E5564, a synthetic lipid A analogue, is a selective, highly active antagonist of endotoxin-mediated activation of immune cells. Preclinical research has indicated that E5564 can block endotoxin-mediated induction of cytokines and endotoxin or Gram-negative bacterial-induced death in animal models. Recent phase I clinical trials have focused on the ability of E5564 to block responsiveness to endotoxin. This was done in two ways: in vivo challenge of human volunteers with 4 ng/kg endotoxin, and by use of an ex vivo assay which utilizes blood drawn from volunteers administered E5564 and challenged with endotoxin at concentrations that ranged from 50 pg/ml to 10 ng/ml. In vivo, 100 μg of E5564 completely blocked signs, symptoms and cytokines induced by concomitantly-administered endotoxin. In contrast, subjects receiving a 50 μg dose of E5564 demonstrated a graded response; cytokines were inhibited 95%, but many signs and symptoms of endotoxemia were still evident. E5564 demonstrated a long pharmacokinetic half-life (> 30 h); however, ex vivo analysis indicated that while single doses of 350 μg induced a nearly complete block of the effects of 1 ng/ml endotoxin immediately upon E5564 administration, antagonistic activity declined rapidly (t 1/2 < 1 h). Similar results were obtained in vivo using a delayed endotoxin challenge. These results have driven us to examine antagonistic activity of E5564 in vivo and ex vivo after administration by continuous infusion or twice-daily dosing. Results from these multiple-dose studies indicate that under these conditions of administration, plasma levels of E5564 can be predictive of long-term pharmacodynamic activity.