Melvyn Lynn

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.

Phase 2 trial of eritoran tetrasodium (E5564), a Toll-like receptor 4 antagonist, in patients with severe sepsis*

Objectives:Endotoxin is a potent stimulus of proinflammatory response and systemic coagulation in patients with severe sepsis. Endotoxin is a component of Gram-negative bacteria that triggers an innate immune response through Toll-like receptor 4 signaling pathways in myeloid cells. We evaluated safety and tolerability of two dose regimens of eritoran tetrasodium (E5564), a synthetic Toll-like receptor 4 antagonist, and explored whether it decreases 28-day mortality rate in subjects with severe sepsis. Design:Prospective, randomized, double-blind, placebo-controlled, multicenter, ascending-dose phase II trial. Setting:Adult intensive care units in the United States and Canada. Patients:Three hundred adults within 12 hrs of recognition of severe sepsis, with Acute Physiology and Chronic Health Evaluation (APACHE) II-predicted risk of mortality between 20% and 80%. Interventions:Intravenous eritoran tetrasodium (total dose of either 45 mg or 105 mg) or placebo administered every 12 hrs for 6 days. Measurements and Main Results:Prevalence of adverse events was similar among subjects treated with 45 mg or 105 mg of eritoran tetrasodium or with placebo. For modified intent-to-treat subjects, 28-day all-cause mortality rates were 26.6% (eritoran tetrasodium 105 mg), 32.0% (eritoran tetrasodium 45 mg), and 33.3% in the placebo group. Mortality rate in the eritoran tetrasodium 105-mg group was not significantly different from placebo (p = .335). In prespecified subgroups, subjects at highest risk of mortality by APACHE II score quartile had a trend toward lower mortality rate in the eritoran tetrasodium 105-mg group (33.3% vs. 56.3% placebo group, p = .105). A trend toward a higher mortality rate was observed in subjects in the lowest APACHE II score quartile for the eritoran 105-mg group (12.0% vs. 0.0% placebo group, p = .083). Conclusions:Eritoran tetrasodium treatment appears well tolerated. The observed trend toward a lower mortality rate at the 105-mg dose, in subjects with severe sepsis and high predicted risk of mortality, should be further investigated.

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.

A lipid A analog, E5531, blocks the endotoxin response in human volunteers with experimental endotoxemia.

BackgroundEndotoxin (lipopolysaccharide [LPS]) has been associated with sepsis and the high mortality rate seen in septic shock. The administration of a small amount of LPS to healthy subjects produces a mild syndrome qualitatively similar to that seen in clinical sepsis. We used this model to test the efficacy of an endotoxin antagonist, E5531, in blocking this LPS-induced syndrome. MethodsIn a placebo-controlled, double-blind study, we randomly assigned 32 healthy volunteers to four sequential groups (100, 250, 500, or 1000 &mgr;g of E5531). Each group of eight subjects (six assigned to E5531, two assigned to placebo) received a 30-min intravenous infusion of study drug. LPS (4 ng/kg) was administered to all subjects as an intravenous bolus in the contralateral arm at the midpoint of the infusion. Symptoms, signs, laboratory values, and hemodynamics (by echocardiogram) were evaluated at prospectively defined times. ResultsIn subjects receiving placebo, LPS caused headache, nausea, chills, and myalgias. E5531 led to a dose-dependent decrease in these symptoms that was statistically significant (p < .05) except for myalgias. The signs of endotoxemia (fever, tachycardia, and hypotension) were consistently inhibited at the three higher doses (250, 500, and 1000 &mgr;g, p < .05). Tumor necrosis factor-&agr; and interleukin-6 blood levels were both lower in those who received E5531 (p < .0001). The C-reactive protein level and white blood cell count response were decreased at all doses (p < .0001). The hyperdynamic cardiovascular state (high cardiac index and low systemic vascular resistance) associated with endotoxin challenge was significantly inhibited at the higher doses of E5531. ConclusionsE5531 blocks the symptoms and signs and cytokine, white blood cell count, C-reactive protein, and cardiovascular response seen in experimental endotoxemia. This agent is a potent inhibitor of endotoxin challenge in humans and may be of benefit in the prevention or treatment of sepsis and septic shock.

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.

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.

Consequences of Interaction of a Lipophilic Endotoxin Antagonist with Plasma Lipoproteins

ABSTRACT E5531, a novel synthetic lipid A analogue, antagonizes the toxic effects of lipopolysaccharide, making it a potential intravenously administered therapeutic agent for the treatment of sepsis. This report describes the distribution of E5531 in human blood and its activity when it is associated with different lipoprotein subclasses. After in vitro incubation of [14C]E5531 with blood, the great majority (>92%) of material was found in the plasma fraction. Analysis by size-exclusion and affinity chromatographies and density gradient centrifugation indicates that [14C]E5531 binds to lipoproteins, primarily high-density lipoproteins (HDLs), with distribution into low-density lipoproteins (LDLs) and very low density lipoproteins (VLDLs) being dependent on the plasma LDL or VLDL cholesterol concentration. Similar results were also seen in a limited study of [14C]E5531 administration to human volunteers. The potency of E5531 in freshly drawn human blood directly correlates to increasing LDL cholesterol levels. Finally, preincubation of E5531 with plasma or purified lipoproteins indicated that binding to HDL resulted in a time-dependent loss of drug activity. This loss in activity was not observed with drug binding to LDLs or to VLDLs or chylomicrons. Taken together, these results indicate that E5531 binds to plasma lipoproteins, making its long-term antagonistic potency dependent on the plasma lipoprotein composition.

Influence of severity of illness on the effects of eritoran tetrasodium (E5564) and on other therapies for severe sepsis

Disease severity varies widely in patients with severe sepsis. Eritoran tetrasodium (E5564), a TLR4 antagonist, blocks the binding of endotoxin and is being evaluated as a novel therapy for severe sepsis. This analysis aimed to assess the efficacy of eritoran based on severity of illness and similar effects in other recent sepsis trials. Prospective covariates from a randomized, double-blind, placebo-controlled, phase 2 trial were analyzed for treatment interaction measured by 28-day mortality. Five statistical interaction methodologies were used. The modified intent-to-treat population (n = 292), all-cause 28-day mortality was as follows: placebo, 33.3% (32/96); eritoran 45 mg/105 mg, 29.6% (58/196). Logistic regression analysis identified Acute Physiology and Chronic Health Evaluation II scores, predicted-risk-of-mortality scores, IL-6, age, sex, race, and eritoran use as associated with survival. Significant treatment interactions were observed (eritoran vs. placebo) for baseline covariates: Acute Physiology and Chronic Health Evaluation II (P = 0.035), predicted-risk-of-mortality scores (P = 0.008), number of organ failures (P = 0.079), international normalized ratio (P = 0.05), and acute physiology score (P = 0.039). I2 analysis showed that 38% of the total eritoran treatment variance was explained by the severity-of-illness heterogeneity rather than by chance. No interactions observed with other variables. Consistent with the finding in this eritoran trial, other sepsis trials (IL-1 receptor antagonist, TNFsr-p55, antithrombin, drotrecogin alfa-activated) also demonstrated significant treatment by severity interaction. Potential survival benefits of eritoran in severe sepsis patients were associated with high severity of illness. These findings were used to design a phase 3 trial. Similar treatment by severity-of-illness interaction was found in most recent sepsis trials.

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 μM 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.