Association of the Endotoxin Antagonist E5564 with High-Density Lipoproteins In Vitro: Dependence on Low-Density and Triglyceride-Rich Lipoprotein Concentrations

Abstract

The objective of this study was to determine the distribution profile of the novel endotoxin antagonist E5564 in plasma obtained from fasted human subjects with various lipid concentrations. Radiolabeled E5564 at 1 M was incubated in fasted plasma from seven human subjects with various total cholesterol (TC) and triglyceride (TG) concentrations for 0.5 to 6 h at 37°C. Following these incubations, plasma samples were separated into their lipoprotein and lipoprotein-deficient fractions by ultracentrifugation and were assayed for E5564 radioactivity. TC, TG, and protein concentrations in each fraction were determined by enzymatic assays. Lipoprotein surface charge within control and phosphatidylinositol-treated plasma and E5564's influence on cholesteryl ester transfer protein (CETP) transfer activity were also determined. We observed that the majority of E5564 was recovered in the high-density lipoprotein (HDL) fraction. We further observed that incubation in plasma with increased levels of TG-rich lipoprotein (TRL) lipid (TC and TG) concentrations resulted in a significant increase in the percentage of E5564 recovered in the TRL fraction. In further experiments, E5564 was preincubated in human TRL. Then, these mixtures were incubated in hypolipidemic human plasma for 0.5 and 6 h at 37°C. Preincubation of E5564 in purified TRL prior to incubation in human plasma resulted in a significant decrease in the percentage of drug recovered in the HDL fraction and an increase in the percentage of drug recovered in the TRL and low-density lipoprotein fractions. These findings suggest that the majority of the drug binds to HDLs. Preincubation of E5564 in TRL prior to incubation in normolipidemic plasma significantly decreased the percentage of drug recovered in the HDL fraction. Modifications to the lipoprotein negative charge did not alter the E5564 concentration in the HDL fraction. In addition, E5564 does not influence CETP-mediated transfer activity. Information from these studies could be used to help identify the possible components of lipoproteins which influence the interaction of E5564 with specific lipoprotein particles.Inflammatory shock as a consequence of lipopolysaccharide (LPS) or endotoxin release from gram-negative bacteria remains a serious clinical concern (3). In humans, inflammatory responses to LPS result in the release of cytokines and other cell mediators from monocytes and macrophages, which can cause fever, shock, organ failure, and death (3). A number of different approaches have been investigated to try to treat and/or prevent the septic shock associated with infections caused by gram-negative bacteria, including blocking of one or more of the cytokines induced by LPS (14). Recently, novel amphipathic compounds E5531 and E5564 have been developed as direct antagonists of LPS at the LPS receptor, TLR4 (5).Our laboratory has previously reported that the majority of E5531 (an analogue of E5564 which has chemical properties and pharmacological activity similar to those of E5531) associates with high-d...

Extended in Vivo Pharmacodynamic Activity of E5564 in Normal Volunteers with Experimental Endotoxemia

“…In vitro analyses of the antagonistic activity of E5564 after infusion into healthy volunteers determined that even though E5564 has a long pharmacokinetic half-life and metabolism of E5564 is not observed, E5564 demonstrates a relatively short pharmacodynamic (PD) half-life (Wong et al, 2003). In vivo research further supports this observation by demonstrating that low doses of E5564 are extremely active when coadministered with LPS in the human endotoxemia model but rapidly loses antagonistic activity , likely due to its interaction with plasma lipoproteins (Wasan et al, 2003). Similar loss in PD activity has been observed in a dog endotoxin infusion model (Suganuma et al, 2000).…”

“…We have previously shown that loss in antagonistic activity can be observed in vitro in human whole blood (Mullarkey et al, 2003) and is likely due to its interaction with plasma lipoproteins (Rose et al, 2000;Wasan et al, 2003). However, higher concentrations of E5564 (Ն1 M) are not quantitatively inactivated in vitro by whole blood, even after overnight incubation (data not shown), indicating that inactivation of E5564 is either time-dependent or is a "saturable" process.…”

“…LPSinduced adverse events are reported by incidence. Pharmacokinetics and distribution of E5564 into plasma lipoproteins was performed as described (Wasan et al, 2003;Wong et al, 2003). Samples for distribution into lipoprotein were available for five high cholesterol and three low cholesterol subjects.…”

Safety, Pharmacokinetics, Pharmacodynamics, and Plasma Lipoprotein Distribution of Eritoran (E5564) during Continuous Intravenous Infusion into Healthy Volunteers

“…As expected from in vitro studies (15), eritoran partitions predominantly (ϳ60%) into HDL in plasma. However, as described in Tables 3 and 4, the percentage of eritoran recovered within the TRL fraction proportionally increases with increasing TRL TG or cholesterol.…”

“…Preliminary work by Wasan et al using radiolabeled E5531, a similarly structured narrow-spectrum LPS antagonist (16), and, more recently, [ 14 C]eritoran (15) suggests that these types of molecules predominantly bind to high-density lipoproteins (HDL) during incubation in human serum and that interaction with lipoproteins occurs within 5 min, with no measurable redistribution between lipoprotein fractions (16). Studies by Rose and coworkers have supported the importance of plasma lipoprotein binding in influencing the long-term effectiveness of E5531 (8).…”