Note: Descriptions are shown in the official language in which they were submitted.
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1
USE OF BACTERICIDAL/PE ARIT.rrv INCREASING PROTEIN OR
BIOLOGICALLY ACTIVE ANA1 OGS THEREOF TO TREAT
LIPOPOLYSACCIiAR_1DE ASSOCLATED GReM NE ATIVE INFEG'TIONS
Background of the Invention
o Gram negative infections are a major cause of morbidity and mortality
especially
in hospitalized and immunocompromised patients. [Duma, RJ., Am. J. of Med.,
78 (Suppl. 6A): 154-164 (1985); and Kreger B.E., D.E. Craven and W.R. McCabe,
Am. J. Med., 68: 344-355 ( 1980)]
~ 5 Although available antibiotics are effective in containing the infection,
they do
nothing to neuualize the pathophysiological effects associated with
lipopolysaccharide (LPS). LPS, or endotoxin, is a major component of the outer
membrane of gram negative bacteria and is released when the organisms are
20 Ivsed. [Ahenep, J.L.. and KA. Morgan, J. Infect. Dis., 150 (3): 380-388 (
1984)]
L.PS released during antibiotic therapy is a potent stimulator of the
inflammatory
response. Many detrimental effects of L.PS in vivo result from soluble
mediators
25 released by inflammatory cells. [Morrison D.C. and RJ. Ulevich, Am. J.
Pathol.,
93 (2): 527-617 (1978)] L.PS induces the release of mediators by host
inflammatory cells which may ultimately result in disseminated intravascular
coagulation (DIC), adult respiratory disuess syndrome CARDS), renal failure,
and
irreversible shock.
35
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Monocytes and neutrophilic granulocytes play a key role in host defense
against
bacterial infections and also participate in the pathology of endotoxemia.
These
cells ingest and kill microorganisms intracellularly and also respond to LPS
in vivo
and in vitro by releasing soluble proteins with microbicidal, proteolytic,
opsonic,
pyrogenic, complement activating and tissue damaging effects. Tumor necrosis
factor (TNF), a cytokine released by LPS stimulated monocytes misics some of
15
the toxic effects of LPS in vivo. Injecting animals with TNF causes fever,
shock
and alterations in glucose metabolism. TNF is also a potent stimulator of
neutrophils.
Soluble LPS causes decreased neutrophil chemotaxis, increased adhesiveness,
elevated hexose monophosphate shunt activity and O= radical. production,
upregulation of surface receptors for complement, and release of granule
proteins
into the surrounding medium. [Morrison and Ulevich ( 1978)]
Both specific and azurophil compartments degranulate in response to LPS.
[Bannatyne, R.M., N.M. Harnett, KY. Lee and W.D. Rigger, J. Infect. Dis., 156
(4): 469-474 ( 1977)] Azurophil proteins released in response to LPS may be
both
2o harmful and beneficial to the host. Neutrophil elastase causes degradation
of
protease inhibitors responsible for suppressing the coagulation cascade. This
results in coagulopathies such as disseminated intravascular coagulation, a
potentially lethal consequence of endotoxemia. Azurophil granules also contain
25 bactericidal molecules such as myeloperoxidase and
Bactericidal/Permeability
Increasing Factor (BPI).
Rabbit BPI was first discovered in 1975. [Weirs, J., R.C. Franson, S.
Becherdite,
K Schmeidler, and P. Elsbach, J. Clin. Invest., 55:33 (1975)] BPI was isolated
from
human neutrophils in 1978. [Weirs, J., P. Elsbach, I. Olson and H. Odeberg, J.
Biol. Chem, 253 (8): 2664-2672 (1978)].
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In 1984 a 57 kD protein with similar properties was isolated from human
neutrophils. [Shafer, W.M., C.E. Martin and J.K Spitznagel, Infect. Immun.,
45:29
( 1984)] This protein is identical to BPI by N-Terminal sequence amino acid
composition, molecular weight and source. Although, the authors were unable to
reproduce the chromatographic isolation procedure used by Elsbach, et al. and
Weiss, et al.
Human BPI is a 57 kD protein which binds to the outer membrane of susceptible
gr~ negative bacteria. [Weiss, et al. ( 1978)] The fact that BPI is a Lipid A
binding protein is evidenced by: (1) rough strains of bacteria are more
sensitive
to both bactericidal and permeability increasing activities of BPI [Weirs, J.,
M.
Hutzler and L Kao, Infect. Immun., 51:594 ( I986)]; (2) mutations in Lipid A
cause decreased binding and increase resistance to bactericidal activity of
both
~ 5 polymyxin B and BPI [Farley, M.M., W.M. Shafer and J.K Spitznagel, Infect.
Immun., 56:1589 ( 1988)]; (3) BPI competes with polymyxin B for binding to ~
~himurium [Farley 1988]; (4) BPI has sequence homology and
immunocrossreactivity to another LPS binding protein Lipopolysaccharide
Binding
Protein (LBP). LBP-LPS complexes have been shown to stimulate the oxidative
burst on neutrophils in response to formylated peptides. High density
lipoprotein
(HDL), another LPS binding protein, found in human serum in complex with LPS
does not show the stimulatory effect on neutrophils. BPI binding disrupts LPS
s~~e, alters microbial permeability to small hydrophobic molecules and causes
cell death (Weirs, et al., 1978). BPI kills bacteria under physiologic
conditions of
pH and ionic strength in vitro indicating that it may be active in vivo
outside the
low pH environment of the phagolysosome. All of the bactericidal and
permeability increasing activities of BPI are present in the N-terminal 25kD
fragment of the protein. [Ooi, C.E., J. Weirs, P. Elsbach, B. Frangione, and
B.
Marrion, J. Biol. Chem., 262: 14891 ( 1987)] Prior to the subject invention.
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however, it has been understood that the beneficial effects of BPI are limited
to
its bactericidal effects.
Despite improvements in antibiotic therapy, morbidity and mortality associated
with endotoxemia remains high. Antibiotics alone are not effective in
neutralizing
the toxic effects of LPS. Therefore, the need arises for an adjunct therapy
with
direct LPS neutralizing activity. Current methods for treatment of endotoxemia
use antibiotics and supportive care. Most available adjunct therapies treat
symptoms of endotoxic shock such as low blood pressure and fever but do not
inactivate endotoxin. Other therapies inhibit inflammatory host responses to
LPS.
As indicated below, present therapies have major limitations due to toxicity,
immunogenicity, or irreproducible efficacy between animal models and human
trials.
~ 5 Polymyxin B is a basic polypeptide antibiotic which has been shown to bind
to,
and structurally disrupt, the most toxic and biologically active component of
endotoxin, Lipid A. Polymyxin B has been shown to inhibit LPS activation of
neutrophil granule release in vitro and is an effective treatment for gram
negative
sepsis in humans. However, because of its systemic toxicity, this drug has
limited
use except as a topical agent.
Combination therapy using antibiotics and high doses of methylprednisolone
sodium succinate (MPSS) has been shown to prevent death in an experimental
model of gram negative sepsis using dogs. Another study using MPSS with
antibiotic in a multicenter, double blind, placebo-controlled, clinical study
in 2'_'3
patients with clinical signs of systemic sepsis concluded that mortality was
not
significantly different between the treatment and placebo groups. Further, the
investigators found that resolution of secondary infection within 14 days was
significantly higher in the placebo group.
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A relatively new approach to treatment of endotoxemia is passive immunization
with endotoxin neutralizing antibodies. Hyperimmune human immunoglobulin
against E.E. Coli JS has been shown to reduce mortality in patients with gram
negative bacteremia and shock by SO%. Other groups have shown promising
5 results in animal models using mouse, chimeric, and human monoclonal
antibodies. Although monoclonal antibodies have advantages over hyperimmune
sera, e.g. more consistent drug potency and decreased transmission of human
pathogens, there are still many problems associated with administering
l~unoglobulin to neutralize LPS. Host responses to the immunoglobulins
themselves can result in hypersensitivity. Tissue damage following complement
activation and deposition of immune complexes is another concern in the use of
therapies involving anti-endotoxin antibodies in septic patients. Also,
immunoglobulins are large molecules, especially the pentameric IgMs currently
in
~ 5 clinical trials, and are rapidly cleared by the reticuloendothelial
system,
diminishing the half-life of the drug.
Endotoxins elicit responses which are beneficial as well as damaging to the
host.
Endotoxemia induces production of LPS binding proteins from the liver and
causes release of microbicidal proteins from leukocytes. In applicants'
studies of
neutrophil proteins involved in host defense, it has been determined that one
of
these proteins, BPI, is not only a potent microbiridal agent in vitro, but it
also
interferes with the ability of LPS to stimulate neutrophils. Specifically, it
has been
demonstrated that BPI binds to soluble LPS and neutralizes its ability to
activate
neutrophils. Accordingly, this invention provides a therapeutic method for the
treatment of LPS toxicity in gram negative-septicemia.
35
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Summary of the Invention
The present invention provides a method of inhibiting lipopolysaccharide
(LPS)-mediated stimulation of cells. This method comprises contacting the
cells,
in the presence of a cell-stimulating amount of lipopolysaccharide, with
Bactericidal/Permeability Increasing Protein (BPI) in an amount effective to
inhibit cell stimulation.
0 The invention further provides a method of treating a gram negative
bacterial
infection. This method comprises contacting the bacterial infection with
purified
20
BPI or a biologically active polypeptide analog thereof in an amount effective
to
inhibit LPS-mediated stimulation of cells and thereby treat the bacterial
infection.
Additionally, the present invention provides for a composition for treatment
of a
gram negative bacterial infection. This composition comprises purified BPI or
a
biologically active polypeptide analog thereof in an amount effective to
inhibit
LPS-mediated stimulation of cells and a suitable carrier.
The present invention additionally provides a method of treating a subject
suffering from endotoxin-related shock caused by a gram negative bacterial
infection which comprises administering to the subject an amount of BPI
effective
to combat the gram negative infection and treat the subject so as to alleviate
the
endotoxin-related shock.
Further, the invention provides a method of treating a subject suffering from
disorder involving disseminated intravascular coagulation. The method
comprises
administering to the subject an amount of BPI effective to alleviate the
symptoms
of disseminated intravascular coagulation and thereby treat the subject.
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Fun per, the present invention provides a method of treating a subject
suffering
from endotoxemia caused by a gram negative infection which comprises
administering to the subject an amount of BPI effective to combat the gram
negative bacterial infection and treat the subject suffering from endotoxemia.
As used herein endotoxemia means a condition in which the blood contains
poisonous products, either those produced by the body cells or those resulting
from microorganisms, i.e. gram negative bacteria.
The invention also provides a method of treating a subject suffering from
endotoxin-related anemia caused by a gram negative bacterial infection which
comprises administering to the subject an amount of BPI effective to combat
the
gram negative bacterial infection and treat the subject so as to alleviate
endotoxin-
related anemia.
The present invention further provides a method of treating a subject
suffering
from endotoxin-related leukopenia caused by a gram negative bacterial
infection.
The method comprises administering to the subject an amount of BPI effective
to
combat the gram negative bacterial infection and treat the subject so as to
alleviate endotoxin-related leukopenia. Further, the invention includes a
method
of treating a subject suffering from endotoxin-related thrombocytopenia caused
by
a gram negative bacterial infection which comprises administering to the
subject
~~ ~°~t of BPI effective to combat the gram negative bacterial
infection and
treat the subject so as to alleviate endotoxin-related thrombocytopenia.
The invention also provides a method of inhibiting a pyrogen which comprises
contacting the pyrogen with an amount of BPI so as to inhibit the pyrogen.
35
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Furthermore, the invention also includes a method of inhibiting
lipopolysaccharide-mediated tumor necrosis factor production by cells which
comprises contacting the cells in the presence of a cell-stimulating amount of
lipopolysaccharide, with BPI in an amount effective to inhibit
lipopolysaccharide-
mediated tumor necrosis factor production by cells.
The invention also includes a method of inhibiting gram negative bacteria-
mediated tumor necrosis factor production by cells which comprises contacting
the
gram negative bacteria with BPI in an amount effective to inhibit gram
negative-
mediated tumor necrosis factor production by cells.
Moreover, the invention includes a composition for the treatment of a subject
suffering from endotoxin-related shock. The composition comprises a purified
BPI
or a biologically active polypeptide analog thereof in an amount effective to
treat
a subject suffering from endotoxin-related shock and a suitable carrier.
Further, the invention includes a composition for the treatment of a subject
suffering from disseminated intravascular coagulation. The composition
comprises
2~ a purified BPI or a biologically active polypeptide analog thereof in an
amount
effective to treat a subject suffering from disseminated intravascular
coagulation
and a suitable carrier.
~~ mention also includes a composition for the treatment of a subject
suffering
from endotoxemia comprising a purified BPI or a biologically active
polypeptide
analog thereof in an amount effective to treat a subject suffering from
endotoxemia and a suitable carrier.
.
The invention additionally provides a composition for the treatment of a
subject
suffering from endotoxin-related anemia comprising purified BPI or a
biologicaliv
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activ~ polypeptide analog thereof in an amount effective to treat a subject
suffering from endotoxin-related anemia and a suitable carrier.
Additionally, the invention provides a composition for the treatment of a
subject
suffering from endotoxin-related leukopenia. The composition comprises
purified
BPI or a biologically active polypeptide analog thereof in an amount effective
to
treat a subject suffering from endotoxin-related leukopenia and a suitable
carrier.
Further provided is a composition for the treatment of a subject suffering
from
endotoxin-related thrombocytopenia. The composition comprises purified BPI or
a biologically active polypeptide analog thereof in an amount effective to
treat a
subject suffering from endotoxin-related thrombocytopenia and a suitable
carrier.
As used herein endotoxin-related leukopenia is a condition associated with a
gram
negative bacterial infection, the manifestation of which is a decrease below
the
normal number of leukocytes in the peripheral blood. Moreover, as used herein
endotoxin-related thromborytopenia is a condition associated with a gram
negative
bacterial infection, the manifestation of which is a decrease below the normal
number of thrombocytes.
Also, the invention provides a composition for inhibiting lipopolysaccharide-
mediated tumor necrosis factor production by cells comprising purified BPI or
a
biologically active polypeptide analog thereof in an amount effective to
inhibit
lipopolysaccharide-mediated tumor necrosis factor production by cells and a
suitable carrier. Moreover, the invention provides a composition for
inhibiting
gram negative bacteria-mediated tumor necrosis factor production by cells
comprising purified BPI or a biologically active polypeptide analog thereof in
an
amount effective to inhibit gram negative bacteria-mediated tumor necrosis
factor
production by cells and a suitable carrier.
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The invention provides a composition for inhibiting a pyrogen. Il~e
composition
comprises purified BPI or a biologically active polypeptide analog thereof in
an
amount effective to inhibit a pyrogen.
Additionally, the invention provides a method of preventing a symptom
associated
5 with a gram negative bacterial infection in a subject which comprises
administering
to the subject an amount of Bactericidal/Permeability Increasing Protein
effective
to prevent the gram negative bacterial infection and thereby prevent the
symptom.
10 Moreover, also provided is a method of preventing a disorder involving
disseminated intravascular coagulation in a subject which comprises
administering
to the subject an amount of Bactericidal/Permeability Increasing Protein
effective
to prevent the symptoms of disseminated intravascular coagulation and thereby
preventing the disorder.
Finally, the invention provides a method of isolating and recovering purified
Bartericidal/Permeability Increasing Protein which comprises: (a) obtaining a
crude sample of Bactericidal/Permeability Increasing Protein; and (b)
separating
2~ the crude sample by column chromatography using de-pyrogenated solutions
thereby isolating and recovering purified Bactericidal/Permeability Increasing
Protein.
30
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11
Figure la:Mean fluorescence intensity of CR1 on freshly isolated neutrophils
was
measured by FACS analysis. Cells were stimulated with varying doses of E. Coli
0111:B4 LPS as described in Materials and Methods. Since mean fluorescence
intensity varies between individuals, the data is expressed as percent of the
maximum response observed. Data shown represents the mean + /- Standard
Error of three experiments.
Figure lb:O111:B4 LPS (10 ng/ml) was preincubated with varying doses of crude
azurophil extract for 30 minutes at 37 ~ C prior to testing for neutrophil
stimulation. Data shown represents the mean + /- Standard Error of duplicates
from a representative experiment. Values are expressed as % inhibition of the
response to LPS alone.
Figure 2:Crude azurophil extract was separated by reverse phase HPLC. Each
peak was collected manually and protein concentrations were determined by
amino acid analysis. An aliquot ( l,ug) of each peak was dried in the presence
of
low endotoxin BSA, then redried in the presence of pyrogen free 0.1% acetic
acid.
Data shown represent the mean + /- Standard Error of duplicates from a
representative experiment.
Figure 3a:BPI purified by size exclusion followed by ration exchange HPLC was
subjected to reverse phase HPLC and fractions were tested for LPS inhibitory
activity.
;Figure 3b:Data show the RPLC profile of the 2X purified material along with
the
inhibitory activity and SDS PAGE analysis of fractions 20,21 and 22.
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Flguri 4:0111:B4 LPS ( 10 ng/ml) was preincubated with varying doses of (A)
purified BPI, and (B) polymyxin B, then tested for neuuophil stimulatory
activity.
Results from two experiments shows inhibition of complement receptor
expression
on neutrophils with Standard Errors for replicate samples.
Figure 5: (a) A bar graph illustrating BPI expression on the surface of
neutrophils stimulated with FMLP, TNF, and L,PS. (b) A bar graph illustrating
maximal CR3 upregulation of human neutrophil cell surface expression.
Figure 6: A bar graph illustrating that BPI and polymyxin B inhibited more
than
70% at time=0 of the neutrophil response to LPS.
Figure 7: A graph illustrating that BPI inhibits LPS activity on LAL assay.
Figure 8: A chromatogram showing a fractionated azurophile granule extract by
cation exchange HPLC (step 1); the dotted line traces LPS inhibitory activity
and
the solid Iine traces protein absorbance.
Figure 9: A chromatogram showing a fractionated azurophile granule extract by
cation exchange hiPLC (step); the dotted line traces LPS inhibitory activity
and
the solid line traces protein absorbance.
Figure 10: A chromatogram showing a fractionated azurophile granule extract by
size exclusion HPLC (step 3); the dotted line traces LPS inhibitory activity
and the
solid line traces protein absorbance.
Figure 11: An SDS-PAGE gel of the azurophil granule extract, the precipitated
extract, and fraction pools from the three chromatographic steps.
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Figure 12: Analysis of purified BPI by microbore reverse phase I-iPLC
identifying
a single major peak which accounts for 97% of the total protein.
Figure 13: A line graph illustrating inhibition of the neutrophil response to
10
ng/ml LPS by BPI.
Figure 14: A line graph showing BPI directly binds to LPS.
Figure 15: A line graph showing BPI binding to immobilized LPS was inhibited
bY PolYmYxin B.
20
Figure 16: A line graph showing that BPI binds to LPS in the presence of
plasma.
Figure 17: A line graph showing BPI binds to LPS in the
presence of serum.
Figure 18: A bar graph showing that BPI modulates pyrogenic response to LPS.
35
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Detailed Descriytion of the Invention
The present invention provides a method of inhibiting lipopolysaccharide
(IrPS)-mediated stimulation of cells. This method comprises contacting the
cells,
in the presence of a cell-stimulating amount of lipopolysaccharide, with BPI
in an
amount effective to inhibit cell stimulation
The amount of BPI effective to inhibit cell stimulation will vary according to
the
conditions present. T'he amount effective to inhibit cell stimulation is
preferably
from about 100 ng to about 100 mg, with the most preferred amount being from
about 10 ~g to about 10 mg.
Neutrophils and monocytes are the cells of greatest importance with regard to
the
~ 5 application of the subject invention. However, other cells such as
endothelial cells
are also affected by LPS and may be used in this invention.
In the preferred embodiment purified BPI is used. BPI also comprises
20 recombinant BPI and biologically active polypeptide analogs thereof. One
suitable
analog 'of BPI comprises a polypeptide which has a molecular weight of about
25kD and corresponds to the h'-terminal amino acid sequence of BPI.
25 ~ used herein a biologically active polypeptide analog of
Bactericidal/Permeability Increasing Protein means a polypeptide which has
substantially the same amino acid sequence as, and the biological activity of,
native or naturally-occurring Bactericidal/Permeability Increasing Protein.
30 ~e ~vention further provides a method of treating a gram negative bacterial
infection. This method comprises contacting the bacterial infection with
purified
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BPI nr a biologically active polypeptide analog thereof in an amount effective
to
inhibit LPS-mediated stimulation of cells and thereby treat the bacterial
infection.
It would be clear to one skilled in the art that gram negative bacterial
infection
includes gram negative sepsis, the most common nosocomial infection which
5 causes death. Generally, gram negative sepsis is a severe toxic, febrile
state
resulting from infection with pyogenic microorganisms, with or without
septicemia.
The gram negative bacterial infection may be associated with endotoxic shock
or
10 an inflammatory condition. The inflammatory condition may, for example, be
associated with disseminated intravascular coagulation (DIC), adult
respiratory
distress syndrome CARDS), or renal failure.
In the preferred embodiment the gram negative bacterial infection is present
in
a subject, most preferably, a human being.
Additionally, the present invention provides for a composition for treatment
of a
gram negative bacterial infection. This composition comprises purified BPI or
a
biologically active polypeptide analog thereof in an amount effective to
inhibit
LPS-mediated stimulation of cells and a suitable carrier.
In the preferred embodiment the BPI or a biologically active polypeptide
analog
thereof is administered in a pharmaceutically acceptable carrier.
Pharmaceutically
acceptable carrier encompasses any of the standard pharmaceutical carriers
such
as sterile solution, tablets, coated tablets and capsules. Typically such
carriers
contain excipients such as starch, milk, sugar, certain types of clay,
gelatin, stensic
acid, talc, vegetable fats or oils, gums, glycols, or other known excipients.
Such
carriers may also include flavor and color additives or other ingredients.
Compositions comprising such carriers are formulated by well known
conventional
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16
methods. However, the composition comprising BPI or a biologically active
polypeptide analog thereof in an amount effective to suppress LPS mediated
stimulation of neutrophils or monocytes is previously unknown.
In this method, the administration of the composition may be effected by any
of
the well known methods, including but not limited to, oral, intravenous,
intramuscular, and subcutaneous administration.
In the practice of the method of this invention the amount of BPI or a
biologically
0 active polypeptide analog thereof incorporated in the composition may vary
widely. Methods for determining the precise amount are well known to those
skilled in the art and depend inter alia upon the subject being treated, the
specific
pharmaceutical carrier and route of administration being employed, and the
frequenry with which the composition is to be administered.
The present invention additionally provides a method of treating a subject
suffering from endotoxin-related shock caused by a gram negative bacterial
infection which comprises administering to the subject an amount of BPI
effective
to combat the gram negative infection and treat the subject so as to alleviate
the
endotoxin-related shock. Endotoxins, as used herein, are substances containing
Iipopolysaccharide complexes found in the cell walls of microorganisms,
principally
gram-negative bacteria.
Further, the invention provides a method of treating a subject suffering from
disorder involving disseminated intravascular coagulation. The method
comprises
administering to the subject an amount of BPI effective to alleviate the
symptoms
of disseminated intravascular coagulation and thereby treat the subject.
As used herein, the term disseminated intravascular coagulation is a complex
disorder of the clotting mechanisms, in which coagulation factors are consumed
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17
at an accelerated rate, with generalized fibrin deposition and thrombosis,
hemorrhages, and further depletion of the coagulation factors. Moreover,
disseminated intravascular coagulation may be acute or chronic.
Further, the present invention provides a method of treating a subject
suffering
from endotoxemia caused by a gram negative infection which comprises
administering to the subject an amount of BPI effective to combat the gram
negative bacterial infection and neat the subject suffering from endotoxemia.
0 As used herein endotoxemia means a condition in which the blood contains
poisonous products, either those produced by the body cells or those resulting
from microorganisms, i.e. gram negative bacteria.
The invention also provides a method of treating a subject suffering from
endotoxin-related anemia caused by a gram negative bacterial infection which
comprises administering to the subject an amount of BPI effective to combat
the
gram negative bacterial infection and treat the subject so as to alleviate
endotoxin-
related anemia.
The present invention further provides a method of treating a subject
suffering
from endotoxin-related leukopenia caused by a gram negative bacterial
infection.
The method comprises administering to the subject an amount of BPI effective
to
26 combat the gram negative bacterial infection and treat the subject so as to
alleviate endotoxin-related leukopenia. Further, the invention includes a
method
of treating a subject suffering from endotoxin-related thrombocytopenia caused
by
a gram negative bacterial infection which comprises administering to the
subject
an amount of BPI effective to combat the gram negative bacterial infection and
xreat the subject so as to alleviate endotoxin-related thrombocytopenia.
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The invention also provides a method of inhibiting a pyrogen which comprises
contacting the pyrogen with an amount of BPI so as to inhibit the pyrogen.
As used herein, a pyrogen is any fever-producing substance; exogenous pyrogens
include bacterial endotoxins, especially of gram-negative bacteria; endogenous
pyrogen is a thermolabile protein derived from such cells as mononuclear
leukocytes which acts on the brain centers to produce fever.
Furthermore, the invention also includes a method of inhibiting
lipopolysaccharide-mediated tumor necrosis factor production by cells which
p comprises contacting the cells in the presence of a cell-stimulating amount
of
lipopolysaccharide, with BPI in an amount effective to inhibit
lipopolysaccharide-
mediated tumor necrosis factor production by cells.
~ 5 The invention also includes a method of inhibiting gram negative bacteria-
mediated tumor necrosis factor production by cells which comprises contacting
the
gram negative bacteria with BPI in an amount effective to inhibit gram
negative-
mediated tumor necrosis factor production by cells.
20 The amount of BPI effective to inhibit cell stimulation will vary according
to the
conditions present. The amount effective to inhibit cell stimulation is
preferably
from about 100 ng to about 100 mg, with the most preferred amount being from
about l0ug to about 10 mg.
In the above-described methods, the BPI comprises recombinant BPI or a
biologically active polypeptide analog thereof. Moreover, the biologically
active
polypeptide analog of BPI comprises a polypeptide which has a molecular weight
,of about 25 kD and corresponds to the N-terminal amino acid sequence of BPI.
Moreover, the invention includes a composition for the treatment of a subject
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19
suffe:-ing from endotoxin-related shock. The composition comprises a purified
BPI
or a biologically active polypeptide analog thereof in an amount effective to
treat
a subject suffering from endotoxin-related shock and a suitable carrier.
Further, the invention includes a composition for the treatment of a subject
suffering from disseminated intravascular coagulation. The composition
comprises
a purified BPI or a biologically active polypeptide analog thereof in an
amount
effective to treat a subject suffering from disseminated intravascular
coagulation
and a suitable carrier.
15
The invention also includes a composition for the treatment of a subject
suffering
from endotoxemia comprising a purified BPI or a biologically active
polypeptide
analog thereof in an amount effective to treat a subject suffering from
endotoxemia and a suitable carrier.
The invention additionally provides a composition for the t~eaunent of a
subject
suffering from endotoxin-related anemia comprising purified BPI or a
biologically
active polypeptide analog thereof in an amount effective to treat a subject
suffering from endotoxin-related anemia and a suitable carrier.
Additionally, the invention provides a composition for the treatment of a
subject
suffering from endotoxin-related leukopenia. The composition comprises
purified
BPI or a biologically active polypeptide analog thereof in an amount effective
to
treat a subject suffering from endotoxin-related leukopenia and a suitable
carrier.
Further provided is a composition for the treatment of a subject suffering
from
endotoxin-related thrombocytopenia. The composition comprises purified BPI or
a biologically acti~re polypeptide analog thereof in an amount effective to
treat a
subject suffering from endotoxin-related thrombocytopenia and a suitable
carrier.
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Also, the invention provides a composition for inhibiting lipopolysaccharide-
mediated tumor necrosis factor production by cells comprising purified BPI or
a
biologically active polypeptide analog thereof in an amount effective to
inhibit
lipopolysaccharide-mediated tumor necrosis factor production by cells and a
suitable carrier. Moreover, the invention provides a composition for
inhibiting
5 gram negative bacteria-mediated tumor necrosis factor production by cells
comprising purified BPI or a biologically active polypeptide analog thereof in
an
amount effective to inhibit gram negative bacteria-mediated tumor necrosis
factor
production by cells and a suitable carrier.
15
The invention provides a composition for inhibiting a pyrogen. The composition
comprises purified BPI or a biologically active polypeptide analog thereof in
an
amount effective to inhibit a pyrogen.
Additionally, the invention provides a method of preventing a condition
associated
with a gram negative bacterial infection in a subject which comprises
administering
to the subject an amount of BactericidaZ/Permeability Increasing Protein
effective
to prevent the gram negative bacterial infection and thereby prevent the
condition.
In the previously described method the condition is any of the conditions
selected
from the group consisting of endotoxin-related shock, endotoxemia, endotoxin-
related anemia, endotoxin-related leukopenia, or endotoxin-related
thrombocytopenia
Moreover, also provided is a method of preventing a disorder involving
disseminated intravascular coagulation in a subject which comprises
administering
to the subject an amount of Bactericidal/Permeability Increasing Protein
effective
to prevent the symptoms of disseminated intravascular coagulation and thereby
preventing the disorder.
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21
Finally, the invention provides a method of isolating and recovering purified
Bactericidal/Permeability Increasing Protein which comprises: (a) obtaining a
crude sample of Bactericidal/Permeability Increasing Protein; and (b)
separating
the crude sample by column chromatography using de-pyrogenated solutions
thereby isolating and recovering purified Bactericidal/Permeability Increasing
Protein. Moreover, in this method the Bactericidal/Permeability Increasing
Protein comprises native Bactericidal/Permeability Increasing Protein or a
biologically active polypeptide analog thereof. Further, the biologically
active
polypeptide analog of Bactericidal/Permeability Increasing Protein comprises a
polypeptide which has a molecular weight of about 25 kD and corresponds to the
N-terminal amino acid sequence of Bartericidal/Permeability Increasing
Protein.
We have found that biological activity level of BPI varies depending on the
method used for obtaining BPI. It appears that depyrogenated BPI, i.e. BPI
isolated and recovered by the above-described method using de-pyrogenated
~ 5 solutions, shows a much higher level of biological activity than pyrogen-
containing
BPI (Table 5 and Figure 4A).
This invention is illustrated in the Experimental Details and Results sections
2o which follow. These sections are set forth to aid in an understanding of
the
invention but are not intended to, and should not be construed to, limit in
any way
the invention as set forth in the claims which follow.
30
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22
Examyie 1
Materials and Methods:
t 0 Lipopolysaccharide from E.E. Coli 0111:B4, S. Syphimurium wild type,
glycolipid
from S~tvnhimurium RE mutant, and Lipid A from ~y~7himurium RE mutant,
and LPS from P. aeru '~tnosa were purchased from RIBI Immmunochem Research,
Inc., Hamilton, MT; Fmet-Leu-Phe (FMLP) and polymyxin B Sulfate from Sigma
Chemical Co., St. Louis, MO; Hank's Balanced Salt Solution without calcium,
magnesium and phenol red (HBSS) from Hazelton Research Products, Denver,
PA; Ficoll-Paque* Percoll~and MacrodeX from Pharmacia Inc., Piscataway, NJ;
TNF and anti-TNF from Endogen, Boston MA; Fluorescein conjugated
goat-anti-mouse IgG from TAGO Inc., Burlingame, CA: IgGl control antibody
from Coulter Immunology, Hialeah, FL; Phycoerythrin (PE) conjugated anti CR3
(Leu-15) and IgG2a control from Becton Dickinson, Mountain View, CA, Anti
CR1 monoclonal antibody, Yz-1, was a kind gift from Dr. Rick Jack at Harvard
University
Granulocytes were isolated from buffy coats obtained from local blood banks.
Buffy coats were. diluted 3-4X in HBSS and granulocytes were separated from
'mononuclear cells by centrifugation through 64% Percoll. The pellet was
subjected to diisopropylfluorophosphate (DFP), washed, and resuspended in ice
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cold lysis buffer ( 10 mM PIPES, pH 6.8, 100 mM KCL, 3mM NaCI, 3.5 mM
MgCl2) aad disrupted by nitrogen cavitation (Purr Instrument Co., Moline, IL).
Azurophil granules were isolated on discontinuous Percoll gradients as
described
by Borregaard. [Borregaard, N., J.M. Heiple, E.R. Simons, and R.A. Clark, J.
Cell.
Biol., 97: 52-61 (1983)] The azurophil granules were collected and Percoll was
removed by centrifugation at 180,000 X G for 2 hours. The granules were lysed
by 4 cycles of freeze-thaw followed by 1 minute of sonication. The lysed
granules
were extracted in an equal volume of 100 mM glyrine, pH 2 by vortexing
intermittently for 1 hour at room temperature. The acid extract was clarified
by
centrifugation at 30,000 X G for 20 minutes and at 200,000 X G for 30 minutes.
Venous blood was drawn from healthy volunteer donors into acid citrate
dextrose
~ 5 anticoagulant and immediately placed on ice. Five parts of blood were
mixed with
1 part of cold Macrodex;' and allowed to settle for 1.5 - 2 hours at 4 ~ C.
Leukocyte-rich plasma was washed 1X in cold HBSS, then resuspended in HBSS
and layered over Ficoll-Paque. ~ If significant erythrocyte contamination was
2o present, the granulocyte pellet was subjected to hypotonic lysis. The cells
were
washed 2X in HBSS and resuspended in HBSS + 2% autologous plasma to give
a final granulocyte concentration of 1 X 1f6/ml in the incubation mixture.
25 ~ BPI Punfication
Approximately 2 mg of crude azurophil granule extract was separated by size on
a Biosil ('1"SK-250) (7.8 mm x 600 mm) high performance size exclusion column
using 50 mM glycine and 100 mM NaCl buffer, pH 2.0, under isocratic conditions
of a flow rate of 1 ml/min. Column fractions with the greatest LPS inhibitory
activity contained a large proportion of the 54 KD species as shown by SDS
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24
PAGE. These TSK fractions were pooled and run over an Aquapore weak cation
exchange (WCX) column (2.1 mm X 30 mm) using 50 mM citrate, pH 5.5, and
eluted in a gradient of 0-75%, of 50 mM citrate and 1 M NaCI (Buffer B) in 25
min, then 75-100% Buffer B in 5 min with a flow rate of 200 ml/min. Material
of 57 KD was recovered from canon exchange and appeared as a single band on
SDS page. In some experiments BPI was further purified by reverse phase HPLC
on a Vydac C4 column loaded for 12 min in 0.1% CH3CN plus 0.1% TFA, in 30
min with a flow rate of 200 ml/min (Raisin Instruments, Emeryville, CA).
0 Neutrophil stimulation
Isolated neutrophils were kept on ice until incubated with and without stimuli
at
37' C for 30 minutes. Following the incubation, cells were washed in a large
volume of cold PBS + 0.05% Na Azide + 2% autologous plasma. Pellets were
t5 divided in two, one stained with SO~uI control IgGl antibody (20~ug/1x106
cells),
the other with 50 ul of 20 pug/ 1x106 cells anti-CR 1 for 30 minutes at 0' C.
Following this incubation the cells were washed 2X with PBS + autologous
plasma, then stained with goat-anti-mouse IgG-FTTC, and in some experiments,
20 20u1 of IgGZa-phycoerythrin (PE) in control wells, and 20A1 Leu-15 PE in
test
wells. Following a 30 minute incubation at 0' C and 2 more washes, the cells
were
analyzed by flow cytometry on a Becton Dickinson FACStar flow cytometer
(Becton Dickinson, Mountain View, CA). Neutrophil stimulation was measured
25 by comparing mean fluorescence intensity of samples which had been
incubated
in HBSS + 2% autologous plasma alone (control) to those incubated with LPS
or LPS which had been preincubated for 30 minutes at 37' C with BPI or
polymyxin B. Data are expressed as % stimulation or % inhibition and were
calculated using the mean fluorescence intensity (Fl), on a log scale,
according to:
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% Stimulation = [(Experimental - Control)/ (Maximum - control)] X
100
and
% Inhibition= 1-[(+Inhibitor)-(Control)]/
[(-Inhibitor)-(Control)] X 100.
5
Vapor phase hydrolysis of BPI and amino acid derivitization was performed
using
a Pico-tag Workstation (Waters, Milford MA) and chromatographic analysis of
the
phenylthiocarbamyl amino acids was performed on an applied Biosystems 130 A
MPL,C using Protocols provided by the manufacturer.
BPI N-terminal sequence was analyzed by automated Edman degradation using
an applied Biosystems 477A pulse liquid phase sequenator (Applied Biosystems,
Foster city, CA). Phenyltheohydantion amino acid analysis was performed on
line
using an applied biosystems Model 120A liquid chromatograph.
Human neutrophils may be stimulated both in vivo and in vitro by
lipopolysaccha,ride. Upon activation, surface expression of receptors for C3b
and
C3bi (CR1 and CR3 respectively), increases. Using the Fluorescence Activated
Cell Sorter (FACS), fluorescence intensity of freshly isolated human
neutrophils
was measured following stimulation with increasing doses of 0111:84 LPS
(Figure
la). Because commonly observed maximum stimulation was at or above 10
ng/ml, experiments testing for inhibition of 0111:84 LPS used 10 ng/ml as the
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stimulatory dose. All experiments were performed in duplicate. In most
experiments, data is shown only for CR1 since we did not observe any condition
where neutrophil stimulation caused upregulation of CR1 or CR3 alone (M.
Marra et al. ( 1990) J. Immunol. L(2):662-666).
To determine whether proteins found in neuuophil azurophil granules could
interfere with the neutrophil response to LPS, crude acid extracts of
azurophil
granules were pre-incubated with LPS for 30 minutes at 37' C. The mixture was
then tested for its ability to stimulate neuuophils. Azurophil protein
(l,ug/ml)
could effectively block stimulation of 1 X 106 polymorphonuclear leukocytes
(PMN)/ml by 10 ng/ml of LPS (Figure lb). This effect was not observed using
glycine extraction buffer preincubated with LPS, nor was there any stimulation
of
neutrophils using crude extract or glycine buffer control (data not shown).
To further investigate which of the proteins in the extract was/were
responsible
for inhibitory effect, crude acid extracts were separated by reverse phase
HPLC;
each peak was assayed separately for LPS inhibitory activity. The identity of
each
of the peaks was previously determined using a two-dimensional purification
2~ approach involving microbore reverse phase HPLC in first dimension followed
by
30
SDS PAGE, electroblotting and microsequencing. The azurophil proteins can be
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TABLE 1
ZUROPHIL GRA NULE
DERIVED
A -
Peak Identity PROTEINS
1 5 10 15
1 Defensins CYCRIPACIAGERRY
(HNP-2)
2 Granulocidin VCSCRLVF~CRRTGLR
(HNP-4)
3 Eosinophil
Cationic Protein
(ECP) XPPQFTRAQWFAIQH
4a Eosinophil-
Derived Neurotoxin
~5 (EDN) KPPQFTXAQXFETQX
4b CathepsinG IIGGRESRPHSRPYM
Sa Lysozyme KVFERXELARTLKRL
2~ Sb Eosinophil
Major Protein
(MBP) TCRYLLVRSLQTFSQ
6 Unlmown IVGGRKARPXQFPFL
25
7 Unknown IVGGHEAQPHSRPYM
8a MyeloperoxidaseV N C E T S C V Q Q P P
C F P
8b Elastase IVGGRRARPHAXPFM
9 Bactericidal/Permeability
Increasing
Protein (BPI) VNPGVVVRISQKGLD
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28
resolved into 10 discrete peaks whose identities are shown in Table 1. The
amino
acid sequences shown are for the first 15 amino acids of the N-terminal.
LPS inhibitory activity of l,ug of each peak is shown in Figure 2. As shown,
peak
9 had the highest LPS neutralizing activity. The major protein species in this
peak
has N-terminal identity with Bactericidal/Permeability Increasing Protein
(BPI)
described previously (Weiss, J., P. Elsbach, I. Olsson and H. Odeberg, J.
Biol.
Chem, 253 (8): 2664-2672 (1978)). BPI has been shown to contain the majority
of the gram negative bactericidal activity in azurophil granule protein
extracts.
0 Cathepsin G showed some inhibition of LPS, but the data between experiments
were not as reproducible as for peak 9. Cathepsin G has been shown to bind to
LPS in vitro and to kill gram negative organisms, although to a lesser extent
than
BPI. Other proteins which have demonstrated microbicidal activity against gram
negative organisms are elastase and the defensins. However, these proteins ( 1
iug/ml) did not inhibit the stimulatory activity of LPS on neutrophils.
LPS inhibitory activity of crude azurophil extracts was further characterized
and
purified using size exclusion and ion exchange followed by reverse phase
chromatography. LPS inhibitory activity comigrates with a pure 57 ICD band
seen
on SDS PAGE (Figure 3b).
Because the buffer used in the RPLC separations [CH3CN and 0.1 plc
trifluoroacetic acid ('IFA)] significantly diminishes the LPS inhibitory
activity of
BPI (data not shown), and since the material purified from ion exchange
chromatography was of high purity as judged by SDS PAGE, size exclusion/ion
exchange material was used to generate a dose response curve (Figure 4a). Data
is shown from two experiments, each performed in duplicate. This size
exclusion/ion exchange purified material was confirmed to be BPI by N-terminal
sequence analysis. Protein concentration was determined by amino acid
analysis.
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As s< en in Ftgnre 4a, about 90 ng/ml of BPI is required for maximal
inhibition
of tse neutrophil response to 10 ng/ml 0111/84 LPS. The neutrophil response
to formulated peptide ( 10''M FMLP) was not inhibited by BPI (data not shown).
Figure 4b shows a similar does response curve for the polypeptide antibiotic
Polymyxin B (PMB). Polymyxin B binds to the Lipid A moiety of LPS and
neutralizes some of its toxic effects both inin vivo and in vitro. polymyxin B
has
been demonsuated to bind to LPS stoichiometrically (Morrison, D.C. and D.M.
Jacobs, Immunochem, 13: 813-818 (1976)). The calculated amount of PMB
required to inhibit 10 ng/ml of smooth LPS is approximately 0.67 nM. In the
subject experiments 0.4 ng/ml, or 0.36 nM of polymyxin B was required to
completely inhibit neutrophil stimulation using 10 ng/ml of LPS. 90 ng/ml, or
1.58 nM BPI was required for 100% inhibition of 10 ng/ml LPS.
Therefore, on a molar basis the amount of BPI required to inhibit LPS
stimulation
of neutrophils in vitro was approximately 4X the amount required for polymyxin
B.
To test whether BPI can inhibit LPS from other gram negative organisms, LPS
2p molecules with varying polysaccharide chain lengths and Lipid A were tested
in
the subject system against 90 ng/ml of 2X purified BPI. Data shown in Table 2
demonstrates that although the stimulatory dose may vary between these
molecules, LPS from both smooth and rough chemotypes as well as Lipid A are
all inhibited by BPI.
35
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10 NG f ML 1 NG~ML
5 E.COLIO111:B4 97 '
WILD TYPE 103 113
S. TYPHIMURIUM
0 RE MUTANT 113 109
S. TYPHIMURIUM
RE MUTANT 33 99
LIPID A
~ 5 P. AERUGINOSA 112 '
' Low to no stimulation tration
at this endotoxin concen
25
35
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As previously discussed, Bacterial/Permeability-Increasing protein (BPI) is a
cationic SO-60,000 m.w. protein first purified from human neutrophil granules
by
Weiss et al. (Weirs, J., P. Elsbach, I. Olsson and H. Odegerg. 1978.1. Biol.
Chem.
253:2664.). BPI alters bacterial cell membrane permeability and has
bactericidal
activity specifically against gram negative organisms. To date, the literature
on
BPI has focused exclusively on its bactericidal activity.
We report that BPI binds to LPS and inhibits both neutrophil and monocyte
responses to soluble LPS in vitro. BPI also inhibits LPS activity in the
Limulus
~ebocyte Lysate assay. Our research has identified BPI as a lead molecule for
the development of novel therapies against endotoxic shock.
In response to LPS, human neutrophils upregulate cell surface expression of
2~ complement receptors CR1 and CR3 (Figures la and Sb). To measure this
neutrophil response to LPS, we incubated freshly isolated human neutrophils
with
E. Coli 0111:B4 LPS (Figure 4a), and showed that maximal CR1 upregulation is
observed using 10 ng/ml LPS (Figure 4). Neutrophil stimulation with LPS was
not
.inhibited by exogenous anti-TNF antibodies, suggesting that LPS acted
directly on
neutrophils in this system.
BPI inhibits the neutrophil response to LPS (Figure 4a). Inhibition of CR
upregulation was complete at a dose of approximately 1.8-3.6 nM (100-200
ng/ml)
; BpI compared to 0.4 nM polymyxin B required to inhibit 10 ng/ml smooth LPS
(approximate m.w. 15,000) is about 0.7 nM, matching closely with the observed
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32
value of 0.4 aM. On a molar basis, the amount of BPI requires ibit LPS
was approximately 5-fold greater than the amount required for poly ..:pcin B.
BPI inhibits LPS-mediated neutrophil stimulation but not stimulation by either
FMLP or TNF (Table 3). These data demonstrate that BPI inhibits LPS directly
and does not disrupt neutrophil mechanisms involved in CR upregulation.
Neutralization of LPS by BPI occurred rapidly. Even without preincubation,
both
BPI (and polymyxin B) inhibited more than 70% of the neutrophil response to
0 LPS (Figure 6). Maximal inhibition was seen following only 5 minutes of
preincubation.
BPI inhibits CR upregulation stimulated by LPS from smooth and rough bacterial
strains, as well as lipid A (Table 4) Because of the broad range of BPI
activity
against these different forms of LPS, among which only lipid A and 2-keto-3-
deoxy-octonate are shared determinants, it is likely that LPS inhibition by
BPI is
affected through lipid A.
gpI inhibits other LPS-mediated activities. At a concentration of
approximately
9 nM, or 500 ng/ml, BPI significantly inhibited LPS activity in the LAL assay
(Figure 7). When LPS and BPI were added together without preincubation no
inhibition was observed (data not shown), indicating that BPI acted on LPS,
and
had no effect on the LAL assay system. BPI also inhibits LPS-mediated TNF
production by human adherent mononuclear cells (Table 5).
.
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TABLE 3
Effect of BPI on Neutrophil
Stimulation by Various Agents
Inhibition of CR Upregulation on Neutrophils
Stimulus Dose % Inhibition % Inhibition
CRl CR3
15
LPS 10 ng/ml ~ 109 102
FMLP 10'' M 9 11
rTNF 50 U/ml 0 0
Neutrophils were incubated with E.E. Coli O111:B4 LPS, FMLP or TNF
preincubated in the presence or absence of 2.7 nM BPI. Data is reported as
percent inhibition of CR expression in response to each stimulus preincubated
with buffer alone.
30
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TABLE 4
Inhibition of LPS and Lipid A induced
Neutrophil Stimulation by BPI
Inhibition of CR Upregulation on Neutrophils
Stimulus Dose CRl CR3
(ng/ml) 96 Inhibition % Inhibition
None - 0 0
E.E. Coli
011:84 LPS 10 100 99
S~yphimurium
Wild Type LPS 10 104 100
S. xryhi
RE Mutant LPS 1 97 95
~ 5
S. yhimurium
RE Mutant
Lipid A 1 111 104
Neutrophils were stimulated with LPS and lipid A preincubated with and without
2.7 nM purified BPI. Results are expressed as percent inhibition of
fluorescence
intensity observed with each type of LPS alone.
30
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TAB LE 5
BPI Inhibits LPS-Induced
TNF Production by Human Monocytes
5
TNF (nQ/mll Produced in Resuonse to LPS Preincubated With':
LPS Medium 100 ng/ml 500 ng/ml 250 ng/ml Buffer
(ng,~ml) ~ one Polymy~dn B BPI $EI n r
0 0 0 0 0 0
0.1 61 0 0 0 81
1 1051 96 0 0 1090
10 2053 2154 1490 1746 2325
s~. Coli 0111:B4 LPS, was preincubated with BPI or polymyxin B (Pr~~l, than
added to adherent peripheral blood mononuclear cells. TNF produc::un was
assayed by ELISA.
30
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36
BPI was first purified by Elsbach and Weiss in 1978. In our initial studies we
isolated BPI from azurophil granule extracts in a single step by reverse phase
HPLC. Recovery of BPI activity from reverse phase was poor, probably due to
the denaturing conditions. Here we show the purification of LPS inhibitory
activity using only non-denaturing steps and demonstrate that most of the
activity
from neutrophils comigrates with BPI. Improvements in the purification have
also
led to very high specific activity material as will be shown in the following
section.
Figures 8-10 show the three chromatographic steps currently employed in our
lab.
The absorbance is traced by the solid line and LPS inhibitory activity on the
dotted lines. Table 6 shows the recovery of activity and protein and the
specific
activity, as measured in~arbitrary LPS neutralizing units (NU). One
neutralizing
unit is that amount of BPI that inhibits OS E.U. LPS by SO% in the LAL test. A
Commassie stained SDS-PAGE gel, of these pools is shown in Figure 11.
Analysis of the purified BPI by microbore reverse phase HPLC (Figure 12)
identified a single major peak which accounted for 97% of the total protein by
integration. Tryptic mapping of . BPI allowed us to sequence several major
fragments which further confirm the identity of the protein. The full length
published sequence for BPI is known (P.W. Gray et al. (1989) J. Biol. Chem.
~4( 16):9505).
30
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37
II. PURIFICATION OF BPI UNDER RIGOROUSLY PYROGEN-FREE
CONDITIONS
Materials and Methods
eaeents USP grade sterile irrigation water was obtained from Travenol
Laboratories Inc., Dee~eld IL; Pyrosart filters from Sartorius GmbH, W.
Germany; CM Sepharose FF from Phasmacia, Upsaza, Sweden; Polyaspartamide
weak ration exchange HPLC column (100 X 4.6mm) from the Nest Group,
Southborough MA; Glycine and Bio-Sil 6250 size exclusion HPLC column (600
X 7.5mm) from Bio-Rad Laboratories, Richmond CA; Polyacrylamide
electrophoresis gels from Novex, Encitas CA; Sequencing and amino acid
analysis
reagents and buffers from Applied Biosystems Inc., Foster City, CA;
Trifluoroacetic acid, constant boiling HCL, hydrolyzate amino acid standard,
and
~ 5 BCA protein assay reagents from Peirce Chemical Co., Rockford, IL; Limulus
Amebocyte Lysate assay from Whittaker Bioprodurts, Inc., Walkersville, MD;
Lipopolysaccharide from RIBI Immunochem Research, Inc., Hamilton, MT;
HPLC grade Acetonitrile from J.T. Baker, Phillipsburg, NJ; all other buffers
and
salts used were reagent grade. 18 megohm purity water was prepared by Lab
Five ultrapure water system from Technic, Seattle, WA. O.SM NaOH for
sanitization was prepared from reagent grade NaOH pellets and USP water.
(~ranuls extracts from Neutroo~ were prepared as described (U.S. Serial No.
199,206, filed May 26, 1988) except that the percoll separation of azurophil
granules was omitted. Instead, whole granule fractions were obtained by
centrifuging the post nuclear supernatant at 17,000 g for 20 minutes. The
granule
pellet was then suspended in a volume of 1 ml of 50 mM glycine pH 2 for every
;4X10E8 cells lysed. Resuspended granules were lysed by five freeze/thaw
cycles
on dry ice ethanol followed by vigorous agitation for one hour at 4 degrees C.
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The soluble extract was obtained by centrifugation at 30,OOOg for 30 minutes.
Limulus Ameboc~r~ysat~ was performed as directed by the manufacturer.
Where necessary the pH of samples was adjusted to neutrality by the addition
of
pyrogen free O.SM phosphate buffer pH 7.4. and salinity was decreased to < 150
mM by dilution with USP water.
LPS neutralization assay: was performed as previously described (M. Marra et
al.
( 1990) J. Immunol. x(2):662-666).
High salt fractionation of granule extracts: 200 mgs of extracted protein were
pooled from various preparations and kept on ice. 1 volume of sterile SM NaCI
was added for every 4 volumes of extract. The resulting precipitate was
pelleted
by centrifugation at 20,000 g for 20 minutes at 4 ~ C. This supernatant was
prepared for CM sepharose chromatography by diluting with 4 volumes of USP
irrigation water and adjusting the pH with enough 1M Tris pH 7.4 to give a
final
concentration of 50 mM. Only fresh, sterile, pyrogen free stock salts and
buffers
were used.
CM Se~harose chromatoQr_aRhv: An XK-16 column (Pharmacia) was packed with
sufficient resin to give a bed volume of Smls. The column was installed on a
gradient FPLC equipped with a Pl pump for sample loading. Prior to use, all
s~~s in contact with the mobile phase were extensively washed with O.SM
NaOH. The column was sanitized by washing at 0.2 mls/min. with O.SM NaOH
for 4 hrs. The column was then re-equilibrated and a blank run was performed.
Fractions from the blank run and eluenu were tested by LAL assay for
pyrogenicity. Prepared exuact was loaded at a flow rate of 400 mls/hr. Once
~ loaded the column was washed with 2 to 3 column volumes of starting buffer.
The
granule extract was kept on ice during loading. The column was run at room
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39
tcm~~erature.
Weak canon exchange HPLC: was performed using an Eldex ternary gradient
pump equipped with a Rheodyne injector and a Gilson model 1118 U.V. detector.
Wettable surfaces were washed with O.SM NaOH followed by extensive rinsing
with USP water to remove all traces of base prior to installing the column.
Blank
fractions and eluents were tested for pyrogenicity as above.
C'~el ~rmeation HPLC: was performed with the same precautions and equipment
0 outlined for weak cation exchange HPLC.
Poly~lamide gel electrophoresis: 8 to 16% acrylamide gradient gels were
purchased from Novex and run according to the manufacturers specifications.
Protein seguence determination: An Applied Biosystems 477A pulsed liquid
phase sequenator equipped with a 120A PTH amino acid analyzer was used for
automated edmund degradation.
Microbore reverse phase HPLC: Material for protein sequencing was prepared
by desalting on a 30 X 2.1 mm Aquapore butyl column. The gradient used was
to 100% B in 30 minutes at a flow rate of 200 ml/minute. Detector settings
were 214 nm wavelength at 2.0 absorbance units full scale (see insert figure
X).
25 pn HP 3396A was used to integrate and plot data.
,; was performed on the system described above using the
PTC column, buffers and separation conditions provided by ABI. Sample
30 hydrolysis and PTC derivatives were prepared using a Pico-Tag workstation
from
the Waters chromatography division of Millipore using the manufacturer's
protocols.
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Protein concentrations were determined using .A method
instructions 23230, 23225 from Peirce Chemical Co. In order to minimize buffer
interference, samples were diluted 1(I fold and the micro reagent protocol was
used.
RESULTS
BPI purified from azurophil granules was previously shown to inhibit
neutrophil
activation by LPS and to inhibit LP;S directly in the LAL assay. In order to
10 ~rther define the role of BPI and investigate the presence of other similar
molecules in both azurophil and specific granules, we undertook the
purification
of LPS inhibitory activity from whole ;granules extracted at acid pH.
Preliminary
studies verified the presence of LPS inhibitory activity in the crude extract.
To identify the endotoxin neutralizing activity we attempted its purification
from
whole granule extracts. Purification. of LPS neutralizing activity was greatly
enhanced by the observation that high concentrations of NaCI ( 1M) caused the
reversible precipitation of about ninety percent of the protein present in the
granule extract. Essentially all of the LPS inhibitory activity remained in
the
soluble supernatant. The soluble fra~aion was then diluted, to reduce the
ionic
strength, and further purified and con<:entrated by CM sepharose cation
exchange
chromatography. A broad peak of activity eluted which was subsequently further
~ p~~d ~~g a polyaspartamide high performance cation exchange column. A
somewhat sharper peak of activity was recovered which comigrated with a major
protein of about 55,000 molecular weight by SDS-PAGE along with several lower
molecular weight proteins. Gel permeation HPLC was used as the final
purification step and identified a peak of activity which eluted with a single
sharp
protein peak. The purified protein migrated as two closely spaced bands on SDS-
PAGE at 55,000 molecular weight. 25% of the total endotoxin neutralizing
CA 02323630 2000-10-26
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41
activity was recovered with a 250 fold purification.
The purified endotoxin neutralizing protein was subjected to reverse phase
HPL,C
followed by N-terminal sequence analysis by automated Edman degradation. The
sequence, shown in figure 6 was identified as bacterial permeability
increasing
protein by virtue of complete homology through 39 residues. In addition the
amino acid composition of the purified molecule was virtually identical to
that of
BPI (data not shown).
To investigate whether both closely spaced bands were BPI we subjected the
purified proteins to western blotting analysis using BPI-specific rabbit
polyclonal
antisera raised against a synthetic peptide comprising amino acids 1-20 of
BPI.
Both bands were immunoreactive. The differences may arise from glycoslyation.
20
30
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. , WO 90/09183 ' PC1"/US90/00837
42
Purification of BPI under rigorously pyrogen-free conditions, as described in
section II resulted in a more potent BPI preparation as shown by the dose
TABLE 6
Recovery Specific
Acts Acts
Extract 100% 100% 0.11 NU/ag
Precipitated 149% 17.3% 0.93 NU/ug
Step 1 35% 1.50 2.51 NU/,ug
Step 2 14% 0.75 1.97 NU,Gtlg
Step 3 18% 0.10% 18.9 NU/,ag
response curve in Figure 13. Inhibition of LPS-mediated CR upregulation was
complete at 25 ag/mI BPI, representing a 4-fold increase in activity compared
to
the material used in section I. On a molar basis this BPI preparation
inhibited
LPS at approximately stoichiometric proportions, equivalent to molar
inhibitory
concentrations of polymyxin B. BPI also inhibited LPS-mediated TNF production
by human adherent mononuclear cells at a lower concentration following
purification under pyrogen-free conditions (Tables 7 and 8).
BPI binds to LPS (Figure 14). In these experiments, 4 ug of LPS/well was
immobilized on 96 well plastic plates, then incubated with varying
concentrations
of BPI, and developed with anti-BPI polyclonal antisera. BPI binding to LPS
was
inhibited by polymyxin B (Figure 15), demonstrating specificity of BPI
binding.
BPI binds to LPS in the presence of both plasma (Figure 16) and serum (Figure
17), demonstrating potential inin vivo efficary of BPI.
W090~09183 CA 02323630 2000-10-26 P~1'~LrS94~0083i
43
TABLE 7
BPI Inhibits LPS-Induced TNF
Ptoduaion by Human Monocyces
T'.v'F lctJml l Ptoduccduottae to LPS Pteincubated
in Res with':
LPS Medium 100 ns/ml 400 ng/mlLSO ng/ml 25 ng/ml Buffer
ng/ml alone PMB BPI BPI BPI Control
0 0 0 0 0 0 0
0.1 48 79 0 0 0 269
1 1150 120'1 0 0 0 1292
10 1370 1270 145 333 559 1413
li 0111:84 LPS, was preincubated
with BPI or poiymyun
B (PMH), then added to
adherent petiphenl blood
mononuclear cells.
T1YF produa~on was assayed
by ELISA.
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44
TABLE 8
INHIBCIZON OP LPS-INDUCED
TNF PRODUCIION BY HUMAN MONO(.YTES
'I~f' /ml) ProduccdResponse to LPS Preincubaced
(nf; in with':
1000 ng/ml100 a8/ml 250 ngJml 50 n8/ml lOng/mlBuffer
LPS PolymyxinPolymycn H BPl BPI HP( Control
B
10 333 18 601 257 270 f 23 270 67 436 6g7
3g 37
100 769101 114073 8343p 6g6+g4 1003+50892+47
1000 844 144 1016 20 1130=10 T78 189 1025=71773
+
88
.S aureus
1685;121 1541 ~ 397 1538~ 139 1268 ~ 374 1554 ~ 324 1423~447
'BPI or polymyxin B sulfate were preincubated with 0-10 ng/ml E. Coli 0111:B4
LPS or 0.1%w/v killed S.S. aureus then added adherent peripheral blood
mononuclear cells. TNF production was assayed by ELISA.
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30
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EXAMPLE 3
5 Stage IA- ogenici~y of Gl3rcine Buffer:
305u1 of Glycine Buffer control (Supplied by Redwood City) was diluted to 7 ml
in PBS (Redwood City) and mixed in polypropylene tubes (pyrogen-free). The
10 rube was labeled with notebook # 1990 and tested in a three rabbit USP
Rabbit
Pyrogen assay at a dose of 2 ml/rabbit (actual injection dose was 2.1
ml/rabbit).
The product was non-pyrogenic; it produced a total temperature rise for all
three
rabbit of 0.4 C.
304 ul of BPI (Lot 78038, dated 8/19/89) was diluted to 7 ml using PBS
(Redwood City) and mixed in polypropylene tubes (pyrogen-free). The Tube was
labeled with notebook #20170 and tested in a three rabbit USP Pyrogen assay at
a dose of 2.0 ml/rabbit.
Th° Product was non-pyrogenic as demonstrated by a total temperature
rise of
02' C.
~Ee II- ,~oEenici of BPI ire-incubated with endotoxin:
; Endotoxin from E. Coli OSS.BS (Sigma Chemicals) was diluted in PBS (Redwood
City) to 4096 EU/ml. This concentration was confirmed by the T .AI - Assay.
CA 02323630 2000-10-26
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46
304 ul of BPI (Lot 78038, dated 8/19/89) was diluted to 7 ml with the PBS
diluted
endotoxin (4096 EU/ml) hereinabove using polypropylene tubes. The tube was
mixed by vortexing to effect mixing. The BPI+Endotoxin and Endotoxin in PBS
were incubated at 37' C in a water bath for 30 minutes. Following incubation
at
37' C the BPI + Endotoxin showed an endotoxin concentration of 122 EU/ml. The
endotoxin diluted in PBS did not show a change in the end point of 4096 EU/ml.
The BPI+Endotoxin and Endotoxin in PBS in PBS were tested in the three rabbit
USP pyrogen assay and were found pyrogenic with total temperature rises of
4.6' C and 7.5' C, respectively.
To achieve improved results with the manipulations of the endotoxin
preparation
we switched from the E.E. coli OSS:BS from Sigma to the Official FDA
References.
A vial of EC-5 was rehydrated with PBS (Redwood City) to 2 ml to give a
concentration of 5000 EU/ml. We verified by the label claim of 10,000 EU/ml
by LAL assay.
The BPI+Endotoxin sample was prepared by adding 38,u1 of PBI (Lot 78038) to
73 ml of PBS plus 320u1 of the 5000 EU/ml of EC-5 endotoxin. The preparation
v~ wed in a polypropylene tube(pyrogen-freed) and mixed well. An 8.0 ml
sample of EC-5 endotoxin was prepared in PBS(Redwood City) to the same
concentration without the addition of BPI. Both samples was incubated at 37' C
for 30 minutes in a water bath.
The two samples were tested for endotoxin activity using the LAL assay. The
BPI + Endotoxin was negative. The endotoxin sample was positive at the target
CA 02323630 2000-10-26
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47
of 2~'J EU/ml (Figure 18).
Both samples were tested in the three rabbit USP Pyrogen Assay at a dose of
2.0
ml/rabbit.
The BPI+Endotoxin was non-pyrogenic and caused a total temperature rise of
1.1' C. The EC-5 endotoxin in PBS was pyrogenic and caused a total temperature
rise of 3.9 ' C.
15
25
35
