Leukotriene B 4 ((5S,12R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB 4 )) 1 is a metabolite of arachidonic acid and is one of the most potent activators of granulocytes and macrophages (1-3). BLT, the LTB 4 -specific G-protein-coupled receptor (GPCR), is a target for anti-inflammatory drugs, and many BLT antagonists have been developed and are being evaluated. No BLT antagonists are yet approved available for clinical use; their lack of efficacy may be due in part to the presence of the other LTB 4 receptors. We cloned BLT1, a high affinity LTB 4 receptor (4), and BLT2, a low affinity LTB 4 receptor (5), and showed that BLT1 is expressed almost exclusively in peripheral leukocytes, whereas BLT2 is expressed ubiquitously with the highest expression in spleen. The structural similarities of these receptors (45% amino acid identity) and the low homologies of BLTs to other known GPCRs suggest that these BLTs comprise a novel receptor family. BLT1 and BLT2 form a gene cluster both in human and mouse genomes.The human BLT2 open reading frame overlaps the promoter region of BLT1 (6), suggesting the expression of these two LTB 4 receptors is tightly intertwined. Human granulocytes, eosinophils, and mononuclear cells express both BLT1 and BLT2 (7), so the precise pharmacological characterization of these two receptors using native cells is difficult. In this paper, we report the inhibitory effects of various BLT antagonists and eicosanoids on LTB 4 binding using CHO cells stably expressing either human BLT1 or BLT2. We also show that several hydroxyeicosanoids other than LTB 4 bind to and activate BLT2 but not BLT1. These findings provide insights into the possible functions of BLT2 and information helpful for the isolation of the other related GPCRs that recognize eicosanoids. (8) and all of the eicosanoids other than LTB 4 were purchased from Cayman Chemical Co. LTB 4 is a generous gift from Ono Pharmaceutical Co. (Osaka, Japan). LY 255283 (9) and LY 223982 (10) were from Lilly Research Laboratories. CP 105696 (11) and CP 195543 (12) are from Pfizer Inc. ZK 158252 is from Schering AG (Berlin, Germany). [ 3 H]LTB 4 (6956 GBq/mmol) was purchased from PerkinElmer Life Sciences. EXPERIMENTAL PROCEDURES Materials-U75302Cell Culture and Flow Cytometry-CHO cells stably expressing FLAG-tagged human BLT1 (CHO-FLAG-BLT1) or hemagglutinin (HA)-tagged human BLT2 (CHO-HA-BLT2) were established as described previously (5, 13). The cells were cloned by limiting dilution and their expression of the receptors confirmed by staining the cells with antibodies against the epitope added to the amino terminus of the receptors. The cells were fixed with PBS(Ϫ) containing 0.5% (w/v) paraformaldehyde for 5 min on ice and blocked with PBS(Ϫ) containing 2% goat serum (Life Technologies, Inc.). The cells were incubated with 30 g/ml anti-FLAG antibody (clone M5, Eastman Kodak), 10 g/ml anti-HA antibody (clone CA12-5, Roche Molecular Biochemicals), or 30 g/ml control mouse IgG (Santa Cruz Biotechnology) in PBS(Ϫ) containing 2% oat seru...

SummaryONO-4641 is a next-generation sphingosine 1-phosphate (S1P) receptor agonist selective for S1P receptors 1 and 5. The objective of the study was to characterize the immunomodulatory effects of ONO-4641 using preclinical data. ONO-4641 was tested in both in-vitro pharmacological studies as well as in-vivo models of transient or relapsing-remitting experimental autoimmune encephalomyelitis (EAE). In vitro, ONO-4641 showed highly potent agonistic activities versus S1P receptors 1 and 5 [half maximal effective concentration (EC50) values of 0·0273 and 0·334 nM, respectively], and had profound S1P receptor 1 down-regulating effects on the cell membrane. ONO-4641 decreased peripheral blood lymphocyte counts in rats by inhibiting lymphocyte egress from secondary lymphoid tissues. In a rat experimental autoimmune encephalomyelitis (EAE) model, ONO-4641 suppressed the onset of disease and inhibited lymphocyte infiltration into the spinal cord in a dose-dependent manner at doses of 0·03 and 0·1 mg/kg. Furthermore, ONO-4641 prevented relapse of disease in a non-obese diabetic mouse model of relapsing-remitting EAE. These observations suggest that ONO-4641 may provide therapeutic benefits in the treatment of multiple sclerosis.

Although IL-17 is a pro-inflammatory cytokine reportedly involved in various autoimmune inflammatory disorders, its role remains unclear in murine models of colitis. Acute colitis was induced by 2.5% dextran sodium sulfate (DSS) treatment for 5 days. A novel sphingosine-1-phosphate receptor agonist W-061, a prototype of ONO-4641, was orally administered daily, and histopathological analysis was performed on the colon. The number of lymphocytes and their cytokine production were also evaluated in spleen, mesenteric lymph node, Peyer's patch and lamina propria of the colon. Daily administration of W-061 resulted in improvement of DSS-induced colitis, and significantly reduced the number of CD4+ T cells in the colonic lamina propria. Numbers of both Th17 and Th1 cells were reduced by W-061 treatment. W-061, however, had no influence on the number of Treg cells in lamina propria. Thus, Th17 and Th1 cells in lamina propria were thought to be the key subsets in the pathogenesis of DSS-induced colitis. In conclusion, W-061 may be a novel therapeutic strategy to ameliorate acute aggravation of inflammatory bowel diseases.

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Many butterflies acquire nutrients from non-nectar sources such as puddles. To better understand how male Papilio butterflies identify suitable sites for puddling, we used behavioral and electrophysiological methods to examine the responses of Japanese Papilio butterflies to Na+, K+, Ca2+, and Mg2+. Based on behavioral analyses, these butterflies preferred a 10-mM Na+ solution to K+, Ca2+, and Mg2+ solutions of the same concentration and among a tested range of 1 mM to 1 M NaCl. We also measured the ion concentrations of solutions sampled from puddling sites in the field. Na+ concentrations of the samples were up to 6 mM, slightly lower than that preferred by butterflies in the behavioral experiments. Butterflies that sipped the 10 mM Na+ solution from the experimental trays did not continue to puddle on the ground. Additionally, butterflies puddled at sites where the concentrations of K+, Ca2+, and/or Mg2+ were higher than that of Na+. This suggests that K+, Ca2+, and Mg2+ do not interfere with the detection of Na+ by the Papilio butterfly. Using an electrophysiological method, tip recordings, receptor neurons in contact chemosensilla inside the proboscis evoked regularly firing impulses to 1, 10, and 100 mM NaCl solutions but not to CaCl2 or MgCl2. The dose–response patterns to the NaCl solutions were different among the neurons, which were classified into three types. These results showed that Japanese Papilio butterflies puddle using Na+ detected by the contact chemosensilla in the proboscis, which measure its concentration.Electronic supplementary materialThe online version of this article (doi:10.1007/s00114-012-0976-3) contains supplementary material, which is available to authorized users.

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The discovery of 1-({6-[(2-methoxy-4-propylbenzyl)oxy]-1-methyl-3,4-dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylic acid 13n (ceralifimod, ONO-4641), a sphingosine-1-phosphate (S1P) receptor agonist selective for S1P and S1P, is described. While it has been revealed that the modulation of the S1P receptor is an effective way to treat autoimmune diseases such as relapsing-remitting multiple sclerosis (RRMS), it was also reported that activation of the S1P receptor is implicated in some undesirable effects. We carried out a structure-activity relationship (SAR) study of hit compound 6 with an amino acid moiety in the hydrophilic head region. Following identification of a lead compound with a dihydronaphthalene central core by inducing conformational constraint, optimization of the lipophilic tail region led to the discovery of 13n as a clinical candidate that exhibited >30 000-fold selectivity for S1P over S1P and was potent in a peripheral lymphocyte lowering (PLL) test in mice (ED = 0.029 mg/kg, 24 h after oral dosing).