alpha-Neup5Ac-(2--3)-beta-D-Galp-(1--4)-[alpha-L-Fucp-(1--3)]-D-GlcpNAc and Peritonitis

Heparin has been used clinically as an anticoagulant and antithrombotic agent for over 60 years. Here we show that the potent anti-inflammatory property of heparin results primarily from blockade of P-selectin and L-selectin. Unfractionated heparin and chemically modified analogs were tested as inhibitors of selectin binding to immobilized sialyl Lewis(X) and of cell adhesion to immobilized selectins or thrombin-activated endothelial cells. Compared with unfractionated heparin, the modified heparinoids had inhibitory activity in this general order: over-O-sulfated heparin > heparin > 2-O,3-O-desulfated > or = N-desulfated/N-acetylated heparin > or = carboxyl-reduced heparin > or= N-,2-O,3-O-desulfated heparin >> 6-O-desulfated heparin. The heparinoids also showed similar differences in their ability to inhibit thioglycollate-induced peritonitis and oxazolone-induced delayed-type hypersensitivity. Mice deficient in P- or L-selectins showed impaired inflammation, which could be further reduced by heparin. However, heparin had no additional effect in mice deficient in both P- and L-selectins. We conclude that (a) heparin's anti-inflammatory effects are mainly mediated by blocking P- and L-selectin-initiated cell adhesion; (b) the sulfate groups at C6 on the glucosamine residues play a critical role in selectin inhibition; and (c) some non-anticoagulant forms of heparin retain anti-inflammatory activity. Such analogs may prove useful as therapeutically effective inhibitors of inflammation.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Adhesion; Dermatitis, Allergic Contact; Disease Models, Animal; Glucosamine; Heparin; Humans; Inflammation; L-Selectin; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Structure; Oligosaccharides; P-Selectin; Peritonitis; Sialyl Lewis X Antigen; Sulfates; U937 Cells

1. We investigated the ability of a newly synthesized sugar derivative, OJ-R9188, [N-(2-tetradecylhexadecanoyl)-O-(L-alpha-fucofuranosyl)-D-seryl]-L-glutamic acid 1-methylamide 5-L-arginine salt, to block binding of selectins to their ligands in vitro and inhibit the infiltration of leukocytes in vivo. 2. OJ-R9188 prevented the binding of human E-, P- and L-selectin-IgG fusion proteins to immobilized sialyl Lewis(x) (sLe(x))-pentasaccharide glycolipid, with IC(50) values of 4.3, 1.3, and 1.2 microM, respectively. 3. In a mouse model of thioglycollate-induced peritonitis, OJ-R9188 at 10 mg kg(-1), i.v. inhibited neutrophil accumulation in the peritoneal cavity. In the IgE-mediated skin reaction, OJ-R9188 at 3 and 10 mg kg(-1), i.v. significantly inhibited extravasation of neutrophils and eosinophils into the inflammatory sites and at 10 mg kg(-1), i.v. also inhibited infiltration caused by picryl chloride-induced delayed-type hypersensitivity in mice. These results suggest that OJ-R9188 may be a useful selectin blocker, with activity against human and mouse E-, P- and L-selectins in vitro and in vivo, and that blocking selectin-sLe(x) binding is a promising strategy for the treatment of allergic skin diseases.

Topics: Animals; Anti-Inflammatory Agents; Binding, Competitive; Deoxyguanosine; Dermatitis; Dexamethasone; Dose-Response Relationship, Drug; E-Selectin; Free Radical Scavengers; Humans; Hypersensitivity, Delayed; Immunoglobulin G; L-Selectin; Male; Mice; Mice, Inbred BALB C; Oligosaccharides; P-Selectin; Peritonitis; Picryl Chloride; Polysaccharides; Recombinant Fusion Proteins; Selectins; Sialyl Lewis X Antigen; Thioglycolates

P-selectin is a cell adhesion molecule of the selectin family. This study evaluated the effects of novel, low molecular weight P-selectin inhibitors in a cell adhesion assay and a murine model of peritonitis.. U937 or HL60 was used for cell adhesion assay. Human polymorphonuclear cells were studied for the production of superoxide. BALB/c mice were used for the in vivo study.. The thioglycollate (TG)-induced accumulation of leukocytes in mice was measured 6 h after the treatment. KF38789 or antibody (1 mg/kg) was injected intravenously prior to TG injection and at 3 h following initial injection.. Low molecular weight, non-carbohydrate inhibitors against P-selectin- mediated cell adhesion were tested. One of the most potent inhibitors, KF38789, inhibited the binding of U937 cells to immobilized P-selectin immunoglobulin G chimeric protein (P-selectin-Ig) with an IC50 value of 1.97 microM. Cell adhesion to both E-selectin-Ig and L-selectin-Ig were not affected even by 100 microM of KF38789. Moreover, KF38789 inhibited P-selectin-induced superoxide production from human polymorphonuclear cells. Intravenously injected KF38789 significantly inhibited the TG-induced accumulation of leukocytes in the mouse peritoneal cavity (p<0.01).. A novel low molecular weight compound, KF38789, specifically inhibited P-selectin-dependent cell adhesion and the leukocyte recruitment in mouse peritonitis.

Topics: Animals; Antibodies, Monoclonal; Cell Adhesion; Humans; Mice; Mice, Inbred BALB C; Neutrophils; Oligosaccharides; P-Selectin; Peritonitis; Pyrones; Sialyl Lewis X Antigen; Superoxides; Thioglycolates; U937 Cells

An early step of the inflammatory response-the rolling of leukocytes on activated endothelial cells-is mediated by selectin/carbohydrate interactions. The tetrasaccharide sialyl Lewis(x) (sLe(x)) 1 is a ligand for E-, P-, and L-selectin and, therefore, serves as a lead structure to develop analogues which allow the control of acute and chronic inflammation. Here we describe the efficient synthesis (10 linear steps) of the potent sLe(x) mimetic 2. Compared to sLe(x), compound 2 showed a 30-fold improved affinity in a static, cell-free E-selectin-ligand binding assay (IC(50) = 36 microM). These data were confirmed by a marked inhibition in an in vitro cell-cell rolling assay which simulates in vivo conditions (IC(50) approximately 40 microM). The assays are predictive for the in vivo efficacy of test compounds as indicated by a marked inhibitory effect of 2 in a thioglycollate induced peritonitis model of acute inflammation in mice (ED(50) approximately 15 mg/kg).

Topics: Acute Disease; Animals; Carbohydrate Sequence; E-Selectin; Ligands; Mice; Molecular Mimicry; Molecular Sequence Data; Oligosaccharides; Peritonitis; Sialyl Lewis X Antigen; Structure-Activity Relationship; Thioglycolates

Neutrophil recruitment into systemic inflammatory sites in vivo is thought to be initiated by selectin-mediated endothelial adherence. The effect of fucoidan (natural sulfated polymer of L-fucose) on the selectin dependent PMN migration into rat peritoneum following the induction of inflammation by peptone injection was studied. Peritonitis was characterized by an increase in the total cell number (from 45.3 x 10(6) to 91.6 x 10(6)/rat), and by highly elevated PMN content (from 0.2% to 58%) in the rat peritoneal cavity 3 h after peptone injection. Intravenous administration of fucoidan was found to reduce, in a dose-dependent manner, neutrophil migration into peritoneum. Fucoidan in a dose as low as 0.8 mg per rat caused 96.8% reduction of neutrophil extravasation. The inhibitory effect of fucoidan was also dependent on the time intervals between the peptone and fucoidan injections. The maximal inhibitory effect of fucoidan was observed within the first 15 min after the induction of peritonitis and it was maintained at a level of 80% during 1.5 h. Administration of fucoidan 2.5 h after peptone injection had practically no effect on PMN extravasation. Since P-selectin is known to play a key role at the earlier stages of PMN extravasation, it was suggested that the inhibitory effect of fucoidan was mostly due to its interaction with P-selectin. The in vitro experiments demonstrated the high affinity of fucoidan for both isolated P-selectin and P-selectin in plasma membranes of activated platelets.

Topics: Animals; Biotinylation; Blood Platelets; CA-19-9 Antigen; Carbohydrate Metabolism; Cell Movement; Female; Gangliosides; Humans; Kinetics; Neutrophils; Oligosaccharides; P-Selectin; Peptones; Peritonitis; Polysaccharides; Rats; Rats, Wistar; Sialyl Lewis X Antigen