Biomimetic polymeric nanoparticle-based photodynamic immunotherapy and protection against tumor rechallenge.

In this study, we sought to design a bionanomaterial that could exert anticancer effects against primary tumors and protect against rechallenged tumors via photodynamic immunotherapy. As a biomaterial, we used an amphiphilic phenylalanine derivative of poly-gamma glutamic acid, which forms nanoparticles by self-assembly. For anticancer effects, we co-entrapped hydrophobic chlorin e6 and monophosphoryl lipid A in the core of the plain amphiphilic phenylalanine nanoparticles (AN), to generate M/C/AN. For comparison, we used plain AN and chlorin e6-loaded AN (C/AN). In vitro studies showed that B16F10 cancer cells treated with C/AN or M/C/AN generated reactive oxygen species and exhibited an enhanced surface display of calreticulin upon exposure to 660 nm light irradiation. C/AN and M/C/AN exerted similar photodynamic anticancer effects; however, M/C/AN, but not C/AN, induced in vitro dendritic cell maturation. Our biodistribution study revealed that C/AN and M/C/AN showed higher accumulation at the tumor tissues compared to that seen in the free chlorin e6-treated group. In B16F10 tumor-bearing mice, the intravenous injection of C/AN or M/C/AN showed similar photodynamic anticancer effects against primary tumors. However, the growth of rechallenged tumors was more significantly inhibited in the M/C/AN group compared to the C/AN group. At day 40 after inoculation of the primary tumor, M/C/AN-treated mice showed 100% survival, whereas the other groups showed 0% survival. In the tumor microenvironment, higher infiltration of CD8+ T cells was observed in the M/C/AN group compared to the other groups. Our results suggest that AN co-loaded with a photosensitizer and an immune stimulant may hold great potential for use in photodynamic immunotherapy to inhibit both primary and metastatic tumors.

Targeted Codelivery of an Antigen and Dual Agonists by Hybrid Nanoparticles for Enhanced Cancer Immunotherapy.

Among approaches of current cancer immunotherapy, a dendritic cell (DC)-targeted vaccine based on nanotechnology could be a promising way to efficiently induce potent immune responses. To enhance DC targeting and vaccine efficiency, we included imiquimod (IMQ), a toll-like receptor 7/8 (TLR 7/8) agonist, and monophosphoryl lipid A (MPLA), a TLR4 agonist, to synthesize lipid-polymer hybrid nanoparticles using PCL-PEG-PCL and DOTAP (IMNPs) as well as DSPE-PEG-mannose (MAN-IMNPS). The spatiotemporal delivery of MPLA (within the outer lipid layer) to extracellular TLR4 and IMQ (in the hydrophobic core of NPs) to intracellular TLR7/8 can activate DCs synergistically to improve vaccine efficacy. Ovalbumin (OVA) as a model antigen was readily absorbed by positively charged DOTAP and showed a quick release in vitro. Our results demonstrated that this novel nanovaccine enhanced cellular uptake, cytokine production, and maturation of DCs. Compared with the quick metabolism of free OVA-agonists, the depot effect of OVA-IMNPs was observed, whereas MAN-OVA-IMNPs promoted trafficking to secondary lymphoid organs. After immunization with a subcutaneous injection, the nanovaccine, especially MAN-OVA-IMNPs, induced more antigen-specific CD8+ T cells, greater lymphocyte activation, stronger cross-presentation, and more generation of memory T cells, antibody, IFN-γ, and granzyme B. Prophylactic vaccination of MAN-OVA-IMNPs significantly delayed tumor development and prolonged the survival in mice. The therapeutic tumor challenge indicated that MAN-OVA-IMNPs prohibited tumor progression more efficiently than other formulations, and the combination with an immune checkpoint blockade further enhanced antitumor effects. Hence, the DC-targeted vaccine codelivery with IMQ and MPLA adjuvants by hybrid cationic nanoparticles in a spatiotemporal manner is a promising multifunctional antigen delivery system in cancer immunotherapy.

Efficient Delivery of Tyrosinase Related Protein-2 (TRP2) Peptides to Lymph Nodes using Serum-Derived Exosomes.

Exosomes (EXO) are considered to be versatile carriers for biomolecules; however, the delivery of therapeutic peptides using EXOs poses several challenges. In this study, the efficiency of serum-derived EXOs in delivering tyrosinase-related protein-2 (TRP2) peptides to lymph nodes is determined. TRP2 peptides are successfully incorporated into EXOs, which show a uniform and narrow size distribution of around 45 nm. The TRP2-incorporated exosomes (EXO-TRP2) are efficiently internalized into macrophages and dendritic cells, and are seen to display a punctate distribution. EXOs loaded with TRP2 together with MPLA, (EXO-MPLA-TRP2) result in a strong release of proinflammatory cytokines (TNF-α and IL-6) from both RAW264.7 and DC2.4 cells. Finally, subcutaneous injection of fluorescently labeled EXO-TRP2 followed by ex vivo imaging using in vivo imaging system (IVIS) show a strong fluorescent signal in the lymph nodes after only 1 h, which is maintained until at least 4 h after injection. Taken together, the findings suggest that serum-derived EXOs can serve as promising carriers to deliver therapeutic peptides to lymph nodes for immunotherapy.

Development of Adjuvanted Solid Fat Nanoemulsions for Pulmonary Hepatitis B Vaccination.

Pulmonary vaccination is one of the most promising routes for immunization owing to its noninvasive nature and induction of strong mucosal immunity and systemic response. In the present study, recombinant hepatitis B surface antigen loaded solid fat nanoemulsions (SFNs) as carrier system and monophosphoryl lipid A as an adjuvant-carrier system was prepared and evaluated as multiadjuvanted vaccine system for deep pulmonary vaccination. Deposition and clearance from the deep lung of rats were determined by gamma scintigraphy. Biodistribution of SFNs was determined by the live animal imaging system. SFNs dispersion showed slower clearance as compared with sodium pertechnetate control solution (∗∗∗p <0.001) from the pulmonary region due to the virtue of particulate and hydrophobic nature of formulations. Humoral (sIgA and IgG) and cellular (IL-2 and IF-γ) immune responses were found to be significant (∗∗∗p <0.001) when compared with naïve antigen (recombinant surface antigen without any excipient) solution. Data indicate that deep pulmonary immunization offers a stronger immune response with balanced humoral, mucosal, and cellular immunization, which further needs to be tested in higher animals to support this hypothesis for clinical translation of this so far neglected yet potential target tissue for immunization.

IL-18 and Subcapsular Lymph Node Macrophages are Essential for Enhanced B Cell Responses with TLR4 Agonist Adjuvants.

Designing modern vaccine adjuvants depends on understanding the cellular and molecular events that connect innate and adaptive immune responses. The synthetic TLR4 agonist glycopyranosyl lipid adjuvant (GLA) formulated in a squalene-in-water emulsion (GLA-SE) augments both cellular and humoral immune responses to vaccine Ags. This adjuvant is currently included in several vaccines undergoing clinical evaluation including those for tuberculosis, leishmaniasis, and influenza. Delineation of the mechanisms of adjuvant activity will enable more informative evaluation of clinical trials. Early after injection, GLA-SE induces substantially more Ag-specific B cells, higher serum Ab titers, and greater numbers of T follicular helper (TFH) and Th1 cells than alum, the SE alone, or GLA without SE. GLA-SE augments Ag-specific B cell differentiation into germinal center and memory precursor B cells as well as preplasmablasts that rapidly secrete Abs. CD169+ SIGNR1+ subcapsular medullary macrophages are the primary cells to take up GLA-SE after immunization and are critical for the innate immune responses, including rapid IL-18 production, induced by GLA-SE. Depletion of subcapsular macrophages (SCMф) or abrogation of IL-18 signaling dramatically impairs the Ag-specific B cell and Ab responses augmented by GLA-SE. Depletion of SCMф also drastically reduces the Th1 but not the TFH response. Thus the GLA-SE adjuvant operates through interaction with IL-18-producing SCMф for the rapid induction of B cell expansion and differentiation, Ab secretion, and Th1 responses, whereas augmentation of TFH numbers by GLA-SE is independent of SCMф.

Continuous pharmacodynamic activity of eritoran tetrasodium, a TLR4 antagonist, during intermittent intravenous infusion into normal volunteers.

BACKGROUND: Eritoran tetrasodium (E5564), a structural analogue of the lipid A portion of endotoxin (lipopolysaccharide or LPS), is an antagonist of LPS and other Toll-like receptor 4 (TLR4) ligands. Eritoran tetrasodium quantitatively blocks LPS response in vivo in animal and human endotoxemia models and demonstrates a long pharmacokinetic half-life, but a short pharmacodynamic half-life. The objective of this study was to assess the safety, and pharmacokinetic and pharmacodynamic profile of E5564 infused twice-daily at three target steady-state plasma levels of approximately 1, 3 and 10 microg/ml in healthy volunteers. RESULTS: Loading and maintenance doses of up to 77 mg over 3 days in females and 105 mg over 6 days in males were safe and well-tolerated except for self-limiting phlebitis at the drug infusion site. Plasma levels reached steady state by 24 h. The C(max), C(min), and C(88), AUC(0 -infinity) were dose proportional and gender independent. Pharmacodynamic activity measured by an ex vivo LPS challenge assay, demonstrated dose-dependence for both E5564 and LPS and plasma levels of approximately 3 microg/ml E5564 or greater blocked up to 1 ng/ml LPS. CONCLUSIONS: Every 12-h dosing of E5564 can replace continuous infusion, while maintaining uninterrupted blocking of high-dose LPS.

Clearance of bacterial lipopolysaccharides and lipid A by the liver and the role of argininosuccinate synthase.

The liver is thought to be involved in the systemic clearance and detoxification of lipopolysaccharide (LPS). Argininosuccinate synthase (AS), a liver cytosolic urea cycle enzyme, has been found to bind to and inactivate LPS and lipid A. To elucidate the participation of AS in the clearance of LPS by liver and hepatocytes, we investigated the correlation between AS content and the removal of lipid A and LPS in vivo and in vitro, tracing levels of biological activity. A hepatotoxic model in which mice were injected with CCl(4) revealed a significant reduction in lipid A clearance along with liver failure on day 1; total body clearance was changed to 0.534 ml/min from 1.42 ml/min. AS content in liver concomitantly decreased to about half and AS leaked to blood at about 6 microg/ml. Total body clearance of i.v. injected AS was estimated at 0.083 ml/min, which predicted about 24-h leakage of AS after CCl(4) injection. The treatment also reduced the clearance of R-type LPSs to a lesser degree the larger its polysaccharide portion. S-type LPS, which has a large O-antigen polysaccharide, exhibited enhancement of clearance on CCl(4) treatment. When pretreated in vitro with AS and injected into normal mice, lipid A and R-type LPS showed a similar pattern of clearance of residual activities to the untreated forms, but S-type LPS exhibited enhancement of clearance. Comparison between different strains of mice revealed a correlation of AS content in liver and lipid A clearance, where the higher AS strain C3H/He mice showed a more rapid clearance than the lower AS strains C57BL/6 and BALB/c. Primary spheroid cultures of hepatocytes treated with 0.1 microM dexamethasone and 1 microM glucagon showed about a 2-fold increase in AS amount and a more rapid clearance of LPS from culture medium than untreated cells. These results suggest that AS in hepatocytes may be involved in the process of lipid A and LPS clearance and the extracellular leakage of AS may also participate in the systemic detoxification.

Development of an injectable formulation for the novel lipid A analog E5531 using a 'pH-jump method'.

In order to design an injectable formulation of E5531, a novel synthetic disaccharide analog of novel lipid A, for the treatment of septic shock, a 'pH-jump method' was developed. In this method, E5531 was dispersed in 0.003 mol/l NaOH (pH 11.0, above pK(a2)) at 50 degrees C (above phase transition temperature) and then mixed with a buffer to neutralize the pH to 7.3. E5531 was dispersed as particles, and the size was approximately 20 nm. The structure of the particles was vesicular. After dispersal, the solution was sterilized using a filter, filled aseptically into vials, and lyophilized. The size of the particles did not change before and after lyophilization. The relationship between the physicochemical properties of the particles and the pharmacokinetics in rats after intravenous administration was investigated. The membrane fluidity of the particles was affected by the dispersal methods, the dispersal time in 0.003 mol/l NaOH in the pH-jump method, and the addition of Ca(2+) to the solution. The membrane fluidity was correlated with the pharmacokinetics in rats.

Safety, pharmacokinetics, pharmacodynamics, and plasma lipoprotein distribution of eritoran (E5564) during continuous intravenous infusion into healthy volunteers.

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 microg) 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 microg/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 (approximately 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 > or =85%, even when the lowest dose of eritoran (500 microg/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.

LPS binding protein does not participate in the pharmacokinetics of E5564.

E5564, a lipid A analogue, is a potent antagonist of lipopolysaccharide (LPS). Clinically, E5564 was developed as a possible therapy for treatment of sepsis and septic shock. Surface plasmon resonance (SPR) analysis indicates that E5564 binds to LPS binding protein (LBP), in a manner similar to LPS. Gel-filtration radioactive chromatograms of [(14)C]-E5564 in plasma revealed that E5564 initially distributes to the lipoprotein fractions, separated from high-density lipoprotein (HDL); the bound fraction is then released and binds to HDL. Similar results were obtained by heparin-manganese precipitation. At doses of E5564 relevant to its clinical use (i.e. 6 microg/ml), antibodies against LBP did not influence either the distribution of E5564 to non-HDL lipoprotein fractions or the transfer of E5564 from non-HDLs to HDL. Under these conditions, transfer of E5564 to HDL occurs similarly in the plasma of LBP knockout (KO) mice as in the plasma from wild-type mice. In addition, plasma clearance of E5564 in LBP KO mice is similar to that of wildtype mice. Thus, LBP binds E5564 in a manner similar to LPS, but does not play a role in E5564 redistribution/binding to lipoprotein and plasma clearance.

Drug evaluation: E-5564.

Eisai Co Ltd is developing the injectable endotoxin antagonist E-5564, for the potential prevention of the pathophysiological effects of endotoxin-mediated responses caused by bacterial infection, including septic shock.

Extended in vivo pharmacodynamic activity of E5564 in normal volunteers with experimental endotoxemia [corrected].

E5564 (alpha-D-glucopyranose) is a synthetic antagonist of bacterial endotoxin that has been shown to completely block human endotoxin response. Low doses of E5564 (0.35-3.5 mg) have a long pharmacokinetic half-life, but a surprisingly short ex vivo and in vivo pharmacodynamic half-life (generally less than several hours). To determine whether extended antagonistic activity can be achieved in vivo, this study assesses the pharmacodynamic activity of 4- and 72-h infusions of E5564 into normal volunteers. Administration of 3.5 mg of E5564/h x 72 h completely blocked effects of endotoxin challenge at the end of dosing (72 h), and at 48 and 72 h postdosing. Similarly, a 4-h infusion of E5564, 3 mg/h completely blocked endotoxin administered 8 h postdosing. A lower dose of E5564, 0.5 mg/h x 4 h, ameliorated but did not block most effects of endotoxin 8 h postdosing (p <0.05). Finally, the effect of varying plasma lipoprotein content on E5564 activity was studied in subjects having high or low cholesterol levels (>180 or <140 mg/dl) after 72-h infusion of 252 mg of E5564. No differences were observed. These results demonstrate that E5564 blocks the effects of endotoxin in a human model of clinical sepsis and indicate its potential in the treatment and/or prevention of clinical sepsis.

Pharmacokinetics of E5564, a lipopolysaccharide antagonist, in patients with impaired hepatic function.

E5564 is a structural analog of the Lipid A portion of lipopolysaccharide (LPS). E5564 has been tested in several in vitro and in vivo models and has demonstrated its effectiveness against LPS. It is intended to be an antagonist of LPS to reduce the morbidity and mortality associated with sepsis syndrome. This study assessed the pharmacokinetics (PK) of E5564 in patients with impaired hepatic function. E5564 was administered via intermittent intravenous infusion every 12 hours for six times to 24 hepatic-impaired patients (12 each to Child-Pugh Classifications A and B) and 24 matching healthy volunteers. Plasma samples were analyzed by LC/MS/MS. A one-compartment model resulted in good and comparable fits for all volunteers. Regardless of liver disease state, none of the PK parameters compared (i.e., Cmax (0-12),tmax (0-12),CL,t1/2, Vss, AUC(0-12), AUC(0-last), AUC(0-infinity), C(ss,min), C(ss,max), and C(ss,av)) exhibited any difference between these two groups. This suggested that the exposure of E5564 in volunteers was independent of hepatic function. Thus, no dose adjustment is needed in patients with hepatic impairment classified as Child-Pugh A and B.

Association of the endotoxin antagonist E5564 with high-density lipoproteins in vitro: dependence on low-density and triglyceride-rich lipoprotein concentrations.

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 microM 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 degrees 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 degrees 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.

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 (i.v.) 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 micrograms). 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-alpha (TNF-alpha) 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.

Disposition of a synthetic analogue of lipid A (E5564) in rats.

1. E5564, a lipid A analogue that potently antagonises lipopolysaccharide, is being developed to treat sepsis caused by Gram-negative bacterial infections. The pharmacokinetic profile of E5564 is independent of dose between 0.1 and 1 mg kg(-1). The distribution volume of E5564 is slightly larger than the total plasma volume, and the terminal elimination half-life is about 5 h. 2. Following (14)C-E5564 administration (0.5 mg kg(-1)), radioactivity rapidly accumulates in the liver and spleen. The half-life of E5564 in the liver is 5.1 h, which is similar to that in the plasma. At 48 weeks after dosing, 35.27% of the administered radioactivity was still present in the liver. Cumulative urinary and faecal excretion of radioactivity for up to 48 weeks after administration were 3.86 and 67.17% of the dose, respectively. 3. The results of mass spectroscopy and nuclear magnetic resonance analysis reveal that the main hepatic metabolite is di-dephosphorylated E5564. The half-life of di-dephosphorylated E5564 in the liver is 87.4 days, which is similar to that for the hepatic radioactivity. 4. The results indicate that E5564 is rapidly taken up by the liver, is metabolized via dephosphorylation pathways to form dephosphorylated E5564 and is mainly excreted in the faeces.

Induction of intratumoral tumor necrosis factor by a synthetic lipid A analog, ONO-4007, with less tolerance in repeated administration and its implication in potent antitumor effects with low toxicity.

To evaluate the anti-tumor characteristics of ONO-4007, a synthetic analog of lipid A, the authors examined its acute toxicity and anti-tumor activity in a mouse MM46 mammary tumor system in comparison with LA-15-PP, an E. coli-type synthetic lipid A and LPS. Systemic and local (tumor site) induction of tumor necrosis factor (TNF) by a single i.v. shot of ONO-4007 and LA-15-PP correlated with manifestation of their toxicity, showing that ONO-4007 is 100-fold less effective than LA-15-PP. However, a protocol of repeated administration (3 shots twice a week) exhibited about 10 times more therapeutic potency of ONO-4007 for cancer therapy than expected in the above experiments. In a dose inducing submaximal systemic and intratumoral TNF production, repeated injections (twice a week) of ONO-4007 (10 mg/kg), LA-15-PP (0.1 mg/kg) and LPS (0.1 mg/kg) commonly generated a tolerant state in the systemic response (serum and liver) to subsequent stimulation. The intratumoral response was retained with this repeated administration of ONO-4007, but was not with LA-15-PP or LPS. TIM (tumor-infiltrating macrophages) isolated from mice pre-injected with ONO-4007 and LA-15-PP were found to lose their response to both substances, but the response was rapidly recovered until 72 h after injection and virtually no difference was observed in their response to either drug. The in vitro treatment of naive TIM with ONO-4007 or LA-15-PP for 2 h depressed the response to both substances and the depression continued for 72 h even in culture with fresh medium. The relatively high efficacy of ONO-4007 in cancer therapy likely depends on the retraction of the tolerant state, especially at the tumor site where the response to ONO-4007 is recovered much more efficiently than that to lipid A. While constant recruitment of macrophages to tumor tissue might be involved in the difference of tolerance recovery between this region and others, selective response to ONO-4007 may not be explained simply by the sensitivity of recruited TIM. Pharmacokinetical experiments revealed that repeated injections of LA-15-PP enhanced its clearance from blood circulation, while the clearance of ONO-4007 was stable after repeated injections. Thus, pharmacokinetical properties of ONO-4007 may also possibly be implicated in this event.

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, > or = 100 microg of E5564 completely blocked signs, symptoms and cytokines induced by concomitantly-administered endotoxin. In contrast, subjects receiving a 50 microg dose of E5564 demonstrated a graded response; cytokines were inhibited > or = 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 microg 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.

Targeting polymerised liposome vaccine carriers to intestinal M cells.

Due to their transcytotic capability, intestinal M cells may represent an efficient potential route for oral vaccine delivery. We previously demonstrated that the lectin Ulex europaeus agglutinin 1 (UEA1, specific for alpha-L-fucose residues) selectively binds to mouse Peyer's patch M cells and targets 0.5 microm polystyrene microparticles to these cells. Using a gut loop model we now demonstrate that covalently-membrane-bound UEA1 similarly targets polymerised liposomes (Orasomes, approximately 200 nm diameter), potential biocompatable oral vaccine delivery vehicles, to mouse M cells. Targeting was inhibited by alpha-L-fucose while the co-entrapped adjuvant, monophosphoryl Lipid A (MPL), failed to exert any detrimental effect on UEA1-mediated M cell targeting. Lectin-mediated M cell targeting may thus permit the efficacy of mucosal vaccines to be enhanced if cellular relationship between particle binding and immune outcome can be established.

The effect of lipid A analog E5531 on membrane properties of dipalmitoylphosphatidylcholine.

The effect of the lipid A analog E5531 on the phospholipid membrane was determined and compared with that of the lipid A from Escherichia coli (EC). E5531 decreased the phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) membrane and increased the fluidity and permeability. On the other hand, EC increased the phase transition temperature and decreased the membrane fluidity and permeability. These results suggest that the reason for the difference of biological effects of E5531 and lipid A from EC would be caused by the differences from the effect on the cell membranes.