Note: Descriptions are shown in the official language in which they were submitted.
2.~0~.~;~3
1
SPECIFICATION
~i
IMMUNOSTIMULATORY AGENT
TECHNICAL FIELD
This invention relates to an immunostimulatory agent
comprising a peptide derived from lactoferrin having activity to
modulate the release of inflammatory mediators from cells of the
immune system and thereby potentiate the cellular immune
response. In particular, the present invention relates to a
peptide derived from lactoferrin having at least the following
activities: (1) promotes the release of leukotriene B4 from
polymorphonuclear neutrophils; (2) promotes the release of
histamine from mast cells.
BACKGROUND ART
Polymorphonuclear neutrophils and mast cells play a
mayor role in the host defense against bacterial, fungal and
viral infections. Microorganisms or their products can interact
directly with these cells and induce the release of inflammatory
mediators such as leukofir'ienes and histamine which have multiple
effects essential to amplification and control of the
inflammatory response. Leukotriene B4 is a potent chemotactic
and chemokinetic factor which attracts neutrophils, eosinophils,
monocytes and macrophages to sites of infection and tissue
CA 02102509 2001-02-26
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trauma. It promotes the aggregation of neutrophils, augments
their adherence to endothelial cells, stimulates the release of
lysozQmal enzymes and the production of superoxide. It also
inhibits the proliferation of lymphocytes via the induction of
suppressor T cells and augments cytotoxic and natural killer
cell activities. On the other hand; leukot~rienes C4, D4, and E4
are the slow-reacting substances of anaphylaxis which cause long-
lasting contraction of smooth muscle, bronchoconstriction,
vasoconstriction, secretion of mucus, and an increase in
vascular permeability. Histamine induces vasodilation, increase
in vascular permeability, stimulation of secretory glands and
contraction of smooth muscle. Furthermore, histamine modulates
certain immune effector functions such as cell-mediated
cytotoxicity, lymphocyte proliferation, lymphokine production,
and immunoglobulin synthesis. In general, the biological
actions of leukotriene B4 and histamine tend to potentiate the
cellular immune response and stimulate the host defense against
microbial infections.
It is widely recognized that the biological actions of
leukotriene B4 relating to recruitment and stimulation of
neutrophils are essential for the host defense against microbial
infections. The migration of polymorphonuclear neutrophils from
circulating blood to the 'focus of infection is one of the
earliest events of the inflammatory process. The cells leave
the circulation and are guided towards sites of infection and
' 21Q~~0~
tissue trauma by chemotaxis, in response to chemotactic factors
released in the vicinity of invading microorganisms.
Leukotriene B4 is the most potent chemotactic stimulus for
neutrophils. The chemotactic stimulus promotes cellular
movement and enables a variety of subsequent steps essential to
the host defense including attachment, engulfment, ingestion,
degranulation, secretion of lysozomal enzymes into
phagolysozomes, and generation of toxic oxygen radicals, which
ultimately lead to killing of invading microorganisms.
Neutrophils control the concentration of leukotriene B4 in their
environment by generating and metabolizing this inflammatory
mediator. Leukotriene B4 is bound by specific receptors on the
cell surface, internalized, and enzymatically oxidized producing
biologically inactive products, 20-OH-leukotriene B4 and 20-COOH-
leukotriene B4. In the case of ma3or injuries, such as severely
burned patients, the chemotactic responsiveness of neutrophils
is impaired. Their production and release of leukotriene B~ is
diminished, the metabolism of leukotriene B4 to inactive 20-OH-
leukotriene B4 and 20-COON-leukotriene B4 is enhanced, and the
expression of leukotriene B4 receptors on the sell surface is
reduced. Decreased production of leukotriene B4 and reduced
responsiveness to leukotriene B4 leads to inadequate recruitment
of phagocytes at sites of tissue injury and precedes the onset
of microbial invasion.
Lactoferrin is an iron-binding glycoprotein present in
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various biological fluids of Mammals including milk, saliua,
tears and mucous secretions, and released from activated
polymorphonuclear neutrophils at sites of inflammation. Large
quantities of bovine lactoferrin can be obtained by extracting
this protein from raw skim milk or cheese whey originating from
the milk of cows and, consequently, bovine lactoferrin is
readily available as a commercial product of the dairy industry.
In its iron-free state, lactoferrin exhibits broad-spectrum
antimicrobial activity which is commonly attributed to its
ability to chelate iron and produce an iron-deficient
environment limiting microbial growth. The present inventors
first discovered that peptides having more potent antimicrobial
properties than lactoferrin are generated upon enzymatic
digestion of this protein (Japanese Patent Application
No.238364/90). The antimicrobial peptide~derived from
lactoferrin appears~to function by a mechanism distinct from
iron binding and is effective at low concentrations against
various species of Gram-negative and Gram-positive bacteria,
yeasts, and molds, including strains known to cause disease in
humans and animals. The effect of this peptide against
microorganisms is lethal causing a rapid loss of colony-forming
ability. Considerable potential exists for the widespread
commercial use of this peptide as a safe and effective
antimicrobial agent and a novel process has been established for
., its large-scale manufacture as described in Japanese Patent
210200
Application No.150604/91.
It has been demonstrated that lactoferrin has activity
to inhibit calcium ionophore-induced release of histamine from
peritoneal mast cells [Theobald, K. et al. (198T) Agents and
Actions 20:10-16], however, the effect of lactoferrin on the
release of other inflammatory mediators such as leukotrienes is
unknown. Whether peptides derived from lactoferrin have
activity to modulate the release of inflammatory mediators from
cells of the immune system has not been studied previously.
The present inventors investigated for the first time
the effects of peptides derived from lactoferrin on the release
of inflammatory mediators from cells of the immune system.
Surprisingly, they discovered that the antimicrobial peptide of
lactoferrin described in Japanese Patent Application
No.238364/90 is an immunostimulatory peptide having excellent
activity to modulate the release of leukotriene B4 and histamine
from polymorphonuclear neutrophils and mast cells, respectively,
and thereby potentiate the cellular immune response. In
contrast, lactoferrin does not exhibit such useful
immunostimulatory activity.
DISCLOSURE OF THE INVENTION
This invention is based on the discovery of a peptide
derived from lactoferrin having the capability to modulate the
release of inflammatory mediators from cells of the immune
210~~~~
6
system and thereby potentiate the cellular immune response of
humans and animals.
It is an object of the present invention to provide an
immunostimulatory agent comprising a peptide derived from
lactoferrin.
It is another object of the present invention to provide
an immunostimulatory composition containing a peptide derived
from lactoferrin as an active ingredient.
These objects of the present invention are achieved
herein by providing an immunostimulatory peptide derived from
lactoferrin having at least the following activities: (1)
promotes the release of leukotriene B4 from polymorphonuclear
neutrophils; (2) promotes the release of histamine from mast
cells.
BEST MODE FOR CARRYING OUT THE TNYENTION
The peptide of the present invention can be produced by
hydrolysis of lactoferrin or generated by conventional methods
of peptide synthesis and purified. Briefly, for example, a
preferred method for obtaining the peptide of the present
invention is as follows. Bovine lactoferrin isolated from skim
milk or cheese whey is dissolved in distilled water at a
concentration of 5% (w/v) and the pH is adjusted to 3.0 by
addition of 1 N HC1.. Pepsin is added to a final concentration
of 3% (w/w of substrate) and hydrolysis is performed at 3T°C for
CA 02102509 2001-02-26
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4 h. The reaction is terminated by heating at 80°C for 15 min
and the pH of the resulting peptide mixture is adjusted to T.0
by addition of 1 N NaOH. Any insoluble peptides are removed
from the starting material by filtration or centrifugation. The
active peptide is purified from the resulting solution, for
example, by contacting the solution with a butyl moiety-
containing hydrophobic interaction chromatography medium, such
as Butyl-Toyopearl 650 M (Tosoh, Japanj, rinsing the medium with
water to remove unbound peptides, desorbing the
immunostimulatory peptide at a constant pH, preferably
pH 4.8-5.2, and desalting the product. Desalting,can be
accomplished, for example, using the same column of Butyl-
Toyopearl*6~0 M. The solution containing the peptide is
adjusted to pH T.O, by addition of 1 N NaOH, and contacted with
_ the hydrophobic medium. The medium is rinsed with water to
remove the buffer salts a.nd finally the active peptide is
desorbed with 10 mM~HCl and freeze-dried. Accordingly, the
peptide of the present invention can be obtained in greater than
99% purity, free of all other biologically active substances, in
any desired quantity.
The present inventors have succeeded in isolating and
purifying a peptide of bovine lactoferrin with defined nature
and function and subsequently have succeeded in demonstrating
for the first time its activity to modulate the release of
potent inflammatory mediators from cells of the immune system.
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2~~2~4)
8
For instance the peptide of the present invention
consists of a single chain of amino acids having the sequence
Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-Pro-
Ser-Ile-Thr-Cys-Val-Arg-Arg-Ala-Phe. It is the same peptide of
lactoferrin described in Japanese Patent Application
No.23836~/90 having broad spectrum antimicrobial activity. That
is, the peptide of the present invention has both
immunostimulatory and antimicrobial properties.
In the specification of the present invention, the amino
acids and peptides are represented by the abbreviations employed
by IUPAC-IUB Committee on Biochemical Nomenclature, such as the
following abbreviations:
Ala-: L-Alanine residue
Arg-: L-Arginine residue
Asn-: L-Asparagine residue
Asp-: L-Aspartic acid residue
Cys-: L-Cysteine residue
Gln-: L-Glutamine residue
Glu-: L-Glutamic acid residue
Gly-: Glycine residue
His-: L-Histidine residue
Ile-: L-Isoleucine residue
Leu-: L-Leucine residue
Lys-: L-Lysine residue
Met-. L-Methionine residue
CA 02102509 2001-02-26
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Phe-. L-Phenylalanine residue
Pro-. L-Proline residue
Ser-. L-Serine residue
Thr-. L-Threonine residue
Trp-. L-Tryptophan residue
Tyr-. L-Tyrosine residue
Val-. L-Valine residue
In consideration of the fact that lactoferrins of mammalian
species are well known to exhibit a high level of amino acid
sequence homology, it is now obvious as a result of their
discovery that immunostimulatory peptides having substantially
the amino acid sequence of the peptide of the present invention,
but having minor substitutions of amino acids which do not
abolish its immunostimulatory properties, may be produced by
hydrolysis of lactoferrins of other mammals- such as human, water
buffalo; sheep, goat, etc. Furthermore; it is now obvious as
the result of their discovery that immunostimulatory peptides
having substantially the amino acid sequence of the peptide of
the present invention, but having minor deletions, additions, or
substitutions of amino acids or other minor chemical
modifications which do not abolish its immunostimulatory
properties, may be produced by conventional methods of peptide
synthesis. Such obvious peptides are contemplated and should
not be considered as being novel or distinct from the present
invention.
z~o~~,o~
The peptide so obtained has at least the following
immunostimulatory activities: (1) promotes the release of
leukotriene B4 from polymorphonuclear neutrophils; (2) promotes
the release of histamine from mast cells. These useful
activities can be readily demonstrated, for example, by the
procedures described in detail later in this specification (see
Test 1-3). By promoting the release of such potent inflammatory
mediators from neutrophils and mast cells the peptide of the
present invention can potentiate the cellular immune response
and stimulate the host defense against infectious disease. Most
importantly, its activity to promote the release of leukotriene
B4 from neutrophils can enhance the recruitment and stimulation
of phagocytes at sites of infection and tissue trauma and
thereby promote the destruction of invading microorganisms.
Such activity may be especially beneficial in the case of major
injuries, such as severely burned patients, in which
polymorphonuclear neutrophils display diminished production of
leukotriene B4 and reduced responsiveness to leukotriene B4.
The immunostimulatory peptide so obtained is included as
an active ingredient at a concentration of at least 1 ppm and
preferably l0 to 100 ppm in order to obtain the
immunostimulatory composition of the present invention.
The compositions may contain pharmaceutically suitable
solvents (e. g. water, ethanol, glycerol, propylene glycol,
liquid polyethylene glycol), flavoring agents, sweetening
CA 02102509 2001-02-26
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agents, binders, isotonic agents, 'coating agents, surfactants,
absorption delaying agents, and the like. The use of such
agents is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, sts use in the~compositions is contemplated.
Supplementary active ingredients can also be incorporated into
the compositions. Of course, any material used in preparing the
compositions should be non-toxic in the amounts employed.
The peptide of the present invention can be administered
to humans or animals, for example, dermatologically,
ophthalmically, otically, nasally, anally, or vaginally as a
powder or as a component of a solution, suspension, cream,
ointment, or spray. The peptide may also be orally
administered, for example, as a powder or aqueous solution,.or
it may be incorporated into capsules, tablets, syrups, elixirs,
or the like, or it may be incorporated directly into food of the
diet.' The peptide may also be administered, for example,
parenterally, intraperitoneally or intramuscularly as a
component of a sterile injectable solution.
The peptide of the present invention can be used, for
example, in medicinal pharmaceutical products (such as eye
medications, mastitis medications, athlete's foot medications),
non-medicinal pharmaceutical products (such as mouth washes),
various cosmetic products (such as skin lotions and creams),
various food products (such as chewing gum),, it can be also be
CA 02102509 2001-02-26
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added to, compounded with, sprayed onto, adhered to, or used for
coating or impregnation of any and all products wherein its
immunostimulatory activity is desired (such as surgical
dressings, bandages, etc.) or otherwise used for treating any
and all products wherein its immunostimulatory activity is
desired.
The present invention is further described by means of
the following Tests.
TEST 1,
This test was performed to study the activity of the
peptide of the present invention to promote the release of
histamine from rat peritoneal mast cells induced by the calcium
ionophore A2318T.
(1) Sample Preparation
Peritoneal cells were collected from Wistar rats after
intraperitoneal injection of phosphate-buffered saline
(consisting of 120 mM NaGI, 10 mM Na2P04, 3 mM KH2P04, pH ~.4).
Their andomens were massaged and the buffer containing the
cells was recovered. The cells were centrifuged at 300 x g for
15 min and washed twice with phosphate-buffered saline. The
cells from several rats were pooled for use in the experiments.
(2) Experimental Methods
Rat peritoneal cells containing about 1 x 105 mast cells
per m~ were used as the target cells. A 500 ~.el volume of the
cell suspension was incubated with 100 ,u1 volume of the test
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13
substance [purified peptide obtained by the method as in Example
1, or human lactoferrin (Sigma Chemical Co.), or bovine
lactoferrin (Morinaga Milk Industry Co.)] at 3~°C for 30 min. ,
Thereafter, histamine release from the mast cells was induced by
the addition of calcium ionophore A2318T (Sigma Chemical Co.) at
a final concentration of 5 ~M. After 10 min of incubation the
cells Were centrifuged at 300 x g for 15 min and the supernatant
was removed and deproteinized by the addition of 2 ml of HC104
(2%). The deproteinized sample was then centrifuged at 2000 x g
for 20 min and the histamine content in the supernatant was
determined by the fluorophotometric analyser technique
(AutoAnalyzer: Technikon). Cells in the presence of phosphate-
buffered saline served as control.
(3) Results
The experimental data shown in Table 1 indicate that the
peptide of the present invention has activity to promote the
release of histamine from mast cells induced by the calcium
ionophore A2318T. It exhibits such activity at low
concentrations within the range of 1 to 100 ppm, and the amount
of histamine released increases in a dose dependent manner
depending on the concentration of the peptide. On the other
hand, the results further indicate that human and bovine
Iactoferrins show little, if any, such activity.
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TABLE 1
Histamine release
Treatment ppm (Helati~e %)
control (saline) - 100
purified peptide 1 135 +10
145 +15
100 175 +20
lactoferrin, human 1 115 +20
10 110 +10
100 100 +10
lactoferrin, bovine 1 105 +25
10 110 +20
100 125 +25
TEST 2
This test was performed to study the activity of the
peptide of the present invention to promote the release of
histamine from rat peritoneal mast celle induced by a-toxin-
producing Staphylococcus aureus cells.
210~~~
(1) Sample Preparation
Staphylococcus aureus 121, an a-toxin-producing strain,
was cultured overnight in Brain Heart Infusion broth at 3T°O.
The bacterial cells were collected by centrifugation at 4000 x g
for 20 min, washed with phosphate-buffered saline, and suspended
in the same buffer for use in the experiment. Rat peritoneal
cells were prepared as in Test 1.
(2) Experimental Method
The same method as in Test 1 was used, except histamine
release from the mast cells was induced by the addition of a-
toxin-producing Staphylococcus aureus cells at a final
concentration of about 1.0 x 108 cells per ml.
(3) Results
The experimental data shown in Table 2 indicate that the
peptide of the present invention has activity to promote the
release of histamine from mast cells induced by a-toxin-
producing Staphylococcus aureus cells. It exhibits such
activity at low concentrations within the range of 1 to 100 ppm,
and the amount of histamine released increases in a dose
dependent manner depending on the concentration of the peptide.
On the other hand, the results further indicate that human and
bovine lactoferrine shown little, if any, such activity.
-., 2102~(9~)
16
TAELE 2
Histamine release
Treatment ppm
(Relative ~)
control (saline) - 100
purified peptide 1 200 +60
250 +50
100 460 +240
lactoferrin, human 1 180 +5
10 115 +15
100 120 +20
lactoferrin, bovine 1 150 +10
10 160 +10
100 110 +15
TEST 3
This test was performed to study the activity of the
peptide of the present invention to promote the release of
leukotriene B4 from human polymorphonuclear neutrophils induced
by the calcium ionophore A2318T.
1T
(1) Sample Preparation
Human polymorphonuclear neutrophils were prepared by
fractionation of leukocytes from heparinized blood of healthy
donors on a Ficoll-metrizoate gradient followed by dextran
sedimentation. The cells were centrifuged at 300 x g, and
washed three times with phosphate-buffered saline, after which
less than 2% of the platelets remained. Erythrocytes were
removed by exposing the cells to hypotonic conditions. The
purity of the polymorphonuclear neutrophil fraction used was
greater than 9T% as confirmed by light microscopy.
(2) Experimental Method
For analysis of leukotriene release, human neutrophils
suspended in phosphate-buffered saline at approximately 1 x lOT
cells per m1 were used as the target cells. The cells were
incubated for 30 min in the presence of test substance
((purified peptide obtained by the method as in Example 1, or
human lactoferrin (Sigma Chemical Co.), or bovine lactoferrin
(Morinaga Milk Industry Co.)I. Leukotriene release from the
neutrophils was then induced by the addition of calcium
ionophore A2318T (Sigma chemical Co.) at a final concentration
of 5 ~M. After 30 min; the supernatants of the stimulated cells
were deproteinized by addition of 2 ml of methanol-acetonitrile
(1:1) overlaid with nitrogen and frozen for 12 h at -TO°C.
After centrifugation at 1000 x g for 15 min the supernatant was
evaporated to dryness in a freeze dryer, suspended in 0.5 ml of
CA 02102509 2001-02-26
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methanol-water (30:T0), overlaid with nitrogen, and stored
overnight at -TO°C. The samples were centrifuged at 1000 x g
and the supernatant was assayed for the presence of leukotrienes
by high pressure liquid chromatography using a reverse-phase
column of Nucleosil*(C18; 5 ~Wn particles) at a column
temperature of 40°C. The solvent system was a mixture of
phosphate buffer (1T mM K2HP04 containing 0.05% EDTA),
acetonitrile, and methanol (50:30:20) adjusted to pH 5.O,with
phosphoric acid. The absorbance at 280 n~ of the column
effluent was monitored and recorded. The concentration of each
eluted leukotriene was determined from.the peak area by means of
a computing integrator. The leukotrienes were identified from
their retention times with reference to known standards. The
minimum detectable quantity was 1 ng in each instance. Because
leukotriene B4 is rapidly metabolized by neutrophils to the
biologically inactive products 20-OH-leukotriene B4 and 20-COOH-
leukotriene B4 under the conditions of this experiment the total
amount of ieukotriene B4 released includes the sum of these
inactive products.
(3) Results ,
The experimental data shown in Table 3 indicate that the
peptide of the present invention promotes the release of
leukotriene B4 from polymorphonuclear neutrophils. It exhibits
such activity at low concentrations within the range of 1 to 100
ppm, and the amount of leukotriene B4 released increases in a
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~102~~~
19
dose-dependent manner depending on the concentration of the
peptide. On the other hand, the results further indicate that
human and bovine lactoferrins have little, if any, such
activity. None of the compounds tested substantially promotes
the release of the spasmogenic leukotriene C4.
TABLE 3
Leukotriene released (ng)
Treatment ppm
B4 20-OH 20-COOH (Total B4) C4
control (saline) - 12 55 31
(98) 18
purified peptide 1 12 TO 51
(133) 23
14 116 61 (191) 1T
100 18 162 104 (284) 23
lactoferrin, human 1 11 80 41
(131) 12
10 10 50 26 (86) 13
100 10 42 26 (T6) 1T
lactoferrin, bovine 9 50 41
1 (100) 1T
10 11 60 36 (10T) 15
100 11 59 35 (105) 15
2102~~
The following examples are merely illustrative of the
invention.
EXAMPLE I
Production of the peptide of the present invention is
exemplified as follows. Bovine lactoferrin (2.0 kg: Morinaga
Milk Industry Co., Ltd; purity, approximately 90%) isolated from
skim milk was dissolved in distilled water at a concentration of
5% (w/v) and the pH was adjusted to 3.0 by addition of 1 N HC1.
Crystalline pepsin (Difco Laboratories) was added to a final
concentration of 3% (w/w of substrate) and hydrolysis was
performed at 3T°C for 4 h. The reaction was terminated by
heating at 80°C for 15 min. The pH of the resulting peptide
mixture was adjusted to T.0 by addition of 1 N NaOH and
insoluble peptides were removed by filtration. The peptide
solution was spray-dried to obtain 1.9 kg of powdered material.
A 600 g portion of the powdered material was dissolved in
distilled water at a final concentration of 5% (w/v). Butyl-
Toyopearl 650 M (Tosoh Corp.) was rinsed and equilibrated with
water before use and approximately 3.0 liters of this
hydrophobic gel were used. The starting material was initially
contacted with the hydrophobic medium in a stirred tank, then
the liquid was collected and the medium was transferred to a
chromatographic column (10 cm x z0 cm 3.d.). The collected
liquid was again contacted with the medium in the column, then
the hydrophobic medium was rinsed with water, at a flow rate of
2192~~~
21
about 400 ml/min, to remove the unbound peptides. Rinsing of
the medium was continued until the protein content of the water
eluted from the medium declined to a low Level, as indicated by
an absorbance at 280 nm of about 0.06. The bound peptides
including the immunostimulatory peptide were desorbed from the
medium with 10 mM HC1 and mixed with an equal volume of
McIlvaine buffer, pH T.0 (prepared by combining solutions of 0.1
M citric acid and 0.2 M
Na2HP04 in a ratio of 1TT:824). The resulting buffered peptide
solution was contacted with the hydrophobic medium and the
medium was rinsed with about 6 liters of the same buffer. The
active peptide was desorbed selectively from the medium at a
constant pH with about 9 liters of McIlvaine buffer, pH 5.0
(prepared by combining solutions of 0.1 M citric acid and 0.2 M
Na2HP04 in a ratio of 485:515). Desalting of the solution thus
obtained was accomplished using the same 3000 m1 of Butyl-
Toyopearl 650 M. The solution was adjusted to pH T.O, by
addition of 1 N NaOH, and contacted with the hydrophobic medium,
then the medium was rinsed with about 30 liters of water to
remove the buffer salts. Finally, active peptide was desorbed
with 10 mM HC1 and freeze-dried to obtain 10.5 grams of powdered
product. The purity of the product was greater than 99% as
estimated by reverse-phase high performance liquid
chromatography.
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EXAMPLE 2
Production of the peptide of the present invention is
further exemplified as follows. The peptide was chemically
synthesized using an LKB Biolynx model 41T0 automated peptide
synthesizer (manufactured by Pharmacies LKB Biotechnology Co.).
390 mg of Fmoc-phenylalanine anhydride were fixed to Ultrosyn A
resin (manufactured by Pharmacies LKB Biotechnology Co.) through
the carboxyl group using dimethylaminopyridine as a catalyst.
Next, the resin was washed with dimethylformamide containing
piperidine and the protecting group of the amine functional -
group of the C-terminal amino acid was removed. 156 mg :of Fmoc-
alanine anhydride of the second amino acid residue from the C-
terminus were then coupled to the unprotected amine group of the
above-mentioned phenylalanine residue. Subsequently the .
successive desired amino acids were fixed in the same manner,
except for cysteine in which an acetamidomethylated Fmoc-amino
acid was used, coupling of a phenylalanine residue which was the
25th from the C-terminal was completed and a peptide chain of
the desired amino acid sequence was formed. Next the protective
groups were removed and the peptide was released with a solvent
(composed of~94% trifluoroacetic acid, 5% phenol and 196
ethandiol), the peptide was purified by high-performance liquid
chromatography, vacuum-dried, and about I50 mg of
acetoamidomethylated peptide were obtained. These i50 mg of
* trade-mark
CA 02102509 2001-02-26
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acetoamidomethylated peptide were dissolved in 10 ml of 90%
acetic acid aqueous solution, 2.5 ml of 1 M hydrochloric acid
were added, the solution was vigorously stirred for 30 minutes,
ml of 1 M sodium thiosulfate aqueous solution were added and
the reaction was stopped, and the solution was concentrated to
about 40 ml with a rotary evaporator. This concentrated
solution was purified using a Sephadex G15 (manufactured by
Pharmacia Co.) column (50 x 500 mm), vacuum-dried, and about TO
mg of the immunostimulatory peptide were obtained.
EXAMPLE 3
20 mg of the immunostimulatory peptide obtained by the
same method as in Example 1 were dissolved in 1000 ml of
purified water, and an immunostimulatory agent was produced.
EXAMPLE 4
50 mg of the immunostimulatory peptide obtained by the
same method as in Example 2 were dissolved in a mixture of 5 g
of methylcellulose and 1000 ml of purified water, and an
immunostimulatory agent was produced.
EXAMPLE 5
100 mg of the immunostimulatory peptide obtained by the
same method as in Example 1 were dissolved in a mixture of 200
ml of ethyl alcohol and 800 ml of purified water, and an
immunostimulatory agent was produced.
EXAMPLE 6
A mouth wash with the following composition was
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--. 21fl~~0~
24
produced. This mouth wash is 50 to 100 times diluted with water
at the time of use.
Ethyl alcohol 20.0 g
Saccharin sodium 3.0 g
Immunostimulatory peptide of Example 1 0.1 g
Purified water
EXAMPLE 2
An eye drop with the following composition was produced.
Boric acid 1.g g
Immunostimulatory peptide of Example l 0.02 g
Methylcellulose 0.5 g
Purified water gq,4 g
EXAMPLE 8
A dermatological spray with the following composition
was produced.
Propylene glycol 0,4 g
Ethyl alcohol 3.5 g
Freon 1l (trademark; manufactured by duPont Co.;
trichlorofluoromethane) 30.0 g
Freon 12 (trademark;; manufactured by duPont co.;
dichlorodifluoromethane) 48.0 g
Diethyl ether 16.0 g
Immunostimulatory peptide of Example 1 0.1 g
~~1.~~~()~
INDUSTRIAL APPLICATION
The peptide is useful as an immunostimulatory agent for
prevention and treatment of bacterial, fungal, and viral
infections in humans and animals.
