Debra G. Goodwin

IL-22 Induces an Acute-Phase Response

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.

Indole Cytosolic Phospholipase A2 α Inhibitors: Discovery and in Vitro and in Vivo Characterization of 4-{3-[5-Chloro-2-(2-{[(3,4-dichlorobenzyl)sulfonyl]amino}ethyl)-1-(diphenylmethyl)-1H-indol-3-yl]propyl}benzoic Acid, Efipladib

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.

Selective functional inhibition of JAK‐3 is sufficient for efficacy in collagen‐induced arthritis in mice

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.

Reactions of Functionalized Sulfonamides: Application to Lowering the Lipophilicity of Cytosolic Phospholipase A2α Inhibitors∥

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.

The DTH effector response and IL-2 are unaffected by cyclosporine A in autoimmune B6D2F1 mice.

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.

The cPLA2α inhibitor efipladib decreases nociceptive responses without affecting PGE2 levels in the cerebral spinal fluid.

The contribution of central PGE(2) levels to the nociceptive response in rats was assessed and the effects of the selective cPLA(2)α inhibitor efipladib, and pain therapies of different classes on these responses was determined. An inflammatory pain model was optimized in rats so that PGE(2) levels in the cerebrospinal fluid (CSF) could be directly correlated to the nociceptive response. Since efipladib appears to have limited permeation of the blood-brain barrier, we used this compound to determine the extent of pain reversal resulting primarily from peripheral, but not central, inhibition of the arachidonic acid (AA) pathway. The nociceptive response was significantly inhibited by orally administered efipladib, yet spinal fluid levels of PGE(2) and temperature measurements were unaffected compared to vehicle-treated animals. Conversely, intrathecal (IT) administration of efipladib reduced PGE(2) levels in the CSF by 45-60%, yet there was no effect on the nociceptive response. With COX-2 selective inhibitors and ibuprofen, a return of the nociceptive response developed over time, despite complete inhibition of PGE(2) in the spinal fluid. The opposite was true with low doses of indomethacin: inhibition of the nociceptive response was observed despite the lack of effect on central PGE(2) levels. Our results demonstrate that levels of PGE(2) in the spinal fluid do not directly correlate with the nociceptive response and that blocking cPLA(2)α in the periphery significantly decreases inflammatory pain.

CRTH2 is Critical to the Development of Colitis Induced by Dextran Sodium Sulfate (DSS)

Chemoattractant Receptor-homologous molecule expressed on Th2 cells [CRTH2] is expressed on granulocytes (eosinophils, basophils and neutrophils), Th2 cells and monocytes. CRTH2 has been implicated in the development of cutaneous inflammation as CRTH2+ CD4+ T cells and CRTH2+ eosinophils are increased in the blood of atopic dermatitis patients. CRTH2 is also up-regulated in circulating neutrophils of psoriatic patients. Interestingly, CRTH2 is detected in the mucosa of patients with Inflammatory Bowel Disease (IBD) ulcerative colitis, where the CRTH2 positive cells localize within and adjacent to regions of inflamed mucosa suggesting that CRTH2 may play a role in IBD. However, the exact downstream inflammatory pathways resulting from CRTH2 activation in colitis are not well characterized. To gain a better understanding of the effects of CRTH2 activation in the pathogenesis of colitis, we have generated a small molecule inhibitor against CRTH2 and its potency was first validated in an oxazolone induced contact dermatitis model. We investigated the consequences of inhibiting CRTH2 in the development of IBD using a Dextran Sodium Sulfate (DSS)-induced colitis mouse model. Compared to the vehicle control group, mice treated with a selective CRTH2 antagonist had reduced disease severity as measured by weight loss, as well as serum acute phase protein, haptoglobin. Furthermore, pro-inflammatory cytokine gene expression for TNFα, IL-1β, IL-6, IL-17A, and IFNγ were reduced in colons of mice that had been treated with the CRTH2 antagonist compared with DSS treated controls that received vehicle. Taken together, our data identify a previously unrecognized role for CRTH2 in the initiation/amplification and/or stabilization of colon inflammation.