U.S. patent application number 10/004139 was filed with the patent office on 2002-08-29 for uses of lipopolysaccharide binding protein.
This patent application is currently assigned to XOMA Corporation. Invention is credited to Carroll, Stephen F., Dedrick, Russell L..
Application Number | 20020119930 10/004139 |
Document ID | / |
Family ID | 25497158 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119930 |
Kind Code |
A1 |
Dedrick, Russell L. ; et
al. |
August 29, 2002 |
Uses of lipopolysaccharide binding protein
Abstract
Novel LBP compositions and therapeutic uses for LBP are provided
for preventing the adverse effects of exposure to endotoxin.
Inventors: |
Dedrick, Russell L.;
(Kensington, CA) ; Carroll, Stephen F.; (Walnut
Creek, CA) |
Correspondence
Address: |
HONEYWELL INTERNATIONAL INC.
101 COLUMBIA ROAD
P O BOX 2245
MORRISTOWN
NJ
07962-2245
US
|
Assignee: |
XOMA Corporation
|
Family ID: |
25497158 |
Appl. No.: |
10/004139 |
Filed: |
October 23, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10004139 |
Oct 23, 2001 |
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09395453 |
Sep 14, 1999 |
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6306824 |
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Current U.S.
Class: |
514/44R ;
514/2.1 |
Current CPC
Class: |
A61K 38/1709
20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 038/17 |
Claims
What is claimed is:
1. A method for inhibiting adverse effects of endotoxin in
circulation comprising the steps of determining the circulating
lipopolysaccharide binding protein (LBP) level of a subject at risk
for exposure to endotoxin, and administering to said subject having
a circulating LBP level within the normal range an amount of LBP
effective to elevate the circulating LBP level to inhibit the
adverse effects of exposure to endotoxin.
2. The method of claim 1 wherein the circulating LBP level of said
subject is elevated to a level from about 15 .mu.g/mL to about 100
.mu.g/mL.
3. A composition comprising lipopolysaccharide binding protein
(LBP) in a solution buffered at about pH 7.5 and containing a
poloxamer surfactant.
4. A composition comprising LBP in a solution buffered at about pH
7.5 and containing a poloxamer surfactant and a polysorbate
surfactant.
Description
[0001] The present invention relates to a novel use of
lipopolysaccharide binding protein (LBP) as a
prophylactic/therapeutic agent in blocking the pathological effects
of lipopolysaccharide (LPS), also known as endotoxin.
BACKGROUND OF THE INVENTION
[0002] LPS is a major component of the outer membrane of
gram-negative bacteria and consists of serotype-specific O-side
chain polysaccharides linked to a conserved region of core
oligosaccharide and lipid A. LPS is a potent inducer of
inflammation, stimulating the expression of many pro-inflammatory
and pro-coagulant mediators in monocytes, macrophages and
endothelial cells. These responses are important in containing and
eliminating a localized infection, however, adverse effects of
systemic exposure to LPS can include induction of an inflammatory
cascade, damage to endothelium, widespread coagulopathies, and
organ damage. Systemic exposure to LPS can arise from direct
infection of gram negative bacteria, leading to the complications
of gram-negative sepsis [Tracey et al., Adv. Surg. 23: 21-56
(1990)]. Alternatively, a variety of conditions and circumstances,
including trauma, can induce changes in gut permeability that
result in translocation of bacteria, and therefore LPS, into
circulating blood. Bacterial LPS translocated from the gut is
thought to play a major role in post-surgical immunosuppression
[Little et al., Surgery 114(1): 87-91 (1993)] and hemorrhagic
shock. Therefore, there exists a need to discover therapies that
can counteract the effects of LPS in pathologic situations.
[0003] Two proteins, CD14 and lipopolysaccharide binding protein
(LBP) [Schumann et al., Science 249: 1429-1431 (1990); Wright et
al., Science 249: 1431-1433 (1990)] have been shown to be required
to generate an inflammatory response to LPS. LPS must bind to CD14
to activate an inflammatory response. CD14 is a 55 kD protein
expressed via a glycosylphosphatidylinositol-anchor on the surface
of macrophages, monocytes and neutrophils (mCD14). Endothelial and
epithelial cells, which do not express the CD14 protein, are
activated by LPS bound to a soluble form of (sCD14) found in serum
or plasma (at a concentration of about 2 .mu.g/mL in normal human
blood). CD14 preferentially binds to LPS monomers [Tobias et al.,
J. Biol. Chem. 270(18): 10482-10488 (1995)]. Since purified LPS
exists in aqueous solution in micelles or aggregates, direct
binding of LPS to CD14 is very slow [Tobias et al. (1995), supra;
Yu and Wright, J. Biol. Chem. 271(8): 4110-4105 (1996)] and only
occurs at high concentrations of LPS [Hailman et al., J. Exp. Med.
179(1): 269-277 (1994)]. Binding of LPS to CD14 is greatly
accelerated by LBP [Hailman et al. (1994), supra; Tobias et al.
(1995), supra; Yu et al. (1996), supra], and LBP is required for
activation of cells by either mCD14 or sCD14 at physiological
concentrations of LPS [Schumann et al. (1990), supra; Wright et al.
(1990), supra].
[0004] LBP is a 60 kD glycoprotein synthesized in the liver and
present in normal human serum. LBP belongs to the group of plasma
proteins called acute phase proteins, including C-reactive protein,
fibrinogen and serum amyloid A, that increase in concentration in
response to infectious, inflammatory and toxic mediators. LBP
expression has been induced in animals by challenge with LPS,
silver nitrate, turpentine and Corynebacterium parvum [Geller et
al., Arch. Surg. 128(1): 22-28 (1993); Gallay et al., Infect.
Immun. 61(2): 378-383 (1993); Tobias et al., i J. Exp. Med. 164:
777-793 (1986)]. However, while administration of silver nitrate
caused LBP levels to increase in several strains of mice, this was
not observed in one strain, C3H/HeJ, in which LPS does not induce
an inflammatory response [Gallay et al. (1993), supra]. Recently,
an analysis of different human disease states has indicated that
increased LBP levels are uniquely correlated with exposure to LPS.
In human patients with presumed gram-negative sepsis, serum LBP
levels can reach from about 50 to about 100 .mu.g/mL [U.S. Pat. No.
5,484,705]. In contrast, in other disease states, such as
rheumatoid arthritis, involving an acute phase response in which
elevated levels of the acute phase proteins CRP and fibrinogen were
measured in patient serum samples, no significant increases in LBP
levels were observed. Elevated, particularly persistently elevated,
LBP levels have been correlated with poor clinical outcome in
septic patients [U.S. Pat. No. 5,484,705, and U.S. Ser. No.
081377,391 filed Jan. 24, 1995, both of which are hereby
incorporated by reference in their entirety]. This has been
confirmed by Schumann et al., 36th Int'l Conf. on Antimicrobial
Agents and Chemotherapy, New Orleans, La., Sep. 15-18, 1996.
[0005] LBP is reported to bind to LPS aggregates (at low LBP to LPS
ratios) or to disaggregate LPS vesicles (at high LBP to LPS ratios)
[Tobias et al. (1995), supra] to form an LBP:LPS complex that
greatly facilitates binding of LPS to either mCD14 or sCD14 [Wright
et al., J. Exp. Med. 173(5): 1281-1286 (1991); Hailman et al.
(1994), supra; Yu et al. (1996), supra; Tobias et al. (1995),
supra]. LBP is reported to act catalytically in facilitating LPS
binding to CD14, a single LBP molecule enabling the transfer over
100 LPS molecules to CD14 [Hailman et al. (1994), supra]. LBP is
also reported to remain associated with LPS aggregates or LPS
coated particles and facilitate binding to cells expressing mCD14
in a phenomenon known as opsonization [Wright et al., J. Exp. Med.
170(4): 1231-1241 (1989); Kirkland et al., J. Biol. Chem. 268(33):
24818-24823 (1993); Gegner et al., J. Biol. Chem. 270(10):
5320-5325 (1995)]. Thus, LBP potentiates the inflammatory activity
of LPS and is recognized as an immunostimulatory molecule.
Functional analysis of the LBP molecule has demonstrated that LPS
binding resides in the approximate N-terminal half of the protein,
but the C-terminal half is required to permit transfer of LPS to
CD14 [U.S. application Ser. No. 08/261,660 filed Jun. 17, 1994;
Theofan et al., J. Immunol. 152(7): 3624-3629 (1994); Han et al.,
J. Biol. Chem. 269(11): 8172-8175 (1994)]. Because of the observed
potentiating effect LBP has on the inflammatory potential of LPS,
blocking or interfering with the immunostimulatory activity of LBP
has been a therapeutic target of interest.
[0006] For example, a polyclonal antibody preparation to murine LBP
has been shown to prevent LBP mediated binding of LPS to murine
macrophages and subsequent induction of TNF expression in vitro,
effectively neutralizing the activity of LBP. This same polyclonal
antibody was able to reduce lethality in a murine model of
endotoxemia [Gallay et al. (1993), supra].
[0007] Several modified forms of LBP have been developed that bind
LPS but lack the ability to transfer the LPS molecule to CD14. U.S.
application Ser. No. 08/261,660 filed Jun. 17, 1994, hereby
incorporated by reference in its entirety, describes novel
biologically active polypeptide derivatives of LBP, including LBP
derivative hybrid proteins, which are characterized by the ability
to bind to LPS and which lack CD14-mediated immunostimulatory
properties, including the ability of LBP holoprotein to mediate LPS
activity via the CD14 receptor, More particularly, these LBP
protein derivatives including LBP derivative hybrid proteins
lacking those carboxy terminal-associated elements characteristic
of the LBP holoprotein which enable LBP to bind to and interact
with the CD14 receptor on monocytes and macrophages so as to
provide an immunostimulatory signal to monocytes and macrophages.
Such 13P protein derivatives included those characterized by a
molecular weight less than or equal to about 25 kD, including an
amino-terminal LBP fragment having amino acid residues 1-197 that
was designated rLBP.sub.25. This recombinant protein corresponding
to the amino-terminal residues 1-197 of LBP has been shown to bind
LPS but could neither facilitate binding of LPS to CD14 nor permit
LPS-induced expression of TNF [see also, Theofan et al. (1994),
supra; Han et al. (1994), supra]. Additionally, this N-terminal
fragment was shown to inhibit LPS-induced expression of TNF that
was mediated by full-length LBP [Han et al. (1994), supra].
rLBP.sub.25 includes amino acid regions comprising LBP residues 17
through 45, 65 through 99 and 141 through 167 which correspond to
respective biologically active (e.g., LPS binding) domains (e.g.,
Domain I--residues 17 through 45; Domain II--residues 65 through
99; and Domain III--residues 142 through 169) of
bactericidal/permeability-increasing protein (BPI). The LBP
derivative hybrid proteins included hybrids of LBP protein
sequences with the amino acid sequences of other polypeptides and
also characterized by the ability to bind to LPS and the absence of
CD14-mediated immunostimulatory properties. Such hybrid proteins
included fissions of LBP amino-terminal fragments with polypeptide
sequences of other proteins such as BPI, immunoglobulins and the
like. Properties of several LBP/BPI fusion proteins have been
described by Abrahamson et al., J. Biol. Chem. 272(4):2149-2155
(1997). In addition, a recombinant hybrid fusion between the
N-terminal 199 amino acid residues of LBP and the C-terminal 257
residues of BPI was shown to be protective in a rodent model of
gram-negative sepsis [Opal et al., Antimicrob. Agents Chemother.
39(12): 2813-2815 (1995)]. U.S. application Ser. No. 08/261,660
filed Jun. 17, 1994 also describes LBP derivatives in the form of
synthetic LBP peptides that are portions of the LBP sequence
corresponding to either Domain II (residues 65-99) or Domain m
(residues 142-169) of BPI. The LBP derivative designated LBP-1
consisted of residues 73 through 99 of LBP. The LBP derivative
designated LBP-2 consisted of residues 140 through 161 of LBP. In
addition, Taylor et al., J. Biol. Chem. 270(30): 17934-17938
(1995), described synthetic peptides corresponding to residues
91-105 or 94-108 of the mature LBP protein that were reported to
compete with LBP for binding to LPS and could inhibit LPS-induced
expression of TNF in vitro.
[0008] In addition to transferring LPS to CD14, LBP can facilitate
the transfer of LPS to serum lipoproteins [Wurfel et al., J. Exp.
Med. 180: 1025-1035 (1994)]. Association with lipoproteins greatly
reduces the inflammatory potential of LPS [Ulevitch and Johnston
(1978), supra]. Thus, LBP itself can also participate in the
neutralization of LPS. The significance of LBP-mediated transfer of
LPS to lipoproteins, however, remains unclear. Specifically,
elevated levels of LBP found in acute phase serum have been
correlated with a reduction of the rate of association of LPS with
lipoproteins [Tobias arid Ulevitch, J. Immunol. 131(4): 1913-1916
(1983); Tobias et al. (1985), supra; U.S. Pat. Nos. 5,245,013 and
5,310,879]. This ability of LBP to inhibit, rather than facilitate,
the transfer of LPS to lipoproteins was exploited in the initial
purification of LBP [Tobias et al. (1986), supra].
[0009] Dedrick et al., J. Endotoxin Research 3(supp. 1): 18
(Abstract I-14) (October 1996) reported in an abstract that
concentrations of 1 ng/mL to 1 .mu.g/mL of rLBP fully potentiated
the induction of TNF expression in serum-free medium by 1 ng/mL LPS
on a human monocytic cell line (THP.1). In medium containing 10%
serum, LBP concentrations of 30 .mu.g/mL or greater inhibited
LPS-induced TNF expression by the THP.1 cells and also inhibited
E-selection expression in human umbilical endothelial cells (HUVEC)
induced by 10 ng/mL LPS. Moreover, it was reported that
administration of 5 mg/kg rLBP also increased survival in mice
challenged with up to 25 mg/kg E. coli LPS. However, human subjects
suffering from disorders involving bacteria and their endotoxin
(such as sepsis) have been shown to exhibit substantially elevated
levels of LBP in circulation (at concentrations of 50 .mu.g/mL to
100 .mu.g/mL of serum), yet these high circulating levels of LBP do
not appear to have inhibited the adverse effects of bacterial
endotoxin in circulation that were experienced by these subjects.
The role of LBP in promoting or alleviating adverse effects of
endotoxin in circulation thus remains unclear.
[0010] Bactericidal/permeability-increasing protein (BPI) is a
basic protein found in the azurophilic granules of
polymorphonuclear leukocytes [Weiss et al., J. Biol. Chem. 253(8):
2664-2672 (1978)]. BPI binds to LPS, resulting in its clearance and
neutralization. The amino acid sequence of BPI is closely related
to that of LBP [Schumann et al. (1990), supra], and like LBP, the
amino-terminal half of BPI has a binding site for LPS [Ooi et al.,
J. Exp. Med. 174: 649-655 (1991)]. However, BPI has a higher
affinity for LPS than does LBP [Gazzano-Santoro et al., Infect.
Immun. 62(4): 1185-1191 (1994);Wilde et al., J. Biol. Chem.
269(26): 17411-17416 (1994)], and cannot transfer LPS to the CD14
molecule. Thus, BPI effectively competes with LBP for LPS binding
[Heumann et al., J. Infect. Dis. 167: 1351-1357 (1993);
Gazzano-Santoro et al. (1994), supra] and blocks the inflammatory
activity of LPS in vitro [Marra et al., J. Immunol. 144(2): 662-666
(1990); Ooi et al. (1991), supra], and in humans [de Winter et al.,
J. Inflamm. 45: 193-206 (1995)].
[0011] It has been suggested that sCD14 could be a useful
therapeutic agent in endotoxin-related disorders [Schutt et al.
(1991), supra; Schutt et al. (1992), supra; Haziot et al. (1994),
supra; Haziot et al. (1995), supra]. The presence of sCD14 reduces
the amount of LPS complexed with LBP [Tobias et al. (1995), supra],
because, although LBP has a higher affinity for LPS than sCD14, the
distribution of LPS between sCD14 and LBP depends on the molar
ratio of the two proteins. sCD14 has been shown to inhibit
responses that depend on mCD14 [Schutt et al., Res. Immunol. 143:
71-78 (1992); Schutt et al.,Allerg. Immunol. 37: 159-164 (1991);
Haziot et al., J. Immunol. 152: 5869-5876 (1994)], and to protect
mice against experimental endotoxemia [Haziot, et al., J. Immunol.
154: 6529-6532 (1995)].
SUMMARY OF THE INVENTION
[0012] The present invention is based on the discovery that
lipopolysaccharide binding protein (IP), an agent previously
thought to be stimulatory of the adverse effects of bacteria and
their endotoxin, can actually reduce the inflammatory potential of
bacteria and their endotoxin when administered to certain subjects
prior to exposure to the bacterial endotoxin. In particular, the
invention relates to the administration of LBP in an amount
effective to inhibit the adverse effects of bacterial endotoxin to
a subject who has a circulating level of LBP in the normal range as
measured by a quantitative LBP assay. The LBP is administered at a
time prior to exposure to bacterial endotoxin (i.e., when the
subject will be at risk of such exposure). Such times of "at risk
of exposure to endotoxin" include circumstances or conditions
associated with increased translocation of gut associated bacteria
and endotoxin, particularly prior to surgery. Thus, these subjects
are administered prophylactic doses of LBP, to raise the
circulating LBP levels to therapeutically effective amounts, in
advance of exposure to bacterial endotoxin. The invention thus
provides a method of protection against the adverse effects of
bacterial endotoxin at a time prior to endotoxin
challenge/exposure.
[0013] Numerous additional aspects and advantages of the invention
will become apparent to those skilled in the art upon consideration
of the following detailed description of the invention which
describes presently preferred embodiments thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A and 1B display the effect of varying concentrations
of LBP on LPS-induced TNF expression by monocytic THP.1 cells in
serum-free and serum-containing medium, respectively.
[0015] FIGS. 2A and 2B display the effect of varying concentrations
of LBP on E-selectin expression by HUVEC induced by LPS and
1L-1.beta., respectively.
[0016] FIGS. 3A, 3B and 3C display the effect of 5 mg/kg LBP on
survival of mice administered lethal doses of 15, 20 and 25 mg/kg
LPS, respectively.
DETAILED DESCRIPTION
[0017] According to the invention, LBP therapy is specifically
useful when prophylactically administered to subjects who have
circulating LBP levels in the normal range but who are at risk for
exposure to bacterial endotoxin. According to the invention, the
LBP levels of the subject may be determined using a sensitive and
specific assay, such as described in Example 2 below, and LBP is
administered to the subject to increase circulating LBP levels to a
prophylactically/therapeutically effective level, for example, from
about 15 to about 100 .mu.g/mL, to prevent/inhibit the adverse
effects of subsequent exposure to bacterial endotoxin.
[0018] Since its identification, LBP has been described as an
immunostimulatory molecule, because it potentiates the biological
effects of LPS/endotoxin. However, in vitro experiments have
demonstrated that the amount of LBP needed to potentiate
LPS-induced expression of inflammatory molecules was far below that
found in normal human serum. In addition, expression of
inflammatory molecules induced by bacterial endotoxin was actually
inhibited in vitro when the concentration of LBP was raised above
normal circulating concentrations. Furthermore, administration of 5
mg/kg recombinant human LBP to mice following a lethal challenge of
endotoxin reduced lethality. Thus, these results suggested that
increased levels of circulating rLBP were protective against
endotoxin. However, circulating LBP levels have been measured and
found to be substantially elevated in human subjects suffering from
such disorders involving exposure to bacteria and their endotoxin,
such as sepsis, meningococcemia and confirmed abdominal infections.
Despite having high circulating levels of LBP (which can rise to
greater than 100 .mu.g/mL), these subjects still experienced the
adverse effects of bacteria and their endotoxin in circulation.
[0019] The present invention is based upon the understanding that
increasing LBP levels by administration of LBP protect a subject
from the adverse effects of endotoxin exposure if the circulating
LBP level is increased before exposure to endotoxin, rather than
after exposure to endotoxin.
[0020] The present invention specifically contemplates a method for
inhibiting adverse effects of endotoxin in circulation, involving
determining the circulating lipopolysaccharide binding protein
(LBP) level of a subject at risk for exposure to endotoxin, and
administering to said subject having a circulating LBP level within
the normal range an amount of LBP effective to elevate the
circulating LBP level to inhibit the adverse effects of exposure to
endotoxin, preferably to a level from about 15 .mu.g/mL to about
100 .mu.g/mL.
[0021] Another aspect of the present invention provides
compositions comprising lipopolysaccharide binding protein (LBP) in
a solution buffered at about pH 7.5 and containing a poloxamer
surfactant, and compositions comprising LBP in a solution buffered
at about pH 7.5 and containing a poloxamer surfactant and a
polysorbate surfactant.
[0022] As used herein, "circulating LBP level within the normal
range," or "normal circulating LBP level," means, for humans, LBP
concentrations in serum or plasma of from about 1 to about 12
.mu.g/mL as measured by the assay described in Example 2 below,
using the rLBP of Example 1 as a standard. The normal range may
vary depending on the assay and the LBP standards utilized, but can
be determined for any assay and LBP standard using a representative
population of normal human sera or plasma.
[0023] As used herein, "at risk for exposure to endotoxin" include
circumstances or conditions, for example, undergoing surgery or
surgical procedures including transplantation, that are associated
with increased translocation of gut-associated bacteria and their
endotoxin.
[0024] It is contemplated that polypeptide derivatives of LBP
(i.e., fragments of LBP, analogs of LBP in which an amino acid has
been deleted, inserted or modified, and fusion proteins comprising
LBP) that retain the ability to bind LPS may be administered
instead of, or in addition to, LBP, and include derivatives that
exhibit CD14-mediated immunostimulatory properties. Such
derivatives may be obtained by any synthetic or recombinant means
known in the art.
[0025] It is contemplated that LBP or derivatives thereof may be
administered according to the invention in a pharmaceutical
composition with pharmaceutically acceptable diluents, adjuvants,
and carriers. A particularly preferred composition of rLBP
comprises 1-2 mg/mL LBP in 10 mM HEPES buffer, 150 mM NaCl, pH 7.5
with 0.2% poloxamer 188 and 0.002% polysorbate 80. A preferred
composition of rLBP.sub.25 is similar to that for rLBP except that
1.0M NaCl instead of 150 mM NaCl is used. According to the
invention, LBP or derivatives thereof may be administered
systemically and most preferably intravenously in amounts broadly
ranging from about 0.1 mg/kg to about 100 mg/kg of body weight of
the treated subject, preferably at dosages ranging from about 1
mg/kg to about 25 mg/kg of body weight. Other systemic routes of
administration include oral, intravenous, intramuscular or
subcutaneous injection (including into a depot for long-term
release), intraocular and retrobulbar, intrathecal, intraperitoneal
(e.g. by intraperitoneal lavage), intrapulmonary (using powdered
drug, or an aerosolized or nebulized drug solution), or
transdermal. The treating physician may find it advantageous to
continue the prophylactic treatment by continuous infusion or
intermittent injection or infusion, at the same, reduced or
increased dose per day.
[0026] It is further contemplated that LBP or derivatives thereof
may be co-administered in conjunction with other agents that bind,
clear and/or neutraize endotoxin. "Concurrent administration," or
"co-administration," as used herein includes administration of
multiple agents, in conjunction or combination, together, or before
or after each other. The LBP or derivative and second agent(s) may
be administered by different routes, e.g., LBP may be given
intravenously while the second agent(s) is(are) administered
intramuscularly. The LBP or derivative and second agent(s) may be
given sequentially in the same intravenous line or may be given in
different intravenous lines. The agents may be administered
simultaneously or sequentially, as long as they are given in a
manner sufficient to allow all agents to achieve effective
concentrations at the site of action.
[0027] The present invention further provides a novel use for LBP
and fragments or derivatives thereof in the manufacture of
medicaments for prophylactically treating subjects at risk of
exposure to endotoxin.
[0028] Example 1 addresses production and purification of rLBP
suitable for use as a therapeutic or for use as a standard in the
LBP assay of Example 2. Example 2 addresses a sensitive and
specific assay for determining LBP levels in human body fluids.
Example 3 addresses in vivo effects of LBP.
EXAMPLE 1
Production and Purification of rLBP
[0029] Recombinant LBP (rLBP), suitable for use as a therapeutic
according to the invention or for use as a standard in the assay of
Example 2 below, was produced and purified as follows. Plasmid
pING4539, containing the DNA encoding full length human LBP (amino
acids 1-452, designated "rLBP," plus the 25 amino acid signal
sequence) [SEQ ID NOS: 1 and 2], was prepared as described in
Example 2 of U.S. application Ser. No. 08/261,660 filed Jun. 17,
1994, hereby incorporated by reference in its entirety.
[0030] CHO-DG44 cells were transfected with linearized pING4539 DNA
(40 .mu.g, digested with PvuI, phenol-chloroform extracted and
ethanol precipitated) using electroporation. Following recovery,
the cells were diluted and 1.times.10.sup.4 cells were plated per
96-well plate well in selective medium consisting of an .alpha.MEM
medium lacking nucleosides (Irvine Scientific) and supplemented
with dialyzed fetal bovine serum (100 mL serum dialyzed against 4L
cold 0.15 NaCl using 6000-8000 cutoff for 16 hours at 4.degree.
C.). Untransfected CHO-DG44 cells were unable to grow in this
medium because they possess the DHFR.sup.- mutation and were
removed during successive feedings with the selective medium. At
1.5-2 weeks, microcolonies consisting of transfected cells were
observed.
[0031] Clones were analyzed for the presence of LBP-reactive
protein in culture by ELISA in Immulon-II 96 well plates
(Dynatech). Supernatant samples were added to the plates and
incubated 46 hours at 4.degree. C., followed by addition of goat
anti-LBP antiserum and peroxidase-labeled rabbit anti-goat
anti-serum. The 21 most productive positive clones were expanded in
selective .alpha.MEM medium and then grown in selective medium
supplemented with 0.05 .mu.M methotrexate. The best producing
amplified clone was chosen based on ELISA of supernatants as
described above and then expanded in .alpha.MEM media containing
0.05 CM methotrexate for growth in roller bottles.
[0032] The transfected CHO-DG44 cells were cultured as follows. All
incubations were performed in a humidified 5% CO.sub.2 incubator
maintained at 37.degree. C. Working stock cultures were grown in
DME/F-12 with 10% FCS, and after four days of growth were seeded
into five 2-liter roller bottles. After another six days of
incubation, these cells were harvested and seeded into 40 2-liter
roller bottles using 1.times.10.sup.7 cells in 500 mL of DME/F-12
with 5% FCS for each bottle. Four days later, the culture
supernatants from each bottle were removed and replaced with 500 mL
of fresh DME/F-12 with 2.5% FCS, and 10 mL of an S-Sepharose
(Pharmacia) ion exchange resin slurry (50% v/v, sterilized by
autoclaving). After four days of incubation, the media containing
S-Sepharose was harvested and replaced with fresh media again
containing S-Sepharose. This process was repeated one more time, to
yield a total of three harvests of S-Sepharose. Each harvest was
processed for purification separately until the LBP was eluted from
the S-Sepharose beads, and then the three eluates were pooled for
the remainder of the purification procedure. Purification methods
using S-Sepharose have been described in U.S. Pat. No. 5,439,807,
hereby incorporated by reference in its entirety.
[0033] All chromatographic resins used in the purification of rLBP
were purchased sterile (in ethanol) and equilibrated with pyrogen
free buffer or were depyrogenated by immersion in 0.2N NaOH, 1 M
NaCl and then rinsed with pyrogen-free water followed by
equilibration with the appropriate buffer. All buffers and reagents
were prepared with bottled, pyrogen-free water for irrigation
(Baxter).
[0034] After the S-Sepharose beads were harvested from the roller
bottles, they were allowed to settle out of the media. The beads
were then batch washed with approximately 800 mL of 20 mM MES, pH
6.8, 150 mM NaCl. After washing with approximately 400 mL of 20 mM
sodium acetate (NaOAc), pH 4.0, 150 mM NaCl, the beads were loaded
into a 2.5.times.50 cm column. The column was washed with 20 mM
NaOAc, pH 4.0, 400 mM NaCl, until the absorbance at 280nm
(A.sub.280) approached zero, which typically requires approximately
600 mL of buffer. The column was then washed with 20 mM NaOAc, pH
4.0, 600 mM NaCl, again until the A.sub.280 reading returned to
zero, which typically requires about 600 mL of buffer. The rLBP was
eluted with 20 mM NaOAc, pH 4.0, 1.0 M NaCl. The column was
additionally washed with 20 mM NaOAc, 1.5 M NaOAc, pH 4.0, 1.5 M
NaCl to insure all the protein was eluted. Column fractions
containing the protein were analyzed by SDS-PAGE, and those
containing rLBP were pooled. The pooled fractions were adjusted to
a final salt concentration of 200 mM NaCl with 20 mM MES, pH 5.0.
This material was then filtered through a 0.2 .mu.m filter.
[0035] The pooled filtrate was applied to a 20 mL Q-Sepharose
column for removal of nucleic acids and then concentrated on a 20
mL S-Sepharose column equilibrated with 20 mM MES, pH 5.0, 200 mM
NaCl. The S-Sepharose column was washed with 20 mM MES, pH 5.0, 400
mM NaCl and again with 20 mM MES, pH 5.0, 550 mM NaCl, using about
200 mL of buffer per wash. The LBP was then eluted with 20 mM MES,
pH 5.0, 1.2 M NaCl in about 40 mL of volume. The LBP was buffer
exchanged into 10 mM HEPES, pH 7.5, 150 mM NaCl using a 500 mL
Sephacryl S-100 column. Fractions were analyzed with SDS-PAGE, and
rLBP-containing fractions were pooled. The final protein
concentration was adjusted to 1.0 mg/mL and the material was
formulated to 0.2% poloxamer 188 (PLURONIC F-68, BASF Wyandotte
Corp., Parsippany, N.J.), 0.002% polysorbate 80 (TWEEN 80, ICI
Americas, Inc., Wilmington, Del.). The formulated protein was
aliquoted for storage and frozen at -70 degrees until use. The
yields from this procedure generally range from about 140 mg to
about 300 mg rLBP, typically about 200-240 mg rLBP.
EXAMPLE 2
Determination of LBP levels in Human Plasma and Sera
[0036] The ranges of circulating LBP levels for healthy human
subjects and for human subjects from a variety of patient
populations was determined by assaying representative samples of
human plasma or sera with a sandwich ELISA.
[0037] The LBP assay was validated by evaluating for interference
by other compounds, recovery, precision and clinical sensitivity
and specificity. The potential for interference by BPI (which
has>45% sequence homology with LBP), LPS, various blood
preservatives commonly used in blood collection and heat treatment
of blood (to 56.degree. C. or 60.degree. C. to inactivate
complement) was investigated. The rLBP prepared as described in
Example 1 was used as a standard; the activity of the formulated
LBP was shown to be constant over time after storage. rBPI and
rBPI.sub.23 (the recombinant expression product of DNA encoding
residues 1 to 199 of BPI) was prepared in the manufacturing
facility of XOMA Corporation, Berkeley, Calif. for clinical trials,
by a process essentially as described on a smaller scale by Horwitz
et al., Protein Expression and Purfication 8:28-40 (1996).
[0038] Affinity-purified rabbit anti-LBP was prepared by procedures
well known in the art. Briefly, two rabbits were hyper-immunized
with LBP, and pooled antisera from these rabbits was diluted with
an equal volume of phosphate buffered saline (PBS), pH 7.2. An
LBP-Sepharose column was prepared by coupling rLBP produced and
purified as described in Example 1 to cyanogen bromide-activated
Sepharose 4B. A portion of the diluted antisera was passed through
the LBP-Sepharose column; the column was then washed and bound
antibodies were eluted with 0.1M glycine, pH 2.5. Collected
fractions were immediately neutralized with 1M sodium phosphate
buffer pH 8.0. Peak fractions were identified by measuring
absorbance at 280 nm. Several sequential column runs were performed
and all peak fractions from each column run were pooled. This pool
of affinity-purified rabbit anti-LBP antibody was assigned a lot
number and qualified based on consistent performance in the
ELISA.
[0039] Biotin-labeled rabbit anti-LBP antibody was prepared by
procedures well known in the art. Briefly, the antibody was biotin
labeled with biotinamidocaproate N-hydroxysuccinimide ester (Sigma)
in 0.1 M sodium bicarbonate, pH 8.3. Unconjugated biotin was
removed and alkaline buffer exchanged by passing the antibody over
a PD-10 column (Pharmacia) equilibrated with PBS containing 0.1%
sodium azide. This biotin-labeled antibody was assigned a lot
number, stored at 2.degree. C. to 8.degree. C., and qualified based
on consistent performance in the ELISA.
[0040] The sandwich ELISA procedure was carried out as follows.
Fifty .mu.L of affinity purified rabbit anti-LBP antibody (2
.mu.g/mL in PBS) was added to each well of Immulon 2 (Dynatech)
microtiter plates and incubated overnight at 2-8.degree. C. The
antibody solution was removed and 200 .mu.l of 1% non-fat milk
(Carnation or equivalent) in PBS was added to all wells. After
blocking the plates for 1 hour at room temperature, the wells were
washed 3 times with 300 .mu.l/well of wash buffer [PBS/0.05% TWEEN
20 (polysorbate 20, Sigma, St. Louis). Standards, samples and
controls were diluted in triplicate with PBS containing 1% bovine
serum albumin, 0.05% TWEEN 20 [PBS-BSA/TWEEN] and 10 units/mL of
sodium heparin (Sigma), in separate 96-well plates. rLBP standard
solutions were prepared as serial two-fold dilutions of
concentrations from 100 to 0.012 ng/mL. Each replicate and dilution
of the standards, samples and controls (50 .mu.l) was transferred
to the blocked microtiter plates and incubated for 1 hour at
37.degree. C. After the primary incubation, the wells were washed 3
times with 300 .mu.l/well of wash buffer. For each assay,
biotin-labeled rabbit anti-LBP antibody was diluted 1/2000 in
PBS-BSA/TWEEN and 50 .mu.l was added to all wells. The plates were
then incubated for 1 hour at 37.degree. C. All wells were washed 3
times with 300/L/well of wash buffer. Alkaline phosphatase-labeled
streptavidin (Zymed) was diluted 1/2000 in PBS-BSA/TWEEN and 50
.mu.L was added to all wells. After incubation for 15 minutes at
37.degree. C., all wells were washed 3 times with 300 .mu.l/well of
wash buffer) and 3 times with 300 .mu.l/well of deionized water and
the substrate p-nitrophenylphosphate (1 mg/mL in 10% diethanolamine
buffer) was added in a volume of 50 .mu.l to all wells. Color
development was allowed to proceed for 1 hour at room temperature,
after which 50 .mu.l of 1 N NaOH was added to stop the reaction.
The absorbance at 405 nm (A.sub.405) was determined for all wells
using a VMAX Plate Reader (Molecular Devices). The mean A.sub.405
for each set of triplicates was calculated. Outliers (data points
deviating more than 20% from the mean) were rejected. A standard
curve was plotted at A.sub.405 versus ng/mL of LBP. The linear
range was selected, a computerized (RS/1 Release 4.4.4, Bolt
Beranek and Newman, Inc.) linear curve fit was performed, and
concentrations were determined for samples and controls by
interpolation from the standard curve. Sample curves are visually
compared to the standard curve to ensure approximately parallel
slopes before data are accepted. Pooled plasma, sera or urine from
normal healthy human donors were used as the appropriate "negative"
controls. An Assay Control Sample (ACS), prepared by adding rLBP to
normal human plasma to a concentration of 50,000 ng/mL, was used as
a "positive" control.
[0041] Western blot analysis was also performed on selected serum
and plasma samples. Briefly, samples were incubated in microtiter
wells coated with rabbits anti-LBP that had been prepared and
blocked as described above for the sandwich ELISA assay. Six
replicate samples were incubated with the rabbit anti-LBP for 1
hour at 37.degree. C. After incubation, LBP was eluted with sample
buffer and pooled. Ten .mu.L of eluate from each sample was run on
a non-reducing 10% gel under the conditions of Laemmli, Nature
227:680-685 (1970). Proteins were transferred to nitrocellulose by
standard techniques [Towbin et al., Proc. Nat'l. Acad. Sci.
76:43504354 (1979)] and probed for immunoreactivity with
biotin-labeled rabbit anti-LBP antibody (diluted 1/2000 in 0.25M
Tris-HCl, pH 7.2, 0.2 M NaCl, 0.3% TWEEN 20) followed by alkaline
phosphatase-conjugated streptavidin (diluted 1/2000 in the same
buffer). Blots were immersed in a 50 .mu.g/mL solution of
5-bromo-4-chloro-3-indolyl phosphate (Sigma) in 0.12M
veronal-acetate buffer, pH 9.8, containing 0.01% (w/v) nitro blue
tetrazolium and 4 mM MgCl.sub.2. Color development was allowed to
proceed for 1 hour at room temperature.
[0042] Parameters for the optimization of the LBP sandwich ELISA
were evaluated, including signal to noise ratio, the concentration
of rabbit anti-LBP and biotin-labeled rabbit anti-LBP antibodies,
and curve fit. The optimal concentrations for the rabbit anti-LBP
and biotin-labeled rabbit anti-LBP antibodies used in these
experiments were 2 .mu.g/mL and a 1:2000 dilution, respectively.
The standard curve demonstrated reproducibility with a consistent
slope and acceptable signal to noise ratio (>10:1). The linear
range for the standard curve was 164-781 pg/mL.
[0043] For assay validation, the LBP sandwich ELISA was
characterized by determining assay precision, recovery and the
least detectable concentration of rLBP. These parameters were
investigated by performing the sandwich ELISA on human plasma
spiked with different concentrations of rLBP and then frozen and
thawed to mimic the processing of clinical samples.
[0044] Assay precision was expressed as the coefficient of
variation for LBP values of the assay control sample (ACS) and
normal human plasma (No) measured more than 40 times. The mean and
standard deviation for ACS and NHP were 39,300.+-.7,930 ng/mL and
2,970 i 349 ng/mL, respectively. The CV for the ACS and NHP were
20% and 12%, respectively. A more recent evaluation of assay
precision produced comparable values for ACS and NHP of
43,220.+-.8543 ng/mL (CV 19.8%) and 4,372.+-.315 ng/mL (CV 7.2%),
respectively. The NHP values are consistent with the endogenous
levels of LBP in normal human sera reported by Leturcq et al., J.
Cell. Biochem., Suppl. 16C:161 (1992) which ranged from 1 to 24
.mu.g/mL, with an average of 7 .mu.g/mL. Acceptance of assay data
is based on the ACS values obtained with each assay.
[0045] Average recovery of LBP spiked into human plasma (defined as
the amount of rLBP measured in spiked samples minus the
concentration in the unspiked control, divided by the actual amount
spiked in the sample) was 68% across a rLBP concentration range of
0 to 168,000 ng/mL. Average recovery of LBP spiked into human urine
was 77% over an rLBP range of 3 to 1000 ng/mL. The lowest
detectable concentration of LBP spiked into human plasma (producing
a discernible signal above background) was 0.164 ng/mL.
[0046] Western blot data clearly demonstrated that the sandwich
ELISA is specific for LBP. When plasma samples from normal and
presumed septic patients were evaluated, all patient samples
exhibited a single 60 kD band similar to the LBP controls. In the
sandwich ELISA, LBP levels for these patients ranged from 3.35 to
113 .mu.g/mL.
[0047] The sandwich ELISA was also performed on blood samples that
had been heat-treated to 56.degree. C. or 60.degree. C. Heat
treatment diminished the amount of LBP detected (compared to sera
maintained at 4.degree. C.) by 34% and 97%, respectively.
[0048] For the interference studies, the sandwich ELISA and Western
blot analysis were performed on donor blood samples collected in
tubes containing acid-citrate-dextrose (ACD),
ethylene-diaminetetraacetic acid (EDTA) or heparin, and on control
samples of donor blood collected without preservatives and control
samples of commercially available normal human serum. Blood
preservatives did not interfere with the detection of LBP using the
sandwich ELISA. Results obtained using the same blood collected
with and without preservatives were comparable. Western blot
analysis revealed a band pattern that was the same as the pattern
for the LBP controls or for commercially available normal human
sera.
[0049] The sandwich ELISA and Western blot analysis were also
performed on samples spiked with varying concentrations of rBPI and
rBPI.sub.23. Because heparin minimizes non-specific adsorption of
BPI to the microtiter plate (see U.S. Pat. Nos. 5,466,580 and
5,466,581, both of which are hereby incorporated by reference in
their entirety), heparin was added to assay diluents in order to
minimize the signal generated by BPI in the sandwich ELISA. The
results of these analyses showed that this sandwich ELISA does not
demonstrate significant cross-reactivity with BPI. The affinity
purified rabbit anti-LBP was not cross-reactive with either rBPI or
rBPI.sub.23 on Western blot analysis. In the sandwich ELISA, the
reactivity of BPI and rBPI.sub.23 at concentrations ranging from
0.78 to 100 ng/mL was comparable to background level, but higher
concentrations of both forms of BPI (greater than 100 ng/mL)
demonstrated some cross-reactivity.
[0050] In addition, the ability of LPS to interfere with detection
of LBP in the sandwich ELISA was evaluated. No interference by LPS
was observed; addition of LPS (at concentrations ranging from 0 to
100 ng/mL) to samples spiked with varying concentrations (7, 16 and
168 .mu.g/mL) of LBP did not diminish the amount of LBP detected in
the ELISA.
[0051] These data confirm that the LBP sandwich ELISA described is
specific for LBP. The standard curve for the assay is consistent
and reproducible, and the assay can detect concentrations of LBP as
low as 164 pg/mL.
[0052] This assay was used to determine LBP levels in normal human
subjects and in a variety of patient populations. See U.S. Pat. No.
5,484,705 and U.S. Ser. No. 08/377,391 filed Jan. 24, 1995, both of
which are hereby incorporated by reference in their entirety.
Plasma or sera samples from healthy human subjects and humans from
a variety of patient populations, including rheumatoid arthritis
(RA), acute graft vs. host disease after bone marrow
transplantation (BM aGvHD), acute lymphocytic leukemia (ALL),
chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma
(CTCL), diabetes, psoriasis, scleroderma, sepsis, abdominal
infection, meningococcemia, Crohn's disease, aplastic anemia (AA),
systemic lupus erythematosus (SLE), acute immunodeficiency syndrome
(AIDS), alcoholic fatty liver, alcoholic hepatitis, alcoholic
cirrhosis, ulcerative colitis and non-alcoholic cirrhosis, were
assayed as described above. Results are shown below in Table 1.
Results for each of the 59 samples from the healthy human
population are displayed in Table 2 below. Of these 59 samples, 58
samples had LIP levels ranging from 1.43 .mu.g/mL to 7.70 .mu.g/mL,
while one sample had an LBP level of 11.20 .mu.g/mL.
1TABLE 1 Mean .mu.g/mL No. of LBP in Plasma Std. Std. POPULATION
Subjects or Sera Dev. Error HEALTHY 59 4.1 1.7 0.2 SEPSIS 390 30.6
17.6 0.9 ABDOMINAL INFECTION 16 52.1 23.9 6.2 MENINGOCOCCEMIA 17
19.7 9.5 2.4 RA 86 7.8 4.8 0.5 BM aGvHD 8 9.1 5.6 2.0 ALL 6 8.7 8.1
3.3 CLL 9 7.7 5.1 1.7 CTCL 12 7.5 4.2 1.2 DIABETES 13 2.8 1.2 0.3
PSORIASIS 13 8.2 4.0 1.1 SCLERODERMA 4 7.2 2.9 1.5 CROHN'S 19 16.1
10.2 2.4 APLASTIC ANEMIA 16 9.5 9.2 2.3 SLE 10 6.7 2.4 0.8 AIDS 15
5.3 2.3 0.6 ALCOHOLIC FATTY LIVER 10 8.5 7.0 2.2 ALCOHOLIC
HEPATITIS 9 8.0 4.8 1.6 ALCOHOLIC CIRRHOSIS 11 10.9 4.6 1.4
ULCERATIVE COLITIS 7 21.0 19.8 7.5 NON-ALCOHOLIC 6 10.7 8.4 3.4
CIRRHOSIS
[0053]
2TABLE 2 LBP Levels in Plasma/Sera Samples of 59 Healthy Human
Subjects .mu.g/ml LBP .mu.g/ml LBP .mu.g/ml LBP .mu.g/ml LBP 2.59
5.09 4.57 3.50 2.75 5.11 4.13 6.40 2.86 5.77 4.11 7.70 2.93 5.95
4.00 3.60 3.19 5.99 4.09 3.60 3.26 7.41 1.52 1.70 3.64 11.20 3.01
3.40 3.67 3.70 2.93 4.00 3.95 2.01 3.62 3.50 3.96 1.99 6.20 2.40
4.37 1.43 2.78 6.60 4.41 2.78 3.60 5.60 4.42 3.62 4.20 5.30 4.91
2.35 4.10 4.40 4.98 2.94 3.20
EXAMPLE 3
In Vivo Effect of LBP
[0054] The following is an exemplary procedure for administration
of LBP according to the invention. A human subject, for example, a
patient about to undergo a surgical procedure, is identified as a
subject at risk for exposure to endotoxin. An LBP assay such as the
assay described above in Example 2 is performed on plasma or serum
from the subject to determine the subject's circulating LBP level.
If the subject's circulating LBP level is normal, then the subject
is treated with an LBP composition such as that described above in
Example 1 at an appropriate time before surgery as determined by
the treating physician, e.g., from 0-24 hours prior to surgery,
preferably prior to induction of anesthesia, and more preferably
from 1-2 hours prior to surgery. The LBP composition is
administered parenterally, e.g., intravenously, and the circulating
prophylactic/therapeutic LBP level after such administration may be
confirmed by a second LBP assay on plasma or serum from the
patient.
[0055] In a separate, related experiment, the effect of LBP on
LPS-induced TNF Expression in a monocytic cell line (THP.1) was
evaluated as follows. THP.1 cells were maintained in RPMI
(GibcoBRL, Gaithersburg, Md.) with 10% fetal bovine serum (FBS) and
were cultured in RPMI with 10% FBS plus 50
ng/mL,1,25-dihydroxy-vitamin D (BIOMOL Research Laboratories Inc,
Plymouth Meeting, Pa.) for three days prior to treatment with LPS
to induce CD14 expression. Before incubation with LPS, cells were
washed three times with RPMI and suspended in either RPMI with 10%
FBS or in serum free medium [RPMI supplemented with 1% HB101
(Irvine Scientific, Santa Ana, Calif.)]. Cells
(5.times.10.sup.4/well) were added to 96 well plates. Aliquots of
rLBP, diluted in the same medium as the cells, were added to final
concentrations of from 0 to 100,000 ng/mL. Expression of TNF was
induced by the addition of E. coli 0128 LPS (Sigma, St. Louis, Mo.)
to a final concentration of 1 ng/mL. Plates were incubated for 3
hours at 37.degree. C. in 5% CO.sub.2, then an aliquot of the
supernatant was removed and assayed for TNF by the WEHI 164
toxicity assay using CellTiter 96.TM. AQ.sub.ueous (Promega Corp.,
Madison, Wis.) to monitor cell viability. Results displayed in FIG.
1A showed that mCD14-dependent induction of TNF expression by 1
ng/mL LPS in lip.1 cells in serum-free medium was potentiated by
low levels of rLBP (<1 ug/mL) but was inhibited by high levels
of rLBP (>10 .mu.g/mL) in vitro. Results displayed in FIG. 1B
showed that the LPS-induced TNF expression in medium with 10% serum
was inhibited by 30 .mu.g/mL amd 100 .mu.g/mL rLBP.
[0056] In another separate, related experiment, the effect of LBP
on E-selectin expression in human umbilical vein endothelial cells
(HUVEC) was also evaluated as follows. HUVEC (Clonetics, San Diego,
Calif.) were maintained in endothelial cell growth medium (EGM,
Clonetics) with 5% FBS. Cells were grown to confluence in 96 well
plates, washed, and medium containing rLBP was added to final
concentrations of from 0 to 50 .mu.g/mL rLBP. The cells were
incubated with 10 ng/mL E. coli O128 LPS or 10 pg/mL
1L-1.beta.(Genzyme) for 4 hours in M199 (GibcoBRL) with 10% FBS.
Cell surface E-selectin expression was measured by ELISA as
described in Huang et al., Inflammation 19:389-404 (1995), and is
displayed in FIGS. 2A and 2B. High levels of rLBP inhibited
sCD14-dependent induction of E-selectin by LPS, but not by
1L-1.beta..
[0057] In yet a further separate, related experiment, the effect of
LBP injected following endotoxin challenge on survival in an mouse
model of lethal endotoxemia was evaluated as follows. CD1 mice
(n=15 per group). were challenged intravenously with 15, 20, 25
mg/kg E. coli 0111 :B4 LPS (Sigma) and then immediately treated
intravenously with 5 mg/kg rLBP or vehicle (rLBP formulation
buffer). Mortality was recorded for 7 days and is displayed in
FIGS. 3A, 3B and 3C. The results show that administration of 5
mg/kg rLBP to these mice was protective following lethal endotoxin
doses of 15 and 20 mg/kg (p<0.05 vs. LPS alone).
[0058] Numerous modifications and variations in the practice of the
invention are expected to occur to those skilled in the art upon
consideration of the foregoing description of the presently
preferred embodiments thereof. Consequently, the only limitations
which should be placed upon the scope of the present invention are
those which appear in the appended claims.
Sequence CWU 1
1
* * * * *