INTRODUCTION AND RATIONALE: The innate immune response is the first line of defense against invading pathogens(1). This tightly regulated system consists of a wide variety of chemokines, cytokines, cell associated receptors and other mediators orchestrating the initial response to infection. Experimental evidence in the past several years has demonstrated that activation of the efferent vagus nerve has an inhibitory effect on the innate immune response (the cholinergic anti-inflammatory pathway, reviewed in (2,3,4)). The anti-inflammatory effect of the vagal nerve is mediated by the α7 nicotinic acetylcholine receptor (α7nAChR) expressed on macrophages and other cytokine-producing cells (9). Activation of the cholinergic anti-inflammatory pathway with vagus nerve stimulation or α7nAChR agonists improves outcome in animal models of endotoxemia, sepsis and experimental arthritis. This cholinergic anti-inflammatory pathway is the efferent arm of a reflex mechanism that is activated by inflammatory mediators in peripheral tissues signaling through the afferent vagus nerve. Thus, the vagus nerve can relay inflammation signals to the central nervous system and activate an opposing efferent response to inhibit excessive inflammation.

GTS-21 (E-3-(2,4)-dimethoxybenzylidene anabaseine) is a highly specific α7nAchR agonist, that has been developed for the treatment of Alzheimer's disease that has been studied in a few inflammation-related models. In a murine pancreatitis model pre-treatment of the animals with GTS-21 decreased pancreatitis severity and was associated with reduced IL-6 levels and decreased pancreatic neutrophil accumulation (11). Pre-treatment of mice with GTS-21 attenuated systemic TNF concentrations after injection with LPS and increases survival (12,13). Similarly, GTS-21 inhibited blood levels of the inflammatory mediator High Mobility Group Box 1 (HMGB1) and improved survival of mice after cecal ligation and puncture.(13). Recently, we have shown that stimulation of the α7nAChR by the highly selective agonist GTS-21 leads to a dramatic dose-dependent inhibition of pro-inflammatory cytokine release induced by stimulation of various Toll-like receptors (TLR) in human whole blood (Kox et al, submitted). This inhibitory effect of GTS-21 was not specific for the TLR-agonist used and GTS-21 inhibited pro-inflammatory cytokine production stronger than nicotine at equimolar concentrations. Moreover, the production of the anti-inflammatory cytokine IL-10 was not inhibited, but even significantly stimulated. Therefore, stimulation of the α7nAChR by GTS-21 results in a profound anti-inflammatory shift of the pro-/anti-inflammatory balance. So far, no data are available on the anti-inflammatory effects of GTS-21 in humans in vivo.

The human endotoxemia model is widely used to study inflammation in humans in vivo. Systemic inflammation is induced by low-dose infusion of Escherichia coli lipopolysaccharide (LPS) in healthy volunteers, resulting in flu-like symptoms, increased production of C-reactive protein and increased concentrations of pro- and anti-inflammatory cytokines. The "human endotoxemia model" permits elucidation of key players in this proinflammatory response in humans in vivo and it serves as a useful tool to investigate potential novel therapeutic strategies in a standardized setting.

GTS-21 was developed as a novel agent in the treatment of Alzheimer's disease. Until date, 87 healthy male volunteers were enrolled in four Phase I studies that assessed the safety, tolerability, pharmacokinetics, and effects on cognitive function of oral administration of GTS-21 of which 77 subjects received GTS-21 and 10 subjects received placebo. GTS-21 was found to be well tolerated up to a dose of 150 mg three times daily (450 mg/day) in healthy male subjects. The most common adverse event was headache, which occurred in 27% of subjects in the GTS-21 group compared to 21% of subjects in the placebo group. There were no serious adverse events, or severe adverse events reported during these studies. In one patient in the GTS-21 group transient mild elevation of liver enzymes was detected, without signs of hepatic dysfunction.

PRIMARY AIM: Primary aim of the present study is to investigate the putative anti-inflammatory effects of oral administration of GTS-21 on the inflammatory response and subsequent subclinical organ dysfunction in the human endotoxemia model. Secondary aim is to measure the effect of LPS administration in the absence and presence of GTS-21 in human volunteers on vagal nerve activity.