![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Vol. 304, Issue 3, 1093-1102, March 2003
Biology Section (M.M., J.R.R., J.B., S.K., M.P., J.C., F.G.) and Chemistry Section (W.J.C.), Eisai Research Institute of Boston, Inc., Andover, Massachusetts; and Tsukuba Research Laboratories (T.K., A.K.), Eisai Co. Ltd., Tsukuba, Japan
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
-D-Glucopyranose,3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-[(3R)-3-methoxydecyl]-6-O-methyl-2-[[(11Z)-1-oxo-11-octadecenyl]amino]-4-O-phosphono-
-D-glucopyranosyl]-2-[(1,3-dioxotetradecyl)amino]-1-(dihydrogen phosphate), tetrasodium salt (E5564) is a second-generation synthetic lipodisaccharide designed to antagonize the toxic effects of endotoxin, a major immunostimulatory component of the outer cell membrane of Gram
negative bacteria. In vitro, E5564 dose dependently (nanomolar concentrations) inhibited lipopolysaccharide (LPS)-mediated activation of primary cultures of human myeloid cells and mouse tissue culture macrophage cell lines as well as human or animal whole blood as measured by production of tumor necrosis factor- and other
cytokines. E5564 also blocked the ability of Gram negative bacteria to
stimulate human cytokine production in whole blood. In vivo, E5564
blocked induction of LPS-induced cytokines and LPS or bacterial-induced lethality in primed mice. E5564 was devoid of agonistic activity when
tested both in vitro and in vivo and has no antagonistic activity
against Gram positive-mediated cellular activation at concentrations up
to 1 µM. E5564 blocked LPS-mediated activation of nuclear factor-
B
in toll-like receptor 4/MD-2-transfected cells. In a mouse
macrophage cell line, activity of E5564 was independent of serum,
suggesting that E5564 exerts its activity through the cell surface
receptor(s) for LPS, without the need for serum LPS transfer proteins.
Similar to
(6-O-{2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-Z-dodec-5-endoyloxydecl]-2-[3-oxo-tetradecanoylamino]--O-phosphono--D-glucopyranose tetrasodium salt (E5531), another lipid A-like antagonist, E5564 associates with plasma lipoproteins, causing low concentrations of
E5564 to be quantitatively inactivated in a dose- and time-dependent manner. However, compared with E5531, E5564 is a more potent inhibitor of cytokine generation, and higher doses retain activity for durations likely sufficient to permit clinical application. These results indicate that E5564 is a potent antagonist of LPS and lacks agonistic activity in human and animal model systems, making it a potentially effective therapeutic agent for treatment of disease states caused by endotoxin.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The
innate immune response has been described as being composed of inherent
abilities to respond to both Gram positive and Gram negative bacteria,
as well as fungi and other pathogens. Response to Gram negative
bacteria is driven, at least in part, by recognizing and responding to
endotoxin (lipopolysaccharide or LPS), a major constituent of the outer
membrane of Gram negative bacteria. LPS is recognized by divergent
pathways. One pathway uses natural antibodies (Reid et al., 1997
) and
lipoproteins (Wurfel et al., 1995; Wurfel and Wright, 1995; Hailman et
al., 1996; Vreugdenhil et al., 2001) to neutralize and clear LPS. A
second pathway triggers a vigorous and complex inflammatory response
involving cellular activation through lipopolysaccharide binding
protein (LBP), cell surface-bound CD14, and toll-like receptor (TLR) 4 (for recent review, see Diks et al., 2001). This latter inflammatory
response enables a sensitive and robust reaction to LPS, using it as a "sentinel" molecule, signaling the presence of a potentially
infectious agent.
In response to blood-borne infection in mammals, LPS is detected by
cells such as monocytes, macrophages, and hepatic Kupffer cells,
triggering them to produce a large variety of cytokines, and other
cellular mediators (Burrell, 1994; Fiuza and Suffredini, 2001) that can
protect the host. However, during and after bacterial killing or when
translocated from the lumen of the intestine, LPS unassociated with
worsening infection can induce inappropriate (toxic) levels of cellular
mediators that trigger pathophysiological events such as hypotension,
fever, shock, and coagulopathies (Bone, 1991a,b; Ulevitch and Tobias,
1995; Morrison, 1998; Norimatsu and Morrison, 1998; Suffredini and
O'Grady, 1999) often leading to multiorgan failure and death
(Brandtzaeg et al., 2001). Intestinal tract-derived endotoxin has been
implicated as the cause of a variety of clinical manifestations such as
postsurgical inflammatory response after abdominal aortic aneurysm
(Roumen et al., 1993; Lau et al., 2000) and coronary artery bypass
grafting (Martinez-Pellus et al., 1993), hepatic diseases such as
alcoholic cirrhosis (Yin et al., 2001), and aggravation of inflammatory
diseases such as graft versus host disease (Cooke et al., 2001) and
inflammatory bowel disease (Gardiner et al., 1995).
To prevent LPS toxicity, we have investigated the possibility of
developing a receptor antagonist to block its activation of cells.
Lipid A is the unique fatty-acylated diphosphorylated diglucosamine
portion of LPS that is a common element of LPS from most pathogenic
bacteria and is its main toxicophore (Galanos et al., 1985a,b; Takada
and Kotani, 1989), making antagonism of the interaction of lipid A with
target cells an attractive target for the treatment of sepsis,
bacteremia, septic shock, and other indications. To this end, we have
designed a series of synthetic analogs of lipid A (Rossignol et al.,
1999). Previously, we described the synthesis and activity of E5531, an
analog of the lipid A from Rhodobacter capsulatus, as an
antagonist of LPS (Christ et al., 1995; Kawata et al., 1995) and showed
that its antagonistic action involves the cell surface receptor for LPS
previously described as TLR4 (Chow et al., 1999).
Although E5531 demonstrated potent inhibition of LPS when added to
blood in vitro and in vivo, activity decreased as a function of time.
This reaction has been shown to be due to interaction of E5531 with
plasma lipoproteins (Wasan et al., 1999; Rose et al., 2000).
This report describes the activity of E5564, a second-generation LPS
antagonist derived from the structure of R. sphaeroides (Rossignol et al., 1999). Compared with E5531, E5564 is structurally and synthetically less complex, yet seems to possess superior activity
and pharmacological characteristics. E5564 is an inhibitor of
LPS-mediated stimulation of responsive cells in vitro and in vivo as
measured by production of cytokines, as well as morbidity and mortality
associated with LPS poisoning in animal models.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Reagents.
E5564
[-D-glucopyranose,3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-[(3R)-3-methoxydecyl]-6-O-methyl-2-[[(11Z)-1-oxo-11-octadecenyl]amino]-4-O-phosphono--D-glucopyranosyl]-2-[(1,3-dioxotetradecyl)amino]-1-(dihydrogen phosphate), tetrasodium salt], formula weight 1401.60) was synthesized by Eisai Research Institute of Boston (Andover, MA). E5564 was dissolved at 6.7 mg/ml in sterile 0.01 N NaOH, sonicated for 3 min with
an ultrasonicator (VW-380; Misonix, Inc., Farmingdale, NY) then diluted
to 100 µM in lactose-phosphate buffer containing (per milliliter)
0.45 mg Na2HPO4 · 7H2O, 0.35 mg
NaH2PO4 · H2O, and 100 mg of lactose; made up in sterile
water. The pH was adjusted to pH 7.8 with 1 N HCl and this buffered
solution of drug stored as aliquots at 
20°C until use. For use,
each aliquot was thawed only once, and serial dilutions were made in
Ca2+/Mg2+-free Hanks'
balanced salt solution (Invitrogen, Carlsbad, CA). For in vivo
use, commercially formulated E5564 was prepared by treatment of E5564
with NaOH as described above, followed by neutralization, addition of
phosphate-buffered lactose, and lyophilization. Vials of 1 mg of E5564
were reconstituted with sterile distilled water and diluted with 5%
dextrose in water. The following LPS strains were purchased from List
Biologicals (Campbell, CA): Escherichia coli (serotype
0111:B4; trichloroacetic acid-extracted), Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella
minnesota (wild type), Salmonella typhimurium,
Serratia marcescens, and Salmonella minnesota
R595. Salmonella entertidis LPS was purchased from
Sigma-Aldrich (St. Louis, MO). E. coli lipid A [serotype
0111:B4, LA-15-PP(506)] was purchased from Daiichi Chemical (Tokyo,
Japan). Whole bacteria Enterobacter aerogenes lot E25081, a
clinical isolate, and lot ATCC13048 (American Type Culture Collection,
Manassas, VA) were grown to late exponential phase in Mueller-Hinton
broth, and E. coli were grown overnight at 37°C in
heart-brain infusion media (Difco, Detroit, MI), harvested, washed by
centrifugation in Dulbecco's phosphate-buffered saline
(Ca2+/Mg2+-free),
resuspended in distilled deionized water, and lyophilized. Each
purified strain of LPS or lyophilized bacteria was solubilized in
sterile water for injection and stored as aliquots (21 mg/ml) [either
as purified LPS or 1 mg/ml (dry weight) whole bacteria], at 80°C.
For use, thawed samples were sonicated for 1 to 2 min as described
above immediately before each experiment. Live E. coli
(E01292) or S. aureus (E31290) were similarly grown and
prepared and used without freezing.
NF-B Reporter Activity in TLR4-Expressing Cells.
HEK293
cells stably carrying plasmids for TLR4, MD-2, and endothelial
leucocyte adhesion molecule-1 (ELAM-1)-luciferase were generated as
described previously (Yang et al., 2000) and shown to be responsive to
LPS plus CD14 (Hawkins et al., 2002). Cells were seeded in 96-well
plates at a density of 20,000 cells/well and maintained in Dulbecco's
modified Eagle's medium plus 10% fetal bovine serum for 24 h.
The next day, cells were incubated with the indicated concentration of
E5564 in the presence of LPS (100 ng/ml) and soluble CD14 (10 nM) for
18 to 20 h. Steady-Glo reagent (Promega, Madison, WI) was added to
the wells and the amount of luciferase activity in each sample was
quantified in a 1450 MicroBetaTrilux counter (PerkinElmer Wallac,
Gaithersburg, MD).
Preparation of Human Whole Blood and Cytokine Assays.
Induction of TNF- in human whole blood has been described previously
(Rose et al., 1995, 2000). Briefly, the concentrations of antagonists
indicated in the text and figures were added as 10× stocks in 50 µl
of 5% dextrose in water followed by 50 µl of LPS (10 ng/ml final
concentration) to 400 µl of heparinized whole blood obtained from
normal volunteers (18-51 years old; 50-105 kg) for a total of 500 µl/well (final concentration of whole blood was 80%). After 3-, 4-, 6-, 9-, or 24-h (as indicated) incubation with gentle shaking at 37°C
in a 5% CO2 atmosphere, plates were centrifuged
at 1000g for 10 min at 4°C and then plasma was drawn off
and frozen at 80°C. Plasma was appropriately diluted and tested for
TNF- or interleukins-1, IL-6, IL-8, and IL-10 and using the
appropriate human Predicta ELISA kit (Genzyme Diagnostics, Cambridge, MA).
as
described above.
Animal Care and Handling. All animals used for harvest of in vitro tissue samples were housed and cared for according to the Guidelines For Care and Use of Laboratory Animals (USDA National Institutes of Health Publication 86-23). Sprague-Dawley male rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA) and 18- to 22-g C57BL/6J male mice from Taconic Farms (Germantown, NY). Three- to 10-week-old Hartley White male guinea pigs were purchased from Elm Hill Breeding Laboratories (Chelmsford, MA). The animal room was maintained at 65 ± 3°F, 45 ± 5% relative humidity with a 12-h light/dark cycle. Animals were fed solid food and tap water ad libitum.
All in vivo experiments were approved by Animal Care and Use Committee of Eisai Co. Ltd. Eight- to 12-week-old C57BL/6 male mice (Japan SLC, Inc., Shizuoka, Japan), 4- to 6-week-old Hartley guinea pigs (Charles River Japan, Inc., Kanagawa, Japan), and 5- to 7-week-old Fischer rats (Japan SLC, Inc.) were housed and cared for in our laboratories. The animal room was maintained at 23 ± 1°C, 45 ± 5% relative humidity with a 12-h light/dark cycle. Animals were fed Agway ProLab (Agway, Inc., Syracuse, NY) as solid food and tap water was given ad libitum. To increase sensitivity of LPS, animals were primed 10 to 12 days before utilization by intravenous injection with 1 to 2 mg/animal of bacillus Calmette-Guerin (BCG; Japan BCG, Inc., Tokyo, Japan) suspended in pyrogen-free saline.Murine Whole Blood Assays.
Heparinized whole blood was
obtained from 8- to 10-week old Sprague-Dawley male rats or 18- to 22-g
C57BL/6J male mice. Mice were "primed" by injecting i.v. with an
attenuated, live preparation of BCG (2 mg/0.2 ml/tail vein). Blood was
collected 10 to 12 days after the injection of BCG from
CO2-euthanized animals by using cardiac puncture
into syringes containing sodium heparin (LyphoMed Inc., Rosemont, IL)
and pooled and stored on ice. One hundred sixty microliters of blood
was transferred to wells of a 96-well plate, followed by 20 µl of
E5564 and either 20 µl of Hanks' balanced salt solution or LPS. The
tissue culture plate was then incubated at 37°C, 5%
CO2 for 2 h, on a rotating mixer. Samples
were centrifuged (900g, 10 min, 4°C) and the supernatants
frozen for subsequent assay for TNF- or IL-6 by ELISA. Mouse TNF-
and IL-6 were assayed using ELISA mini-kits (Pierce Endogen, Rockford,
IL), TNF- was measured in rat plasma samples by ELISA using a rat
TNF- (BioSource International, Camarillo, CA), and IL-6 was measured
in rat plasma samples by a proliferative assay using IL-6-dependent B9
cells (LeMay et al., 1990).
Preparation of Peritoneal Macrophages and Incubations with LPS
and E5564.
Peritoneal macrophages were isolated from rats, mice,
and guinea pigs treated with 2 mg of a cell wall preparation from
Mycobacterium bovis (BCG; Ribi Immunochem Research Inc.,
Hamilton, MT) as described previously (Kobayashi et al., 1998).
Adherent cells were treated with 10 ng/ml E. coli LPS and
the indicated amount of E5564 was added to cultures of rat peritoneal
macrophages to achieve the concentrations indicated. After a 3-h
incubation, plates were centrifuged, and the resulting supernatant
samples were stored at 80°C until the cytokine assays were
performed. Murine IL-6 and TNF- was measured as described for plasma
(see above). TNF- in guinea pig peritoneal macrophage cultures was
quantified by cytotoxicity (LeMay et al., 1990), whereas IL-6 was
measured by bioassay as described above.
Statistical Analysis. Unless noted, in vitro experiments were done three times using triplicate determinations in each experiment. Mean and S.E. were calculated using standard calculations available in the Microsoft Excel spreadsheet. The E5564 concentration that inhibited 50% of the induced production of cytokine (the 50% inhibitory concentration; IC50) was calculated by a log-linear interpolation between the two points that span the 50% value. For in vivo studies, statistical analyses between the control groups and groups treated with E5564 were performed by one-way analysis of variance or the Fisher's exact test followed by Tukey's multiple comparison test or Dunnett's multiple comparison test. A value of 5% (two-sided) was considered statistically significant.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
E5564 Inhibition of LPS-Induced Cytokines in Human Monocytes and
Blood.
Assays measuring inhibition of TNF- induced by 10 ng/ml
LPS in adherent human monocytes in the presence of 10% human serum (Christ et al., 1995) indicated that the resultant
IC50 value for E5564 in this system was 0.36 ± 0.2 nM.
and other cytokines. As in other
model systems, 10 ng/ml LPS generated a near-maximal response for
TNF- that peaked at approximately 3 h. As shown in Fig.
1, response was inhibited 100% by 10 nM
E5564 with an IC50 value of approximately 1 nM.
In three assays the mean IC50 value for
inhibition of this response was 1.6 ± 0.3 nM (Table 1).
|
|
rose to 1,077.9 ± 181.4 pg/ml, IL-8 to over 20,000 pg/ml, and
IL-10 to over 450 pg/ml.
E5564 (10 nM) inhibited TNF- and IL-1 production by 100% at all
times tested. Similarly, LPS-induced IL-6 production was inhibited
greater than 94% by 100 nM E5564, with a mean
IC50 value of less than 2 nM (Table 1). Complete
inhibition of IL-8 production required slightly higher E5564
concentrations. LPS-induced IL-8 production was inhibited >70 to 90%
by 100 nM E5564 with an IC50 value of 13 nM over
the 24-h incubation time. IL-10 production was inhibited 100% by 10 nM
E5564, and the resulting IC50 of <1 nM E5564
against this cytokine (Table 1).
Potency of E5564 was dose-dependent for agonist. Other in vitro assays
in whole blood, indicated that if LPS concentration was reduced
10-fold, the IC50 value of E5564 was reduced
approximately 2- to 4-fold (data not shown).
The possibility that E5564 demonstrated agonistic activity in human
blood was tested by addition of 10 µM E5564 alone for up to 9 h.
In all experiments, resultant TNF- and/or IL-6 levels with were at
or below basal values, indicating that E5564 possesses no LPS-like
agonistic activity.
Antagonistic Effects of E5564 on TNF- Production Induced by LPS
from Different Strains of Bacteria, Whole Bacteria, and E.
coli Lipid A.
Lipopolysaccharides derived from various
strains of Gram negative bacteria induced TNF- in human blood. These
LPSs demonstrated different dose dependencies, so for comparison
purposes, their concentrations were adjusted to stimulate approximately
similar amounts of TNF- to that induced by the standard E. coli LPS (strain 0111:B4) at 10 ng/ml (Table
2).
|
(Table 2). E5564 similarly
antagonized this activation with IC50 values of
0.65 to 1.5 nM, indicating that E5564 can potently block activation by
whole Gram negative bacteria.
TNF- induced by 10 ng/ml lipid A was inhibited 77% by 10 nM E5564
and 100% by 100 nM E5564, with an average IC50
value of 1.2 ± 0.7 nM (Table 2).
Antagonism of Cellular Activation by Bacteria in Human Blood.
To determine whether cellular activation by live bacteria is inhibited
by E5564, antagonism of live Gram negative bacteria (E. coli) and live Gram positive bacteria (S. aureus) was
tested. As shown in Fig. 2, E5564
inhibited induction of TNF- by high doses of Gram negative bacteria,
but was inactive against Gram positive (S. aureus) bacteria
at concentrations up to 1 µM. These results indicate that E5564 is
active against cellular activation by Gram negative bacteria, but not
Gram positive bacteria (i.e., activation through TLR2).
|
Time-Dependent Inhibition of Antagonistic Activity by Serum.
Side-by-side comparisons of E5564 to E5531 (our "first-generation"
antagonist) indicated that E5564 is nearly 7-fold more potent an
inhibitor of TNF- production than E5531 [IC50
value for E5564 = 1.5 ± 0.37 versus 10.4 ± 3.1 nM for
E5531 (n = 7 assays)].
|
Antagonistic Activity of E5564 in Murine and Guinea Pig Macrophages
and Blood.
Mouse peritoneal macrophages incubated with 10 ng/ml
E. coli endotoxin for 2 h released 3315 ± 318 pg/ml TNF- and 5.0 ± 0.53 ng/ml IL-6. In this assay, E5564 at
a concentration of 100 nM inhibited release of TNF- by 95% and IL-6
by 89%. The IC50 value for E5564 was 20.4 ± 12.5 nM against TNF- and 16.6 ± 6.7 nM against IL-6 (Table
3).
|
was not robust or reproducible. However, IL-6
was found to be dose dependently stimulated by LPS with maximal
response at 10 µg/ml LPS, and greatest dose dependence at 1 to 100 ng/ml LPS. This response was dependent on time of incubation with LPS
for only up to 2 h with little or no further increase seen
thereafter (data not shown). After 2-h incubation with 10 ng/ml LPS,
IL-6 concentrations rose to 13 ± 0.18 ng/ml (n = 3) compared with 0.158 ± 0.013 ng/ml in samples that were not
treated with LPS. E5564 at a concentration of 100 nM inhibited the IL-6
increases by 85%, and the IC50 value for E5564
was 20.2 ± 7.0 nM in these combined experiments (Table 3).
To test for inhibition of nitric oxide production, cultured mouse
macrophages (RAW 264.7) were incubated overnight with 10 ng/ml LPS,
which induced accumulation of more than 20 µM nitrite in the culture
medium. E5564 dose dependently inhibited this induction [IC50 = 91 ± 36 nM (mean ± S.E.;
n = 5)] with >95% inhibition observed at 1 µM
E5564 (data not shown).
Rat peritoneal macrophages stimulated with 10 ng/ml LPS induced
2867 ± 326 pg/ml TNF-, whereas induction of IL-6 was robust but variable with release of between 23 and 163 ng of IL-6 per milliliter of culture medium. E5564 at 100 nM inhibited TNF- and
IL-6 production by 89%. The IC50 value for E5564
was 7 ± 5.6 nM for TNF- and 16.2 ± 17.5 nM (28.6 and 3.9 nM) for IL-6 (Table 3).
E5564 was significantly less active in rat blood than in rat peritoneal
macrophages. At a concentration of 1000 nM, E5564 inhibited LPS-induced
TNF- increases by 86%, and the IC50 for E5564
was 136 ± 61 nM in these combined experiments. In the same incubations where TNF- was measured, 10 µM E5564 inhibited the LPS-induced IL-6 production by 70% with an average
IC50 value of ~2400 nM.
Guinea pig macrophages incubated with 10 ng/ml E. coli LPS
for 3 h released 1897 ± 348 pg/ml TNF- (n = 3) and 3.0 ± 0.43 ng/ml IL-6 (n = 2). E5564 (10 nM) inhibited LPS-induced TNF- and IL-6 by >98%, with a resultant
IC50 value for E5564 of 0.30 ± 0.15 nM for
TNF- and 0.5 ± 0.3 nM for IL-6 (Table 3).
Inhibition of LPS Induced TNF- Release in Vivo.
E5564 or
vehicle was injected intravenously into BCG-primed mice along with 100 µg/kg LPS, a lethal dose. One hour after administration, blood was
collected and TNF- levels measured (Fig.
4). E5564 administered at 30, 100, 300, or 1000 µg/kg suppressed plasma TNF- concentrations by 24, 38, 81, and 93%, respectively.
|
levels (Fig. 4). Administration of 10, 30, 100, and 300 µg/kg E5564 suppressed LPS-induced plasma TNF- concentrations by 29, 57, and 94%,
respectively. The ED50 value for this model
system was estimated to be 37 µg/kg E5564.
Similarly, BCG-primed rats were intravenously administered E5564 or
vehicle along with 3 µg/kg LPS (a nonlethal dose), and blood was
collected 1 h later for assay of TNF concentrations (Fig. 4).
Administration of E5564 inhibited induction of TNF- by 84, 97, and
100%, by 10, 100, and 1000 µg of E5564/kg, respectively.
Effect of E5564 on LPS-Induced Lethality in Mice.
To evaluate
the ability of E5564 to prevent LPS-induced mortality, BCG-primed mice
were injected intravenously with 100 µg/kg LPS along with the
indicated doses of E5564 or vehicle, and incidence of mortality was
monitored for 72 h (Fig. 5). Whereas
the administration of 100 µg/kg LPS alone resulted in 90% mortality
by 72 h, coinjection of E5564 significantly and dose dependently
reduced the incidence of mortality.
|
Effects of E5564 on Septic Shock Caused by Bacterial Infection in
Mice.
The objective of the study was to examine the use of E5564
in conjunction with the -lactam antibiotic latamoxef to prevent mortality in mice injected with E. coli. A suspension of
E. coli (3.23 × 107 cfu/animal)
was injected intraperitoneally into BCG-primed mice. One hour later,
the mice were injected intravenously with vehicle, E5564 alone (5 mg/kg), latamoxef alone (30 mg/kg), or E5564 and latamoxef together.
The incidence of mortality was recorded for 72 h after infection.
|
Lack of Agonistic Activity of E5564 in Vivo.
E5564 was
intravenously injected into BCG-primed animals (3000 µg/kg into mice,
300 µg/kg into guinea pigs, and 100 and 1000 µg/kg into rats) and
blood was collected for analysis of TNF- 1 h later. Neither
E5564 nor vehicle alone caused any marked induction of TNF-,
indicating that E5564 is devoid of endotoxin-like activity in this assay.
How Does E5564 Work?
Interaction of E5564 at the TLR4 receptor
for LPS has been investigated by measuring response to LPS (expression
of NF-B-driven luciferase) by HEK293 cells made responsive to LPS by
transfection with TLR4 and MD-2. As shown in Fig.
7, luciferase expression in this system
is dependent on LPS plus soluble CD14 and inhibited by E5564
(IC50 = 32 nM). In contrast, E5564 did not inhibit
heat-killed S. aureus activation of TLR2-transfected
HEK293 cells at concentrations up to 1 µM (J. Chow, unpublished
data). These results suggest that E5564 interferes with LPS signaling
at the TLR4 receptor and/or soluble CD14.
|
release after various
drug pretreatment regimens. Pilot experiments indicated that treatment
of cells with E5564 followed by rapid, extensive cell washing and
addition of LPS along with serum or LBP for 3 h resulted in clear
dose-dependent inhibition of LPS activity. As shown in Table
4, addition of E5564 in the absence of
serum followed by washing (three times) and addition of serum plus LPS,
resulted in IC50 values of 0.8 ± 0.1 nM
(n = 3). If serum is included in this E5564
pretreatment, the resultant IC50 value was
unchanged at 1.1 nM (n = 2), indicating that serum did
not increase or dramatically decrease the potency of E5564. Similarly,
purified LBP used in the place of serum had no effect on E5564 activity
(data not shown).
|
release. The resulting
IC50 values for E5564 were unchanged after 30-min incubation in serum-free medium (0.7 ± 0.1 nM) and increased
slightly (2.5 nM) when serum was included in the incubation medium.
Extending the "washout" period to 2 h in the presence of serum
further increased the IC50 value to 24 to 25 nM,
indicating a slow loss of "surface-associated" antagonistic activity.
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
A variety of pathologies have been attributed to responses to
endotoxin even in cases where identification of its source is unclear.
In the clinic, antibiotic treatment to control bacterial infection has
been reported to result in the release of endotoxin and consequent
aggravation of septic shock. Although the source of endotoxin during
infection by Gram negative bacteria may seem obvious, endotoxemia has
also been reported to occur during Gram positive and fungal infection
(Opal et al., 1999). In addition, it has been reported that endotoxin
is translocated from the intestinal tract to the splanchnic circulation
under a variety of conditions. Perhaps more definitive proof that
pathological outcomes are truly due to endotoxin awaits the therapeutic
application of an effective endotoxin antagonist.
Mechanism of Action of E5564
It is not clear exactly how E5564 works to block LPS signaling. Because E5564 is a structural analog of the lipid A portion of LPS, it is logical to hypothesize that the antagonist interacts with the same signaling components that interact with LPS such as the soluble serum proteins LBP and sCD14, as well as membrane-associated CD14 and perhaps the TLR4/MD-2 receptor complex. In the present study, E5564 blocked LPS/sCD14-induced reporter activity in TLR4/MD-2-expressing HEK293 (Fig. 7), but not TLR2-mediated signaling by heat-killed S. aureus. These findings indicate that E5564 selectively inhibits LPS signaling via TLR4/MD-2. However, a limitation to this model system is that LPS requires the presence of sCD14 for cellular activation, making it difficult to determine whether E5564 blocks LPS binding to sCD14 or TLR4/MD-2. Results from experiments described in Table 4 indicated that serum components neither increased nor dramatically decreased the potency of E5564, indicating that they are not critical to E5564 antagonistic activity.
Further support of the hypothesis that interaction of E5564 at CD14
does not play a key role in its activity comes from a previous study by
Lien et al. (2000) describing the activity of novel synthetic acyclic
lipid A-like agonists that activate TLR4/MD-2 in the absence of CD14.
E5564 inhibited the actions of these agonists under serum-free
conditions. Taken together, these lines of evidence make it tempting to
speculate that E5564 binds to TLR4/MD-2 complex, thereby blocking LPS
binding or transmembrane signaling.
The downstream effect of inhibiting the initial signaling by LPS seems
to be an inhibition of all LPS-induced cytokines measured, including
TNF-, IL-1, IL-6, IL-8, IL-10, and nitric oxide, which was
measured in cultured cells, whole blood, and in vivo.
The likelihood that E5564 does not inhibit the interaction of LPS with
soluble receptors may increase its value as a therapeutic agent.
Because endotoxemia and sepsis induce acute phase in the liver, plasma
levels of proteins such as LBP change dramatically (Opal et al., 1999).
Therapy targeted toward these serum proteins would require dosing
adjustments to accommodate increased synthesis or turnover of these proteins.
Comparisons of antagonistic potency in cells cultured in 10% serum
versus whole blood allow us to determine whether the high concentration
of proteins/lipoproteins present in serum inhibit E5564 activity. In
all systems but the rat, antagonistic activity of E5564 in cultured
cells was within 4-fold that measured in high serum (blood) compared
with assays done in low-serum conditions (cultured cells or monocytes).
This indicates that serum has little or no inhibitory effect on
antagonistic activity under these in vitro conditions. However,
extended incubations in whole blood demonstrated that activity of E5564
was measurably reduced. Other studies (K. M. Wasan, O. Sivak, R. A. Cote, A. I. MacInnes, K. D. Boulanger, M. Lynn, W. J. Christ, L. D. Hawkins, and D. P. Rossignol, manuscript in preparation)
indicate that like E5531, E5564 is not rapidly metabolized, but binds
to lipoproteins, and time dependently loses antagonistic activity. The
observation that lipoproteins reduce drug activity may explain the poor
activity of E5564 in rat blood that has a relatively high lipoprotein
content (Segrest and Albers, 1986).
Is Endotoxin the Major Component from Gram Negative Bacteria That Activates Cytokine Response in Blood?
It has been postulated that bacterial components other than
endotoxin may activate cells in whole blood, and recently, different subtypes of toll-like receptors have been implicated in responses to
these different components. However, this differentiation of response
to different receptors does not indicate the relative importance of
these components in cellular stimulation. E5564 seems to be a specific
antagonist for the TLR4 receptor and is inactive against TLR2-directed
agonists (Heine et al., 2000). Based on the ability of E5564 to inhibit
cellular activation by LPS from a variety of Gram negative bacteria,
whole killed or live bacteria, but not Gram positive bacteria, it is
likely that E5564 is an effective antagonist for activation of blood
cells specifically by Gram negative bacteria. Furthermore, the
observation that E5564 is a potent and complete (or nearly complete)
inhibitor of Gram negative bacteria in the bacterial sepsis model helps clarify the role of endotoxin in activation of cells in blood. Effective reduction of inflammatory response and death due to bacterial
infection by E5564 indicates that endotoxin or other agents that bind
to TLR4 drive response to Gram negative bacteria.
Therapeutic Potential of E5564 as an Endotoxin Antagonist
E5564 Is Being Developed as an Endotoxin Antagonist for Human
Therapeutic Use.
Safe treatment with E5564 requires that it
generate no LPS-like response on its own (Rossignol et al., 1999). When
added alone to whole blood or injected into BCG-primed animals, E5564
was devoid of agonistic activity (cytokine generation or overt
physiological effects) at concentrations as high as 100 µM in vitro
or up to 1000 µg/animal (the highest concentrations and doses tested).
; Kobayashi et al., 1998); however, we have not measured
plasma endotoxin levels in treated animals in this study because E5564
is a potent activator of the limulus assay, likely due to its
structural similarities to Lipid A (data not shown).
Both our LPS-challenge model and infection model use animals that have
been sensitized or primed to LPS by previous infection with BCG,
increasing cytokine response and lowering the threshold lethal dose of
endotoxin. All animal models of sepsis and infection have been
criticized for their inability to closely mimic human sepsis. However,
we believe that the primed model is the most relevant to the study of
endotoxin antagonists such as E5564. It is well known that compared
with humans, unprimed rodents such as rats and mice and primates
demonstrate a profound insensitivity to endotoxin, requiring endotoxin
doses as high as milligrams per kilogram, whereas humans demonstrate
reproducible response to endotoxin at doses as low as 2 ng/kg. This
argues that either LPS contributes only weakly to the inflammatory
process in animal models, or that response to infection occurs only
after the level of infection is very high, representing a process
different from that in more LPS-sensitive species such as humans.
Even in primed animal models, lethal doses of LPS in are high,
approximately 100 µg/kg, generating estimated plasma concentrations of ~1 µg/ml. These plasma levels are still >100-fold that found in
even the most extreme cases of human sepsis (Opal et al., 1999). Because the dose of E5564 required to protect against LPS is
proportional to the LPS challenge dose, studying E5564 in these animal
models indicates that E5564 can be a safe and effective antagonist even under these extraordinary conditions. E5564 is approximately 10-fold better in human blood than mouse blood (IC50 = 1.6 nM in human whole blood; Table 2 versus ~20 nM in mouse whole
blood; Table 3). Taken together, comparison of in vitro and in vivo
results leads us to believe that in clinical use, we may be able to
readily establish plasma concentrations of E5564 that will provide us with a wide margin of efficacy against even very high plasma
concentrations of LPS.
Complete block of cytokine response by 10 nM E5564 in blood
extrapolates to a human dose of ~100 µg in a 70-kg individual. Recent studies have supported this extrapolation by finding that a dose
of 100 µg of E5564 given to normal volunteers over 30 min completely
blocks response (signs, symptoms, and cytokines) to a dose of 4 ng/kg
endotoxin administered at the midpoint of the E5564 infusion (Lynn et
al., 2003).
In vitro and ex vivo assays have found that low concentrations of E5564
time dependently lose ability to inhibit LPS response. In light of
these observations, it is perhaps not surprising that low doses of
E5564 demonstrate a time-dependent loss of activity after
administration into normal volunteers. This loss in activity is
overcome when E5564 doses are increased (D. P. Rossignol, K. M. Wasan,
N. Wong, L. Q. Ngyen, J. Rose, J. Moran, and M. Lynn, manuscript in
preparation). Phase I clinical safety and tolerability assays
indicate that E5564 is safe and except for the occurrence of phlebitis,
well tolerated at doses up to 252 mg administered over 72 h. At
this dose, in vivo antagonistic activity is retained for at least an
additional 72 h after discontinuing infusion. This leads us to
believe that sufficient therapeutic activity can readily be
administered to patients.
In conclusion, we have found that E5564 is a highly active antagonist
of LPS in vitro in human and animal systems. This activity translates
into effective in vivo antagonism of endotoxin in animal models,
resulting in enhancement of survival after challenge with endotoxin or
bacterial infection. This consistent, potent activity combined with a
clear lack of "LPS-like" agonistic activity leads us to believe
that E5564 can be a safe clinical therapeutic for the treatment of
endotoxemia, including sepsis and septic shock.
| |
Acknowledgments |
|---|
We thank Donna Young for excellent technical assistance. Whole bacteria Enterobacter aerogenes was obtained from Dr. Naoaki Watanabe (Tsukuba Research Laboratories). Lot ATCC13048 was obtained from American Type Culture Collection (Manassas, VA), and Lot E25081 was a clinical isolate.
| |
Footnotes |
|---|
Accepted for publication November 15, 2002.
Received for publication September 17, 2002.
1 Current address: Harvard Medical School, Department of Membrane Transport, Boston, MA 02115.
2 Current address: Eisai Medical Research Inc., Glenpointe Centre W., 500 Frank W. Burr Blvd., Teaneck, NJ 07666-6741.
DOI: 10.1124/jpet.102.044487
Address correspondence to: Daniel P. Rossignol, Eisai Medical Research Inc., Glenpointe Centre West 5th Floor, 500 Frank W. Burr Blvd., Teaneck, NJ 07666-6741. E-mail: dan_rossignol{at}eisai.com
| |
Abbreviations |
|---|
LPS, lipopolysaccharide;
LBP, lipopolysaccharide binding protein;
TLR, toll-like receptor;
NF-B, nuclear factor-B;
HEK, human embryonic kidney;
TNF-, tumor
necrosis factor-;
IL, interleukin;
ELISA, enzyme-linked
immunosorbent assay;
BCG, bacillus Calmette-Guerin;
cfu, colony-forming
unit;
E5531, (6-O-{2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-Z-dodec-5-enoyloxydecyl]-2-[3-oxo-tetradecanoylamido]--O-phosphono--D-glucopyranose tetrasodium salt.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
B translocation in CHO cells can not be blocked by LPS antagonists.
J Endotoxin Res
6:
139.This article has been cited by other articles:
|
|
 |
|
  L. A. J. O'Neill, C. E. Bryant, and S. L. Doyle Therapeutic Targeting of Toll-Like Receptors for Infectious and Inflammatory Diseases and Cancer Pharmacol. Rev., June 1, 2009; 61(2): 177 - 197. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Mencin, J Kluwe, and R F Schwabe Toll-like receptors as targets in chronic liver diseases Gut, May 1, 2009; 58(5): 704 - 720. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Roger, C. Froidevaux, D. Le Roy, M. K. Reymond, A.-L. Chanson, D. Mauri, K. Burns, B. M. Riederer, S. Akira, and T. Calandra Protection from lethal Gram-negative bacterial sepsis by targeting Toll-like receptor 4 PNAS, February 17, 2009; 106(7): 2348 - 2352. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. P. Rossignol, N. Wong, R. Noveck, and M. Lynn Continuous pharmacodynamic activity of eritoran tetrasodium, a TLR4 antagonist, during intermittent intravenous infusion into normal volunteers Innate Immunity, December 1, 2008; 14(6): 383 - 394. [Abstract] [PDF]
|
|
|
|
 |
|
 B. Zhang, G. Ramesh, S. Uematsu, S. Akira, and W. B. Reeves TLR4 Signaling Mediates Inflammation and Tissue Injury in Nephrotoxicity J. Am. Soc. Nephrol., May 1, 2008; 19(5): 923 - 932. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Munford Sensing Gram-Negative Bacterial Lipopolysaccharides: a Human Disease Determinant? Infect. Immun., February 1, 2008; 76(2): 454 - 465. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Tse and A. A Horner Update on toll-like receptor-directed therapies for human disease Ann Rheum Dis, November 1, 2007; 66(suppl_3): iii77 - iii80. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ehrentraut, S. Frede, H. Stapel, T. Mengden, C. Grohe, J. Fandrey, R. Meyer, and G. Baumgarten Antagonism of Lipopolysaccharide-Induced Blood Pressure Attenuation and Vascular Contractility Arterioscler. Thromb. Vasc. Biol., October 1, 2007; 27(10): 2170 - 2176. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. T. Figueiredo, P. L. Fernandez, D. S. Mourao-Sa, B. N. Porto, F. F. Dutra, L. S. Alves, M. F. Oliveira, P. L. Oliveira, A. V. Graca-Souza, and M. T. Bozza Characterization of Heme as Activator of Toll-like Receptor 4 J. Biol. Chem., July 13, 2007; 282(28): 20221 - 20229. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Into, J.-i. Dohkan, M. Inomata, M. Nakashima, K.-i. Shibata, and K. Matsushita Synthesis and Characterization of a Dipalmitoylated Lipopeptide Derived from Paralogous Lipoproteins of Mycoplasma pneumoniae Infect. Immun., May 1, 2007; 75(5): 2253 - 2259. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Bennett-Guerrero, H. P. Grocott, J. H. Levy, K. A. Stierer, C. W. Hogue, A. T. Cheung, M. F. Newman, A. A. Carter, D. P. Rossignol, and C. D. Collard 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 Anesth. Analg., February 1, 2007; 104(2): 378 - 383. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Shimamoto, T. H. Pohlman, S. Shomura, T. Tarukawa, M. Takao, and H. Shimpo Toll-Like Receptor 4 Mediates Lung Ischemia-Reperfusion Injury Ann. Thorac. Surg., December 1, 2006; 82(6): 2017 - 2023. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. F. P. Berbee, C. C. van der Hoogt, R. Kleemann, E. F. Schippers, R. L. Kitchens, J. T. van Dissel, I. A. J. M. Bakker-Woudenberg, L. M. Havekes, and P. C. N. Rensen Apolipoprotein CI stimulates the response to lipopolysaccharide and reduces mortality in Gram-negative sepsis FASEB J, October 1, 2006; 20(12): 2162 - 2164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Shimamoto, A. J. Chong, M. Yada, S. Shomura, H. Takayama, A. J. Fleisig, M. L. Agnew, C. R. Hampton, C. L. Rothnie, D. J. Spring, et al. Inhibition of Toll-like Receptor 4 With Eritoran Attenuates Myocardial Ischemia-Reperfusion Injury Circulation, July 4, 2006; 114(1_suppl): I-270 - I-274. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. W. Moretti, M. F. Newman, L. H. Muhlbaier, D. Whellan, R. P. Petersen, D. Rossignol, C. B. McCants Jr, B. Phillips-Bute, and E. Bennett-Guerrero Effects of Decreased Preoperative Endotoxin Core Antibody Levels on Long-term Mortality After Coronary Artery Bypass Graft Surgery Arch Surg, July 1, 2006; 141(7): 637 - 641. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Roelofs, W. C. Boelens, L. A. B. Joosten, S. Abdollahi-Roodsaz, J. Geurts, L. U. Wunderink, B. W. Schreurs, W. B. van den Berg, and T. R. D. J. Radstake Identification of Small Heat Shock Protein B8 (HSP22) as a Novel TLR4 Ligand and Potential Involvement in the Pathogenesis of Rheumatoid Arthritis. J. Immunol., June 1, 2006; 176(11): 7021 - 7027. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Chen, D. B. Rosenbluth, M. A. Mintun, and D. P. Schuster FDG-PET imaging of pulmonary inflammation in healthy volunteers after airway instillation of endotoxin J Appl Physiol, May 1, 2006; 100(5): 1602 - 1609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ii, N. Matsunaga, K. Hazeki, K. Nakamura, K. Takashima, T. Seya, O. Hazeki, T. Kitazaki, and Y. Iizawa A Novel Cyclohexene Derivative, Ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), Selectively Inhibits Toll-Like Receptor 4-Mediated Cytokine Production through Suppression of Intracellular Signaling Mol. Pharmacol., April 1, 2006; 69(4): 1288 - 1295. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Visintin, K. A. Halmen, E. Latz, B. G. Monks, and D. T. Golenbock Pharmacological Inhibition of Endotoxin Responses Is Achieved by Targeting the TLR4 Coreceptor, MD-2 J. Immunol., November 15, 2005; 175(10): 6465 - 6472. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Zhou, J. Kozlowski, A. L. Goodrich, N. Markman, D. L. Chen, and D. P. Schuster Molecular imaging of lung glucose uptake after endotoxin in mice Am J Physiol Lung Cell Mol Physiol, November 1, 2005; 289(5): L760 - L768. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Zhou, J. Kozlowski, and D. P. Schuster Physiologic, Biochemical, and Imaging Characterization of Acute Lung Injury in Mice Am. J. Respir. Crit. Care Med., August 1, 2005; 172(3): 344 - 351. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Savov, D. M. Brass, B. L. Lawson, E. McElvania-Tekippe, J. K. L. Walker, and D. A. Schwartz Toll-like receptor 4 antagonist (E5564) prevents the chronic airway response to inhaled lipopolysaccharide Am J Physiol Lung Cell Mol Physiol, August 1, 2005; 289(2): L329 - L337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. S. Zamboni, M. A. Campos, A. C. T. Torrecilhas, K. Kiss, J. E. Samuel, D. T. Golenbock, F. N. Lauw, C. R. Roy, I. C. Almeida, and R. T. Gazzinelli Stimulation of Toll-like Receptor 2 by Coxiella burnetii Is Required for Macrophage Production of Pro-inflammatory Cytokines and Resistance to Infection J. Biol. Chem., December 24, 2004; 279(52): 54405 - 54415. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. P. Rossignol, K. M. Wasan, E. Choo, E. Yau, N. Wong, J. Rose, J. Moran, and M. Lynn Safety, Pharmacokinetics, Pharmacodynamics, and Plasma Lipoprotein Distribution of Eritoran (E5564) during Continuous Intravenous Infusion into Healthy Volunteers Antimicrob. Agents Chemother., September 1, 2004; 48(9): 3233 - 3240. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B Safieh-Garabedian, G M Mouneimne, W El-Jouni, M Khattar, and R Talhouk The effect of endotoxin on functional parameters of mammary CID-9 cells Reproduction, March 1, 2004; 127(3): 397 - 406. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Latz, J. Franko, D. T. Golenbock, and J. R. Schreiber Haemophilus influenzae Type b-Outer Membrane Protein Complex Glycoconjugate Vaccine Induces Cytokine Production by Engaging Human Toll-Like Receptor 2 (TLR2) and Requires the Presence of TLR2 for Optimal Immunogenicity J. Immunol., February 15, 2004; 172(4): 2431 - 2438. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Stover, J. Da Silva Correia, J. T. Evans, C. W. Cluff, M. W. Elliott, E. W. Jeffery, D. A. Johnson, M. J. Lacy, J. R. Baldridge, P. Probst, et al. Structure-Activity Relationship of Synthetic Toll-like Receptor 4 Agonists J. Biol. Chem., February 6, 2004; 279(6): 4440 - 4449. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Lynn, Y. N. Wong, J. L. Wheeler, R. J. Kao, C. A. Perdomo, R. Noveck, R. Vargas, T. D'Angelo, S. Gotzkowsky, F. G. McMahon, et al. Extended in Vivo Pharmacodynamic Activity of E5564 in Normal Volunteers with Experimental Endotoxemia J. Pharmacol. Exp. Ther., January 1, 2004; 308(1): 175 - 181. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Liang, Y. N. Wong, I. Allen, R. Kao, M. Marino, and C. DiLea Pharmacokinetics of E5564, a Lipopolysaccharide Antagonist, in Patients with Impaired Hepatic Function J. Clin. Pharmacol., December 1, 2003; 43(12): 1361 - 1369. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Sabroe, R. C. Read, M. K. B. Whyte, D. H. Dockrell, S. N. Vogel, and S. K. Dower Toll-Like Receptors in Health and Disease: Complex Questions Remain J. Immunol., August 15, 2003; 171(4): 1630 - 1635. [Full Text] [PDF]
|
|
|
|
|
|
Y. N. Wong, D. Rossignol, J. R. Rose, R. Kao, A. Carter, and M. Lynn Safety, Pharmacokinetics, and Pharmacodynamics of E5564, a Lipid A Antagonist, during an Ascending Single-Dose Clinical Study J. Clin. Pharmacol., July 1, 2003; 43(7): 735 - 742. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
, 
), IL-1
, 
), IL-6 (
, 
), IL-8 (
, 
), or
IL-10 (
, 
). Complete inhibition of release of all cytokines was
observed in the presence of 100 nM E5564 (data not shown). Incubation
of unstimulated blood samples for up to 24 h induced less than 1%
of peak levels for TNF-
