ABSTRACT
Delayed-type hypersensitivity (DTH) is classically defined as inflammation involving activated Th1 cells and cytokine production. DTH paw swelling, along with the cytokines IL-2, IFNγ, MCP-1 and TNFα, were inhibited in Balb/c mice by cyclosporine A (CsA). Surprisingly, the DTH response in the B6D2F1 mice was unaffected by CsA, despite a decrease in TNFα and IFNγ levels. IL-2 levels, however, were not decreased. To determine if the IL-2 production in the B6D2F1 strain is occurring through CD28-mediated costimulation, both CsA and CTLA-4Ig were administered. Paw swelling and IL-2 levels were decreased, indicating a role for costimulation. Co-administration of temsirolimus and CsA also reduced DTH and IL-2 levels in B6D2F1 mice, demonstrating involvement of the mTORC1 pathway. These results indicate that the cell activation pathways responsible for DTH differ with mouse strain. It is important to understand these differences in order to accurately interpret the results using potential therapeutic agents.
Subject(s)
Autoimmunity/immunology , Cyclosporine/pharmacology , Hypersensitivity, Delayed/immunology , Immunity, Cellular/drug effects , Immunity, Cellular/immunology , Interleukin-2/immunology , Abatacept , Animals , Antibodies/immunology , Antibodies/pharmacology , Autoimmunity/drug effects , CD28 Antigens/metabolism , Cell Movement/drug effects , Cell Movement/immunology , Cyclosporine/pharmacokinetics , Cytokines/metabolism , Erythrocytes/immunology , Female , Foot/pathology , Hypersensitivity, Delayed/pathology , Immunoconjugates/pharmacology , Interleukin-17/antagonists & inhibitors , Interleukin-17/immunology , Lymph Nodes/cytology , Lymph Nodes/immunology , Lymphocyte Activation/immunology , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Inbred BALB C , Mice, Inbred Strains , Multiprotein Complexes , NFATC Transcription Factors/antagonists & inhibitors , NFATC Transcription Factors/metabolism , Protein Kinase Inhibitors/pharmacology , Proteins/antagonists & inhibitors , Sheep , Signal Transduction/drug effects , Signal Transduction/immunology , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , Species Specificity , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , TOR Serine-Threonine Kinases , VaccinationABSTRACT
IL-22 is made by a unique set of innate and adaptive immune cells, including the recently identified noncytolytic NK, lymphoid tissue-inducer, Th17, and Th22 cells. The direct effects of IL-22 are restricted to nonhematopoietic cells, its receptor expressed on the surface of only epithelial cells and some fibroblasts in various organs, including parenchymal tissue of the gut, lung, skin, and liver. Despite this cellular restriction on IL-22 activity, we demonstrate that IL-22 induces effects on systemic biochemical, cellular, and physiological parameters. By utilizing adenoviral-mediated delivery of IL-22 and systemic administration of IL-22 protein, we observed that IL-22 modulates factors involved in coagulation, including fibrinogen levels and platelet numbers, and cellular constituents of blood, such as neutrophil and RBC counts. Furthermore, we observed that IL-22 induces thymic atrophy, body weight loss, and renal proximal tubule metabolic activity. These cellular and physiological parameters are indicative of a systemic inflammatory state. We observed that IL-22 induces biochemical changes in the liver including induction of fibrinogen, CXCL1, and serum amyloid A that likely contribute to the reported cellular and physiological effects of IL-22. Based on these findings, we propose that downstream of its expression and impact in local tissue inflammation, circulating IL-22 can further induce changes in systemic physiology that is indicative of an acute-phase response.
Subject(s)
Acute-Phase Reaction/immunology , Acute-Phase Reaction/physiopathology , Interleukins/immunology , Animals , Mice , Mice, Inbred C57BL , Mice, Knockout , Interleukin-22ABSTRACT
OBJECTIVE: All gamma-chain cytokines signal through JAK-3 and JAK-1 acting in tandem. We undertook this study to determine whether the JAK-3 selective inhibitor WYE-151650 would be sufficient to disrupt cytokine signaling and to ameliorate autoimmune disease pathology without inhibiting other pathways mediated by JAK-1, JAK-2, and Tyk-2. METHODS: JAK-3 kinase selective compounds were characterized by kinase assay and JAK-3-dependent (interleukin-2 [IL-2]) and -independent (IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF]) cell-based assays measuring proliferation or STAT phosphorylation. In vivo, off-target signaling was measured by IL-22- and erythropoietin (EPO)-mediated models, while on-target signaling was measured by IL-2-mediated signaling. Efficacy of JAK-3 inhibitors was determined using delayed-type hypersensitivity (DTH) and collagen-induced arthritis (CIA) models in mice. RESULTS: In vitro, WYE-151650 potently suppressed IL-2-induced STAT-5 phosphorylation and cell proliferation, while exhibiting 10-29-fold less activity against JAK-3-independent IL-6- or GM-CSF-induced STAT phosphorylation. Ex vivo, WYE-151650 suppressed IL-2-induced STAT phosphorylation, but not IL-6-induced STAT phosphorylation, as measured in whole blood. In vivo, WYE-151650 inhibited JAK-3-mediated IL-2-induced interferon-gamma production and decreased the natural killer cell population in mice, while not affecting IL-22-induced serum amyloid A production or EPO-induced reticulocytosis. WYE-151650 was efficacious in mouse DTH and CIA models. CONCLUSION: In vitro, ex vivo, and in vivo assays demonstrate that WYE-151650 is efficacious in mouse CIA despite JAK-3 selectivity. These data question the need to broadly inhibit JAK-1-, JAK-2-, or Tyk-2-dependent cytokine pathways for efficacy.
Subject(s)
Arthritis, Experimental/drug therapy , Janus Kinase 3/antagonists & inhibitors , Analysis of Variance , Animals , Arthritis, Experimental/metabolism , Blotting, Western , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Flow Cytometry , Janus Kinase 1/antagonists & inhibitors , Janus Kinase 1/metabolism , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/metabolism , Janus Kinase 3/metabolism , Mice , Mice, Inbred BALB C , Phosphorylation/drug effects , Signal Transduction/drug effectsABSTRACT
The cPLA(2)alpha inhibitors we reported earlier were potent in both isolated enzyme and rat whole blood assays but have high plogD(7.4). To address these issues, reactions of electrophilic sulfonamides 9-12 were employed to incorporate various heterocyclic or heteroatom-based reagents into cPLA(2)alpha inhibitors. For example, reactions of 9 with sulfur nucleophiles such as thiophenol allowed rapid assembly of thioether analogues that were converted into the corresponding sulfoxides to afford less lipophilic derivatives. Reactions of 10 and 11 with various nitrogen nucleophiles, including aromatic heterocycles and aliphatic amines, provided an efficient way to introduce polarity into cPLA(2)alpha inhibitors. Finally, we report the first application of (2-formylphenyl)methanesulfonyl chloride, 13. Reductive amination of 2-formylphenylmethane sulfonamides allowed the introduction of various nitrogen nucleophiles. Several inhibitors obtained herein have plogD(7.4) values 3-4 units lower than previously synthesized compounds and yet maintain in vitro potency.
Subject(s)
Enzyme Inhibitors/chemistry , Group IV Phospholipases A2/antagonists & inhibitors , Hydrophobic and Hydrophilic Interactions , Sulfonamides/chemistry , Animals , Enzyme Inhibitors/pharmacology , Rats , Structure-Activity Relationship , Sulfides , Sulfonamides/pharmacologyABSTRACT
The optimization of a class of indole cPLA 2 alpha inhibitors is described herein. The importance of the substituent at C3 and the substitution pattern of the phenylmethane sulfonamide region are highlighted. Optimization of these regions led to the discovery of 111 (efipladib) and 121 (WAY-196025), which are shown to be potent, selective inhibitors of cPLA 2 alpha in a variety of isolated enzyme assays, cell based assays, and rat and human whole blood assays. The binding of these compounds has been further examined using isothermal titration calorimetry. Finally, these compounds have shown efficacy when dosed orally in multiple acute and chronic prostaglandin and leukotriene dependent in vivo models.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Benzoates/chemical synthesis , Group IV Phospholipases A2/antagonists & inhibitors , Sulfonamides/chemical synthesis , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Arthritis, Experimental/drug therapy , Benzoates/chemistry , Benzoates/pharmacology , Biological Availability , Bronchoconstriction/drug effects , Calorimetry , Carrageenan , Cell Line , Cyclooxygenase 2 Inhibitors/chemical synthesis , Cyclooxygenase 2 Inhibitors/chemistry , Cyclooxygenase 2 Inhibitors/pharmacology , Edema/chemically induced , Edema/drug therapy , Humans , In Vitro Techniques , Isoenzymes/antagonists & inhibitors , Male , Mice , Protein Binding , Rats , Rats, Sprague-Dawley , Sheep , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
The synthesis and structure-activity relationship of a series of benzenesulfonamide indole inhibitors of cPLA(2)alpha are described. Substitution of the benzenesulfonamide led to analogues with 50-fold improvement in potency versus the unsubstituted benzenesulfonamide lead compound. Rat pharmacokinetics in a minimal formulation was used to prioritize compounds, leading to the discovery of a potent inhibitor of cPLA(2)alpha with oral efficacy in models of rat carrageenan paw edema and Ascaris suum airway challenge in naturally sensitized sheep.
Subject(s)
Group IV Phospholipases A2/antagonists & inhibitors , Group IV Phospholipases A2/metabolism , Indoles/pharmacology , Sulfonamides/chemistry , Administration, Oral , Animals , Ascariasis/drug therapy , Ascariasis/parasitology , Ascaris suum/physiology , Calorimetry , Humans , Indoles/chemistry , Indoles/therapeutic use , Molecular Structure , Rats , Sheep , Structure-Activity Relationship , Temperature , BenzenesulfonamidesABSTRACT
The synthesis and structure-activity relationship of a series of indole inhibitors of cytosolic phospholipase A2alpha (cPLA2alpha, type IVA phospholipase) are described. Inhibitors of cPLA2alpha are predicted to be efficacious in treating asthma as well as the signs and symptoms of osteoarthritis, rheumatoid arthritis, and pain. The introduction of a benzyl sulfonamide substituent at C2 was found to impart improved potency of these inhibitors, and the SAR of these sulfonamide analogues is disclosed. Compound 123 (Ecopladib) is a sub-micromolar inhibitor of cPLA2alpha in the GLU micelle and rat whole blood assays. Compound 123 displayed oral efficacy in the rat carrageenan air pouch and rat carrageenan-induced paw edema models.
