title: Inhibition of endotoxin response by e5564, a novel Toll-like receptor 4-directed endotoxin antagonist.
abstract: Alpha-D-glucopyranose,3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-[(3R)-3-methoxydecyl]-6-O-methyl-2-[[(11Z)-1-oxo-11-octadecenyl]amino]-4-O-phosphono-beta-D-glucopyranosyl]-2-[(1,3-dioxotetradecyl)amino]-1-(dihydrogen phosphate), tetrasodium salt (E5564) is a second-generation synthetic lipodisaccharide designed to antagonize the toxic effects of endotoxin, a major immunostimulatory component of the outer cell membrane of Gram negative bacteria. In vitro, E5564 dose dependently (nanomolar concentrations) inhibited lipopolysaccharide (LPS)-mediated activation of primary cultures of human myeloid cells and mouse tissue culture macrophage cell lines as well as human or animal whole blood as measured by production of tumor necrosis factor-alpha and other cytokines. E5564 also blocked the ability of Gram negative bacteria to stimulate human cytokine production in whole blood. In vivo, E5564 blocked induction of LPS-induced cytokines and LPS or bacterial-induced lethality in primed mice. E5564 was devoid of agonistic activity when tested both in vitro and in vivo and has no antagonistic activity against Gram positive-mediated cellular activation at concentrations up to 1 microM. E5564 blocked LPS-mediated activation of nuclear factor-kappaB in toll-like receptor 4/MD-2-transfected cells. In a mouse macrophage cell line, activity of E5564 was independent of serum, suggesting that E5564 exerts its activity through the cell surface receptor(s) for LPS, without the need for serum LPS transfer proteins. Similar to (6-O-[2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-Z-dodec-5-endoyloxydecl]-2-[3-oxo-tetradecanoylamino]-beta-O-phosphono-alpha-D-glucopyranose tetrasodium salt (E5531), another lipid A-like antagonist, E5564 associates with plasma lipoproteins, causing low concentrations of E5564 to be quantitatively inactivated in a dose- and time-dependent manner. However, compared with E5531, E5564 is a more potent inhibitor of cytokine generation, and higher doses retain activity for durations likely sufficient to permit clinical application. These results indicate that E5564 is a potent antagonist of LPS and lacks agonistic activity in human and animal model systems, making it a potentially effective therapeutic agent for treatment of disease states caused by endotoxin.
authors.affiliation: Biology Section, Eisai Research Institute of Boston, Inc., Andover, Massachusetts, USA.
authors.forename: Maureen
authors.initials: M
authors.lastname: Mullarkey
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Rose
authors.forename: John
authors.initials: J
authors.lastname: Bristol
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kawata
authors.forename: Akufumi
authors.initials: A
authors.lastname: Kimura
authors.forename: Seiichi
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Melinda
authors.initials: M
authors.lastname: Przetak
authors.forename: Jesse
authors.initials: J
authors.lastname: Chow
authors.forename: Fabian
authors.initials: F
authors.lastname: Gusovsky
authors.forename: William J
authors.initials: WJ
authors.lastname: Christ
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Rossignol
mesh_terms: D000818:Animals; D001769:Blood / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D004195:Disease Models, Animal; D029721:Drosophila Proteins; D004347:Drug Interactions; D004731:Endotoxins / Q000037:antagonists & inhibitors*; D004926:Escherichia coli / Q000737:chemistry; D006168:Guinea Pigs; D006801:Humans; D008050:Lipid A / Q000031:analogs & derivatives* / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012772:Shock, Septic / Q000378:metabolism / Q000401:mortality; D013997:Time Factors; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D016207:Cytokines; D029721:Drosophila Proteins; C404742:E5564; D004731:Endotoxins; D008050:Lipid A; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha