Inhibition of Endotoxin Response by E5564, a Novel Toll-Like Receptor 4-Directed Endotoxin Antagonist

Abstract

␣-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-␤-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-␣ 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 M. E5564 blocked LPSmediated activation of nuclear factor-B 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, 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.

Protection from lethal Gram-negative bacterial sepsis by targeting Toll-like receptor 4

“…An anti-TLR4 treatment strategy also is supported by recent data obtained with eritoran (E5564), a synthetic LPS antagonist that binds to MD-2 (12, 31), and TAK-242, a cyclohexene derivative that inhibits TLR4-mediated signal transduction, which prevented lethality in experimental models of LPS shock or bacterial sepsis in rodents (32,33). At a time when most antisepsis clinical trials have yielded frustratingly negative results (2, 34), our experimental data lend strong support to TLR4-targeted therapy (i.e., eritoran and TAK-242) currently under development in patients with Gram-negative sepsis.…”

Inhibition of toll‐like receptor 4 breaks the inflammatory loop in autoimmune destructive arthritis

“…Inhibition of the TLR-4 pathway using this TLR-4 antagonist substantially suppressed clinical and histologic manifestations of CIA. The dose of the antagonist in the prophylactic CIA study (400 g/kg body weight) was based upon the reported dose of other TLR-4 antagonists with similar structure (lipid A analog E5564) (41). For therapeutic treatment, we enhanced the dosage and shortened the intervals, because we expected generation and release of more inflammationand damage-associated TLR-4 agonists during existing disease.…”

Toll-Like Receptors in Health and Disease: Complex Questions Remain

“…TLR4 Ϫ/Ϫ mice are resistant to the effects of systemic endotoxin. LPS or lipid A from Rhodobacter sphaeroides, and the synthetic lipodisaccharide, E5564, prevent activation of human TLR4 by LPS, offering the possibility of new treatments for septic shock (70,71). Microbial targeting of intracellular signaling illustrates other approaches that could be adapted to become viable pharmaceuticals.…”