Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are outer-membrane constituents of Gram-negative bacteria. Lipopolysaccharides play a key role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient. Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist at the present time. We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS and, using animal models of sepsis, have shown that the sequestration of circulatory LPS by small molecules is a therapeutically viable strategy. In this paper, the interactions of a series of acylated homologated spermine compounds with lipopolysaccharide (LPS) have been characterized. The optimal acyl chain length for effective sequestration of LPS was identified to be C 16 for the mono-acyl compounds. The most promising of these compounds, 4e, binds LPS with an ED 50 of 1.37 μM. Nitric oxide production in murine J774A.1 cells, as well as TNF-α in human blood, are inhibited in a dose-dependent manner by 4e at concentrations orders of magnitude lower than toxic doses. Administration of 4e to D-galactosamine-sensitized mice challenged with supralethal doses of LPS provided significant protection against lethality. Potent anti-endotoxic activity, low toxicity, and ease of synthesis render this class of compounds candidate endotoxin-sequestering agents of potential significant therapeutic value.

The role of lipopolysaccharide (LPS) in the pathogenesis of Gram-negative septic shock is well established. The corresponding proinflammatory and immunostimulatory molecule(s) on the Gram-positive bacteria is less well understood, and its identification and characterization would be a key prerequisite in designing specific sequestrants of the Gram-positive endotoxin(s). We report in this paper the comparison of NF-kappaB-, cytokine- and chemokine-inducing activities of the TLR2 ligands, lipoteichoic acid (LTA), peptidoglycan (PGN), and lipopeptides, to LPS, a prototype TLR4 agonist, in murine macrophage cell-lines as well as in human blood. In murine cells, di- and triacyl liopopeptides are equipotent in their NF-kappaB inducing activity relative to LPS, but elicit much lower proinflammatory cytokines. However, both LPS and the lipopeptides potently induce the secretion of a pattern of chemokines that is suggestive of the engagement of a TLR4-independent TRIF pathway. In human blood, although the lipopeptides induce p38 MAP kinase phosphorylation and CD11b upregulation in granulocytes at ng/ml concentrations, they do not elicit proinflammatory cytokine production even at very high doses; LTA, however, activates neutrophils and induces cytokine secretion, although its potency is considerably lower than that of LPS, presumably due to its binding to plasma proteins. We conclude that, in human blood, the pattern of immunostimulation and proinflammatory mediator production elicited by LTA parallels that of LPS.

Lipopolysaccharide (LPS), or endotoxin, a structural component of gram-negative bacterial outer membranes, plays a key role in the pathogenesis of septic shock, a syndrome of severe systemic inflammation which leads to multiple-system organ failure. Despite advances in antimicrobial chemotherapy, sepsis continues to be the commonest cause of death in the critically ill patient. This is attributable to the lack of therapeutic options that aim at limiting the exposure to the toxin and the prevention of subsequent downstream inflammatory processes. Polymyxin B (PMB), a peptide antibiotic, is a prototype small molecule that binds and neutralizes LPS toxicity. However, the antibiotic is too toxic for systemic use as an LPS sequestrant. Based on a nuclear magnetic resonance-derived model of polymyxin B-LPS complex, we had earlier identified the pharmacophore necessary for optimal recognition and neutralization of the toxin. Iterative cycles of pharmacophore-based ligand design and evaluation have yielded a synthetically easily accessible N 1 ,mono-alkyl-mono-homologated spermine derivative, DS-96. We have found that DS-96 binds LPS and neutralizes its toxicity with a potency indistinguishable from that of PMB in a wide range of in vitro assays, affords complete protection in a murine model of LPS-induced lethality, and is apparently nontoxic in vertebrate animal models.Endotoxin, or lipopolysaccharide (LPS), a structural component of the outer membrane of most gram-negative bacteria (31), plays a pivotal role in septic shock, a syndrome of systemic toxicity which occurs frequently as a sequel to serious systemic gram-negative infections (23). The activation by LPS of the innate immune response, mediated via toll-like receptor 4 (TLR4) (39), leads to a dysregulated production of numerous inflammatory mediators, including tumor necrosis factor alpha (TNF-␣), interleukin-1␤ (IL-1␤), and IL-6 (11), gamma interferon (IFN-␥), and IL-12, which appears to be inadequately compensated for by the production of anti-inflammatory cytokines, such as IL-10 and transforming growth factor ␤ (6). The resultant systemic inflammatory response progresses to the frequently fatal syndrome of multiple-system organ failure (3). Despite continuing advances in antimicrobial chemotherapy, the incidence of sepsis has risen almost threefold from 1979 through 2000 (25), emphasizing an urgent, unmet need to develop therapeutic options specifically targeting the pathophysiology of sepsis.The toxicity of LPS resides in its structurally highly conserved glycolipid component called lipid A (22), which is composed of a hydrophilic, bis-phosphorylated diglucosamine backbone, and a hydrophobic domain comprised of acyl chains in amide and ester linkages (14). Polymyxin B (PMB) is a membrane-active peptide antibiotic (37) known to sequester LPS and abrogate its toxicity (12, 16). The otoand nephrotoxicity of PMB limit its systemic use and have led to the development of an extracorporeal hemoperfusion cartridge based on PMB covalently immobilized on a polysty...

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