets p38α, Syk, Src, and Lck, blocked the rise in p38α expression in hyperosmolar Chang cells, and potently reduced inflammatory cytokine release in cellular models of innate and adaptive immunities. In the EIU model, TOP1362 dose-dependently attenuated the LPS-induced rise in inflammatory cell infiltration and ocular cytokine levels with efficacy comparable to that of dexamethasone. Conclusions: TOP1362 is a potent inhibitor of kinases upregulated in DED and markedly attenuates proinflammatory cytokine release in vitro and in vivo, highlighting the therapeutic potential of NSKIs for treating ocular inflammation, such as that observed in DED.

Effect of Narrow Spectrum Versus Selective Kinase Inhibitors on the Intestinal Proinflammatory Immune Response in Ulcerative Colitis.

BACKGROUND: Kinases are key mediators of inflammation, highlighting the potential of kinase inhibitors as treatments for inflammatory disorders. Selective kinase inhibitors, however, have proved disappointing, particularly in the treatment of rheumatoid arthritis and inflammatory bowel disease. Consequently, to improve efficacy, attention has turned to multikinase inhibition. METHODS: The activity of a narrow spectrum kinase inhibitor, TOP1210, has been compared with selective kinase inhibitors (BIRB-796, dasatinib and BAY-61-3606) in a range of kinase assays, inflammatory cell assays, and in inflamed biopsies from patients with ulcerative colitis (UC). Effects on recombinant P38α, Src, and Syk kinase activities were assessed using Z-lyte assays (Invitrogen, Paisley, United Kingdom). Anti-inflammatory effects were assessed by measurement of proinflammatory cytokine release from peripheral blood mononuclear cells, primary macrophages, HT29 cells, inflamed colonic UC biopsies, and myofibroblasts isolated from inflamed colonic UC mucosa. RESULTS: TOP1210 potently inhibits P38α, Src, and Syk kinase activities. Similarly, TOP1210 demonstrates potent inhibitory activity against proinflammatory cytokine release in each of the cellular assays and the inflamed colonic UC biopsies and myofibroblasts isolated from inflamed colonic UC mucosa. Generally, the selective kinase inhibitors showed limited and weaker activity in the cellular assays compared with the broad inhibitory profile of TOP1210. However, combination of the selective inhibitors led to improved efficacy and potency in both cellular and UC biopsy assays. CONCLUSIONS: Targeted, multikinase inhibition with TOP1210 leads to a broad efficacy profile in both the innate and adaptive immune responses, with significant advantages over existing selective kinase approaches, and potentially offers a much improved therapeutic benefit in inflammatory bowel disease.

Highly sensitive fluorescence detection system for microfluidic lab-on-a-chip.

We demonstrate a compact, low cost and practical fluorescence detection system for lab-on-a-chip applications. The system comprises a commercially available InGaN light emitting diode (501 nm) as light source, an organic or silicon photodiode detector, absorptive dye coated colour filters and linear and reflective polarisers. An injection moulded polystyrene microfluidic chip is used as the platform for fluorescence immunoassays for cardiac markers myoglobin and CK-MB. The optical limit of detection (LOD) is measured using a TransFluoSphere® suspension at 5.6 × 10(4) beads µl(-1) which can be equated to ∼3 nM fluorescein equivalent concentration. The LOD for the human plasma immunoassays is measured as 1.5 ng ml(-1) for both myoglobin and CK-MB.

Monoclonal antibodies directed to CD20 and HLA-DR can elicit homotypic adhesion followed by lysosome-mediated cell death in human lymphoma and leukemia cells.

mAbs are becoming increasingly utilized in the treatment of lymphoid disorders. Although Fc-FcgammaR interactions are thought to account for much of their therapeutic effect, this does not explain why certain mAb specificities are more potent than others. An additional effector mechanism underlying the action of some mAbs is the direct induction of cell death. Previously, we demonstrated that certain CD20-specific mAbs (which we termed type II mAbs) evoke a nonapoptotic mode of cell death that appears to be linked with the induction of homotypic adhesion. Here, we reveal that peripheral relocalization of actin is critical for the adhesion and cell death induced by both the type II CD20-specific mAb tositumomab and an HLA-DR-specific mAb in both human lymphoma cell lines and primary chronic lymphocytic leukemia cells. The cell death elicited was rapid, nonapoptotic, nonautophagic, and dependent on the integrity of plasma membrane cholesterol and activation of the V-type ATPase. This cytoplasmic cell death involved lysosomes, which swelled and then dispersed their contents, including cathepsin B, into the cytoplasm and surrounding environment. The resulting loss of plasma membrane integrity occurred independently of caspases and was not controlled by Bcl-2. These experiments provide what we believe to be new insights into the mechanisms by which 2 clinically relevant mAbs elicit cell death and show that this homotypic adhesion-related cell death occurs through a lysosome-dependent pathway.

Induction of cytosolic calcium flux by CD20 is dependent upon B Cell antigen receptor signaling.

The anti-CD20 monoclonal antibody (mAb) rituximab is now routinely used for the treatment of non-Hodgkins lymphoma and is being examined in a wide range of other B-cell disorders, such as rheumatoid arthritis. Despite intensive study, the mechanism of action still remains uncertain. In the current study, anti-CD20 mAb-induced calcium signaling was investigated. Previously, we grouped anti-CD20 mAbs into Type I (rituximab-like) and Type II (B1-like) based upon various characteristics such as their ability to induce complement activation and redistribute CD20 into detergent-insoluble membrane domains. Here we show that only Type I mAbs are capable of inducing a calcium flux in B cells and that this is tightly correlated with the expression of the B-cell antigen receptor (BCR). Inhibitor analysis revealed that the signaling cascade employed by CD20 was strikingly similar to that utilized by the BCR, with inhibitors of Syk, Src, and PI3K, but not EGTA, p38, or ERK1/2, completely ablating calcium flux. Furthermore, binding of Type I but not Type II mAbs caused direct association of CD20 with the BCR as measured by FRET and resulted in the phosphorylation of BCR-specific adaptor proteins BLNK and SLP-76. Crucially, variant Ramos cells lacking BCR expression but with unchanged CD20 expression were completely unable to induce calcium flux following ligation of CD20. Collectively, these data indicate that CD20 induces cytosolic calcium flux through its ability to associate with and "hijack" the signaling potential of the BCR.

The biology of CD20 and its potential as a target for mAb therapy.

CD20 is a 33-37 kDa, non-glycosylated phosphoprotein expressed on the surface of almost all normal and malignant B cells. It is also the target for rituximab, the most effective anti-cancer monoclonal antibody developed to date. Rituximab has now been given to over 300,000 lymphoma patients in the last decade and interestingly is now being explored for use in other disorders, such as autoimmune conditions including rheumatoid arthritis and systemic lupus erythematosus. Despite the success in immunotherapy, knowledge about the biology of CD20 is still relatively scarce, partly because it has no known natural ligand and CD20 knockout mice display an almost normal phenotype. However, interesting insight has come from work showing that CD20 is resident in lipid raft domains of the plasma membrane where it probably functions as a store-operated calcium channel following ligation of the B cell receptor for antigen. In the current review, these and data relating to its activity as a therapeutic target will be discussed in depth. It is clear that a greater understanding of CD20 biology and the effector mechanisms, such as antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and growth regulation, which operate with anti-CD20 mAb in vivo will allow more efficient exploitation of CD20 as a therapeutic target.

CD20-induced lymphoma cell death is independent of both caspases and its redistribution into triton X-100 insoluble membrane rafts.

Rituximab is routinely used for the treatment of neoplasia, although the mechanism of action remains uncertain. In the current study, CD20-induced apoptosis was investigated with a panel of anti-CD20 monoclonal antibodies (mAb) in a wide range of cell lines. A hierarchy of mAb activity was apparent, with the B1 mAb generally the most potent. Apoptosis through CD20 was dependent on the nature of mAb binding and correlated with the extent of homotypic cell adhesion induced. However, using anti-CD20 mAb, which vary in the extent to which they redistribute wild-type and mutant CD20 molecules to membrane rafts, we showed that CD20-induced apoptosis was independent of translocation to TX-100 insoluble rafts. Using crmA-transfected cells and caspase inhibitors, we showed that phosphatidylserine translocation and mitochondrial permeability transition evoked during CD20-induced apoptosis appeared caspase independent. Furthermore, in cytoplasts which lack mitochondria and in Bcl(2)-transfected cells, phosphatidylserine was still translocated to the cell surface after CD20 stimulation. Together, these data imply that CD20 can evoke apoptosis without the involvement of mitochondria and caspases and irrespective of redistribution into TX-100 insoluble membrane rafts.