Daniel P. Rossignol

Effect of Eritoran, an Antagonist of MD2-TLR4, on Mortality in Patients With Severe Sepsis: The ACCESS Randomized Trial

IMPORTANCE Eritoran is a synthetic lipid A antagonist that blocks lipopolysaccharide (LPS) from binding at the cell surface MD2-TLR4 receptor. LPS is a major component of the outer membrane of gram-negative bacteria and is a potent activator of the acute inflammatory response. OBJECTIVE To determine if eritoran, a TLR4 antagonist, would significantly reduce sepsis-induced mortality. DESIGN, SETTING, AND PARTICIPANTS We performed a randomized, double-blind, placebo-controlled, multinational phase 3 trial in 197 intensive care units. Patients were enrolled from June 2006 to September 2010 and final follow-up was completed in September 2011. INTERVENTIONS Patients with severe sepsis (n = 1961) were randomized and treated within 12 hours of onset of first organ dysfunction in a 2:1 ratio with a 6-day course of either eritoran tetrasodium (105 mg total) or placebo, with n = 1304 and n = 657 patients, respectively. MAIN OUTCOME MEASURES The primary end point was 28-day all-cause mortality. The secondary end points were all-cause mortality at 3, 6, and 12 months after beginning treatment. RESULTS Baseline characteristics of the 2 study groups were similar. In the modified intent-to-treat analysis (randomized patients who received at least 1 dose) there was no significant difference in the primary end point of 28-day all-cause mortality with 28.1% (366/1304) in the eritoran group vs 26.9% (177/657) in the placebo group (P = .59; hazard ratio, 1.05; 95% CI, 0.88-1.26; difference in mortality rate, -1.1; 95% CI, -5.3 to 3.1) or in the key secondary end point of 1-year all-cause mortality with 44.1% (290/657) in the eritoran group vs 43.3% (565/1304) in the placebo group, Kaplan-Meier analysis of time to death by 1 year, P = .79 (hazard ratio, 0.98; 0.85-1.13). No significant differences were observed in any of the prespecified subgroups. Adverse events, including secondary infection rates, did not differ between study groups. CONCLUSIONS AND RELEVANCE Among patients with severe sepsis, the use of eritoran, compared with placebo, did not result in reduced 28-day mortality. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00334828.

The TLR4 Antagonist, Eritoran, Protects Mice from Lethal Influenza Infection

There is a pressing need to develop alternatives to annual influenza vaccines and antiviral agents licensed for mitigating influenza infection. Previous studies reported that acute lung injury caused by chemical or microbial insults is secondary to the generation of host-derived, oxidized phospholipid that potently stimulates Toll-like receptor 4 (TLR4)-dependent inflammation. Subsequently, we reported that Tlr4−/− mice are highly refractory to influenza-induced lethality, and proposed that therapeutic antagonism of TLR4 signalling would protect against influenza-induced acute lung injury. Here we report that therapeutic administration of Eritoran (also known as E5564)—a potent, well-tolerated, synthetic TLR4 antagonist—blocks influenza-induced lethality in mice, as well as lung pathology, clinical symptoms, cytokine and oxidized phospholipid expression, and decreases viral titres. CD14 and TLR2 are also required for Eritoran-mediated protection, and CD14 directly binds Eritoran and inhibits ligand binding to MD2. Thus, Eritoran blockade of TLR signalling represents a novel therapeutic approach for inflammation associated with influenza, and possibly other infections.

Blocking of Responses to Endotoxin by E5564 in Healthy Volunteers with Experimental Endotoxemia

E5564 is a second-generation synthetic analogue of the lipid A component of endotoxin (lipopolysaccharide [LPS]). The ability of E5564 to block the toxic activity of LPS was assessed in a double-blind, placebo-controlled study. A bolus infusion of endotoxin (4 ng/kg) was administered to healthy subjects to induce a mild transient syndrome similar to clinical sepsis. Single E5564 doses of 50-250 microg ameliorated or blocked all of the effects of LPS in a dose-dependent manner. All E5564 dose groups had statistically significant reductions in elevated temperature, heart rate, C-reactive protein levels, white blood cell count, and cytokine levels (tumor necrosis factor-alpha and interleukin-6), compared with placebo (P<.01). In doses of > or = 100 microg, E5564 acted as an LPS antagonist and completely eliminated these signs. E5564 also blocked or ameliorated LPS-induced fever, chills, headache, myalgia, and tachycardia (P<.01). These results demonstrate that E5564 blocks the effects of LPS in a human model of clinical sepsis and indicate its potential in the treatment and/or prevention of clinical sepsis.

Inhibition of endotoxin response by synthetic TLR4 antagonists.

Endotoxin, from the outer membrane of Gram-negative bacteria, has been implicated as the etiological agent of a variety of pathologies ranging from relatively mild (fever) to lethal (septic shock, organ failure, and death). While endotoxin (also known as lipopolysaccharide or LPS) is a complex heterogeneous molecule, the toxic portion of LPS (the lipid A portion) is relatively similar across a wide variety of pathogenic strains of bacteria, making this molecule an attractive target for the development of an LPS antagonist. Research over the past fifteen years focused on the design of various lipid A analogs including monosaccharide, acyclic and disaccharide compounds has lead to the development of E5564, an advanced, unique and highly potent LPS antagonist. E5564 is a stable, pure LPS antagonist that is selective against endotoxin-mediated activation of immune cells in vitro and in animal models. In Phase I clinical trials, we have developed an ex vivo endotoxin antagonism assay that has provided results on pharmacodynamic activity of E5564 in addition to the more typical safety and pharmacokinetic evaluations. Results from these assays have been reinforced by analysis of in vivo antagonistic activity using a human endotoxemia model. Results from all of these studies indicate that E5564 is an effective in vivo antagonist of endotoxin, and may prove to be of benefit in a variety of endotoxin-mediated diseases. This review discusses the evolution of synthetic LPS antagonists with emphasis on the SAR and development of E5564 and its precursors.

Antagonism of in vivo and ex vivo response to endotoxin by E5564, a synthetic lipid A analogue

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.

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

ABSTRACT Eritoran, a structural analogue of the lipid A portion of lipopolysaccharide (LPS), is an antagonist of LPS in animal and human endotoxemia models. Previous studies have shown that low doses (350 to 3,500 μg) of eritoran have demonstrated a long pharmacokinetic half-life but a short pharmacodynamic half-life. The present study describes the safety, pharmacokinetics and pharmacodynamics, and lipid distribution profile of eritoran during and after a 72-h intravenous infusion of 500, 2,000, or 3,500 μg/h into healthy volunteers. Except for the occurrence of phlebitis, eritoran administration over 72 h was safe and well tolerated. Eritoran demonstrated a slow plasma clearance (0.679 to 0.930 ml/h/kg of body weight), a small volume of distribution (45.6 to 49.8 ml/kg), and a relatively long half-life (50.4 to 62.7 h). In plasma, the majority (∼55%) of eritoran was bound to high-density lipoproteins. During infusion and for up to 72 h thereafter, ex vivo response of blood to 1- or 10-ng/ml LPS was inhibited by ≥85%, even when the lowest dose of eritoran (500 μg/h) was infused. Inhibition of response was dependent on eritoran dose and the concentration of LPS used as an agonist. Finally, in vitro analysis with purified lipoprotein and protein fractions from plasma obtained from healthy volunteers indicated that eritoran is inactivated by high-density but not low-density lipoproteins, very-low-density lipoproteins, or albumin. From these results, we conclude that up to 252 mg of eritoran can be safely infused into normal volunteers over 72 h and even though it associates extensively with high-density lipoproteins, antagonistic activity is maintained, even after infusion ceases.

A Phase II, double-blind, placebo-controlled, ascending-dose study of eritoran (E5564), a lipid a antagonist, in patients undergoing cardiac surgery with cardiopulmonary bypass

BACKGROUND: Lipid A, the toxic moiety of endotoxin, is linked to multiple complications after cardiac surgery, including fever, vasodilation, and pulmonary and renal dysfunction. The lipid A antagonist eritoran (or E5564) prevents endotoxin-induced systemic inflammation in animals and humans. In this study we assessed the safety of eritoran administration in patients undergoing cardiac surgery and obtained preliminary efficacy data for the prophylaxis of endotoxin-mediated surgical complications. METHODS: A double-blind, randomized, ascending-dose, placebo-controlled study was conducted at nine hospitals. Patients undergoing coronary artery bypass graft and/or cardiac valvular surgery with cardiopulmonary bypass were enrolled. Patients received a 4-h infusion of placebo (n = 78) vs 2 mg (n = 24), 12 mg (n = 26), or 28 mg (n = 24) of eritoran initiated approximately 1 h before cardiopulmonary bypass. RESULTS: No significant safety concerns were identified with continuous safety monitoring, and enrollment continued to the highest prespecified dose (28 mg). No statistically significant differences were observed in most variables related to systemic inflammation or organ dysfunction/injury. CONCLUSIONS: This Phase II safety study suggests that the administration of the novel lipid A antagonist, eritoran, is not associated with overt toxicity in cardiac surgical patients. Blocking lipid A with eritoran does not appear to confer any clear benefit to elective cardiac surgical patients.

E5531, a synthetic non-toxic lipid A derivative blocks the immunobiological activities of lipopolysaccharide

The major pathological responses to Gram‐negative bacterial sepsis are triggered by endotoxin or lipopolysaccharide. As endotoxin is shed from the bacterial outer membrane, it induces immunological responses that lead to release of a variety of cytokines and other cellular mediators. As part of a program aimed at developing a therapeutic agent for septic shock, we have developed E5531, a novel synthetic lipopolysaccharide antagonist. As measured by release by tumour necrosis factor‐α, human monocytes or whole blood can be activated by lipopolysaccharide, lipid A, and lipoteichoic acid (from Gram‐positive bacteria). E5531 potently antagonizes activation by all these agents while itself being devoid of agonistic activity. The inhibitory activity of E5531 was dependent on time of addition. When 10 nM E5531 was added simultaneously with lipopolysaccharide or 1–3 h before addition of lipopolysaccharide, production of tumour necrosis factor‐α was inhibited by more than 98%. The addition of E5531 1 h after lipopolysaccharide reduced the efficacy of E5531 by 47%. Antagonistic activity of E5531 was specific for lipopolysaccharide as it was ineffective at inhibiting interferon‐γ mediated NO release of RAW 264.7 cells, phorbor 12‐myristate 13‐acetate stimulated superoxide anion production in human neutrophils, concanavalin A stimulated mitogenic activity in murine thymocytes and tumor necrosis factor‐α induced E‐selectin expression in human umbilical vein endothelial cells. E5531 as well as MY4, an anti‐CD14 antibody, inhibited radiolabelled lipopolysaccharide binding in human monocytes. These results support our contention that E5531 is a potent antagonist of lipopolysaccharide‐induced release of tumour necrosis factor‐α and other cellular mediators and may be an effective therapeutic agent for human septic shock due to Gram‐negative bacteria.

Safety, Pharmacokinetics, and Pharmacodynamics of E5564, a Lipid A Antagonist, during an Ascending Single-Dose Clinical Study

E5564, a structural analog of the lipid A portion of lipopolysaccharide (LPS), is a potent antagonist of the biochemical and physiologic effects of LPS in several in vitro and in vivo models and is currently under clinical development as a possible therapeutic for the treatment of sepsis and septic shock. The objectives of this study were to (1) assess the safety and tolerability of E5564 following a 30‐minute intravenous (IV) infusion, (2) evaluate the pharmacokinetic profile of E5564, and (3) measure the ability of E5564 to block LPS stimulation ex vivo in blood taken from subjects up to 8 hours after ending the infusion. Healthy male volunteers (n= 7/dose group) were randomly assigned to each of four dose levels (350, 1000, 2000, or 3500 μg). Within each dose group, 5 subjects received drug and 2 received placebo. E5564 or matching placebo was administered by a 30‐minute infusion, and blood samples were collected at predetermined time points. All doses of E5564 were demonstrated to be safe and well tolerated. E5564 plasma concentrations were determined using a validated LC/MS/MS method. The Cmax and AUC of E5564 increased in a dose‐proportional manner. E5564 pharma‐ cokinetics were characterized by a slow clearance (0.67–0.95 mL/h/kg), a small volume of distribution (41–54 mL/kg), and a relatively long elimination half‐life (42–51 h). As measured in the ex vivo assay, E5564 inhibited LPS‐induced tumor necrosis factor–α (TNF‐α) in a dose‐dependent manner, and at the higher doses (2 and 3.5 mg), antagonistic activity was measurable up to 8 hours postinfusion. E5564 lacked LPS‐like agonist activity at doses up to 3.5 mg. Taken together, we believe that E5564 is a safe, potent antagonist of LPS in blood and will likely benefit patients in the treatment of LPS‐related diseases.

Lower Endotoxin Immunity Predicts Increased Cognitive Dysfunction in Elderly Patients After Cardiac Surgery

Background and Purpose— Although coronary artery bypass graft surgery (CABG) improves the quality of life and functional capacity for numerous patients, many also exhibit impairment in cognitive function immediately after surgery. Although the etiology of this cognitive decline is multifactorial, the inflammatory response to the primary insult may modulate the extent of dysfunction. Patients with low preoperative levels of anti-endotoxin core antibody (EndoCAb) are more likely to experience adverse outcomes, suggesting that decreased immunity to endotoxin causes a heightened release of inflammatory mediators. We therefore sought to determine the association of decreased EndoCAb and the incidence of postoperative cognitive decline. Methods— EndoCAb levels were measured before surgery in 460 patients undergoing elective CABG. Cognitive function was measured preoperatively and 6 weeks postoperatively. Multivariable analysis accounted for the effects of age, Parsonnet score, sex, body mass index, baseline cognition, years of education, history of hypertension, bypass time, cross-clamp time, and number of grafts. Results— At 6-week follow-up, 122 patients (36%) showed cognitive decline. Lower preoperative EndoCAb levels were associated with a greater incidence and severity of postoperative cognitive decline. The elderly with decreased endotoxin immunity are particularly susceptible to this decline (relative risk=1.97 for age >64). Conclusions— Reduced preoperative endotoxin immunity is a predictor of increased postoperative cognitive dysfunction in patients undergoing CABG, particularly in those >60 years old. Interventions that increase IgM EndoCAb levels might improve cognitive function after cardiac surgery.