␣-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.

Shock due to Gram-negative bacterial sepsis is a consequence of acute inflammatory response to lipopolysaccharide (LPS) or endotoxin released from bacteria. LPS is a major constituent of the outer membrane of Gram-negative bacteria, and its terminal disaccharide phospholipid (lipid A) portion contains the key structural features responsible for toxic activity. Based on the proposed structure of nontoxic Rhodobacter capsulatus lipid A, a fully stabilized endotoxin antagonist E5531 has been synthesized. In vitro, E5531 demonstrated potent antagonism of LPS-mediated cellular activation in a variety of systems. In vivo, E5531 protected mice from LPS-induced lethality and, in cooperation with an antibiotic, protected mice from a lethal infection of viable Escherichia coli.

The Mycobacterium bovis bacillus Calmette-Guérin (BCG) cell wall skeleton (CWS) consists of mycolic acids, arabinogalactan, and peptidoglycan (PGN) and activates Toll-like receptor 2 (TLR2) and TLR4. Here we investigated the ability of the essential portion of highly purified BCG CWS to support the TLR agonist function by using the following criteria: myeloid dendritic cell (DC) maturation, i.e., tumor necrosis factor alpha (TNF-␣) production and CD83/CD86 up-regulation. The purified PGN region was sufficient to activate TLR2 and TLR4 in mouse DCs and macrophages; in TLR2 and TLR4 double-knockout cells the BCG PGN-mediated TNF-␣ production ability was completely impaired. Likewise, stimulation with BCG CWS of HEK293 cells expressing either human TLR2 or TLR4, MD-2, and CD14 resulted in NF-B activation as determined by a reporter assay. Notably, specific blockers of extracellular human TLR2 (an original cocktail of monoclonal antibodies TLR2.45 and TH2.1) and TLR4 (E5531) inhibited BCG CWS-mediated NF-B activation by 80%. Using this human TLR blocking system, we tested whether human myeloid DC maturation was TLR2 and TLR4 dependent. BCG PGN-mediated DC maturation was blocked by 70% by suppression of both TLR2 and TLR4 and by 30 to 40% by suppression of either of these TLRs. Similar but less profound suppression of BCG CWS-mediated DC maturation was observed. Hence, the presence of BCG PGN is a minimal requirement for activation of both TLR2 and TLR4 in human DCs, unlike the presence of PGNs of gram-positive bacteria, which activate only TLR2. Unexpectedly, however, BCG PGN, unlike BCG CWS, barely activated NF-B in HEK293 cells coexpressing TLR2 plus TLR1, TLR2 plus TLR4, TLR2 plus TLR6, or TLR2 plus TLR10, suggesting that PGN receptors other than TLR2 and TLR4 present on human DCs but not on HEK293 cells are involved in TLR signaling for DC activation.Phagocytosis of Mycobacterium tuberculosis by antigen-presenting cells is usually accompanied by activation of the transcription factor NF-B, secretion of inflammatory and initial cytokines, release of the reactive nitrites, including NO, and secretion of several chemokines (9, 16). These responses involve the outputs of the signaling of pattern recognition receptors for microbes (16, 34). More than 10 members of the mammalian Toll-like receptor (TLR) family in the innate immune system have been identified as representatives of such receptors that primarily respond to microbial constituents to elicit the immune response in macrophages and dendritic cells (DCs) (25,34). M. tuberculosis-mediated adjuvant activity may be expressed through TLRs on DCs.Two of the human TLRs, TLR2 and TLR4, are involved in M. tuberculosis-mediated intracellular signaling in vitro (22,41). Means et al. (22) demonstrated that viable M. tuberculosis bacilli contain distinct ligands that activate cells via TLR2 and TLR4, while heat-killed M. tuberculosis fails to activate cells via TLR4. Several purified mycobacterial ligands have now been identified as TLR2 agonists. Underhill et al. (43) ...

Novel adenosine antagonists, 2-alkynyl-8-aryl-9-methyladenine derivatives, were synthesized as candidate hypoglycemic agents. These analogues were evaluated for inhibitory activity on N-ethylcarboxamidoadenosine (NECA)-induced glucose production in primary cultured rat hepatocytes. In general, aromatic moieties at the 8-position and alkynyl groups at the 2-position had significantly increased activity compared to unsubstituted compounds. The preferred substituents at the 8-position of adenine were the 2-furyl and 3-fluorophenyl groups. In modifying the alkynyl side chain, change of the ring size, cleavage of the ring, and removal of the hydroxyl group were well tolerated. The order of the stimulatory effects of adenosine agonists on rat hepatocytes was NECA > CPA > CGS21680, which is consistent with involvement of the A(2B) receptor. In Chinese hamster ovary cells stably transfected with human A(2B) receptor cDNA, one of the compounds potent in hepatocytes, 15o (IC(50) = 0.42 microM), antagonized NECA-induced stimulation of cyclic AMP production (IC(50) = 0.063 microM). This inhibitory effect was much more potent than those of FK453, KF17837, and L249313 which have been reported to be respectively A(1), A(2A), and A(3) selective antagonists. These findings agree very well with the result that, compared to 15o, these selective antagonists for each receptor subtype showed only marginal effects in rat hepatocytes. These results suggest that adenosine agonist-induced glucose production in rat hepatocytes is mediated through the A(2B) receptor. Furthermore, 15o showed hypoglycemic activity in an animal model of noninsulin-dependent diabetes mellitus, the KK-A(y) mice. It is possible that inhibition of hepatic glucose production via the A(2B) receptor could be at least one of the mechanisms by which 15o exerts its in vivo effects. Further elaboration of this group of compounds may afford novel antidiabetic agents.

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