CA2102509C - Immunostimulatory agent - Google Patents

Abstract

There is disclosed an immunostimulatory agent comprising a peptide derived from lactoferrin having activity to modulate the release of inflammatory mediators from cells of the immune system. The peptide promotes the release of leukotriene B4 from polymorphonuclear neutrophils induced by activators such as the calcium ionophor A23187. It also promotes the release of histamine from mast cells induced by activators such as .alpha.-toxin-producing Staphylococcus aureus cells or the calcium ionophor A23187. The peptide is effective at low concentrations within the range of 1 to 100 ppm. By promoting the release of such inflammatory mediators the peptide can potentiate the cellular immune response and stimulate the host defense against infectious disease. This newly discovered immunostimulatory agent is useful as an active component of pharmaceuticals, hygiene products, clinical foods, etc., for prevention and treatment of bacterial, fungal, and viral infections in humans and animals.

Description

2.~0~.~;~3 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 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 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 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~~~~

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 T
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.
* trade-mark ~\
2~~2~4) 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 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.

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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 IZ
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 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 r.

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.
* trade-mark 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 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~) 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 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.

(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 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 * trade-mark ---.
~102~~~

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.

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~~~

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.

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 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.

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.

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.

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.

A mouth wash with the following composition was trade-mark --. 21fl~~0~

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 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 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.

Claims (13)

WHAT IS CLAIMED IS:

1. A medicament for promoting the release of leukotriene B4 from polymorphonuclear neutrophils or histamine from mast cells in animals or humans comprising effective amounts of a substantially purified and isolated peptide having the amino acid 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 to accomplish said release of leukotriene B4 and histamine, or derivatives of said peptide having minor amino acid substitutions which do not abolish the capacity of the peptide to release leukotriene B4 and histamine, and a pharmacologically acceptable carrier therefor.

2. A medicament according to claim 1 wherein the peptide or derivative thereof is present in the medicament in a concentration of at least 1 ppm by weight.

3. A medicament according to claim 2 wherein the peptide or derivative thereof is present in a concentration of 10 to 100 ppm by weight.

4. The use of effective amounts of a substantially purified and isolated peptide having the amino acid 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 or derivatives of said peptide having minor amino acid substitutions which do not abolish the capacity of the peptide to release leukotriene B4 and histamine, for promoting the release of leukotriene B4 from polymorphonuclear neutrophils or histamine from mast cells.

5. A use according to claim 4 wherein the substantially purified and isolated peptide or derivative thereof is an active ingredient in a medicament in combination with a pharmaceutically acceptable carrier.

6. A use according to claim 5 wherein the substantially purified and isolated peptide or derivative thereof is in a concentration of at least 1 ppm by weight, based on the weight of the medicament.

7. A use according to claim 6 wherein the substantially purified and isolated peptide or derivative thereof is in a concentration of 10 to 100 ppm by weight.

8. An immunostimulatory agent comprising a peptide derived from lactoferrin, said peptide having the amino acid 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, and said peptide promoting the release of leukotriene B4 from polymorphonuclear neutrophils and promoting the release of histamine from mast cells.

9. An immunostimulatory agent according to claim 8, wherein the peptide is present at a concentration of at least 1 ppm (by weight).

10. An immunostimulatory composition containing the peptide referred to in any one of claims 8 and 9 as an active ingredient at a concentration of at least 1 ppm (by weight) and a pharmaceutically acceptable carrier.

11. The use of a substantially purified and isolated peptide from lactofernn having the amino acid 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 or a derivative thereof for the manufacture of a medicament for promoting release of leukotriene B4 from polymorphonuclear neutrophils or histamine from mast cells.

12. A use according to claim 11, wherein said peptide or derivative thereof is contained in said medicament at a concentration of at least 1 ppm (by weight) as an active ingredient.

13. A use according to claim 12, wherein said peptide or derivative thereof is contained in said medicament at a concentration of 10 to 100 ppm (by weight) as an active ingredient.