JP2000143696A - Peptide having physiological activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a prophylactic and therapeutic agent for various kinds of diseases having sthenic or inhibiting action of active oxygen production of neutrophil. SOLUTION: This peptide is represented by an amino acid sequence of the formula Asn-Val-His-Ser-Ser-Leu-Arg or Val-His-Ser-Ser-Leu-Arg and useful as a promoter for active oxygen production of neutrophil, an immunopotentiator or an infectious disease-preventing agent. This peptide is further represented by an amino acid sequence of the formula His-Ser-Ser-Leu-Arg and useful as an inhibitor for active oxygen production of neutrophil, a prophylactic and therapeutic agent for inflammatory diseases, rheumatism, septicemia, tumors, disseminated intravascular coagulation, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide having a physiological activity. More specifically, the present invention relates to a novel peptide having a neutrophil active oxygen production promoting or inhibiting action.

[0002]

2. Description of the Related Art Conventionally, egg white has been used for mice, guinea pigs,
Increases macrophage activity in pigs (Japanese Patent Publication 6-993)
No. 13), an effect of enhancing the antibody-producing ability of a vaccine (Japanese Patent Application Laid-Open No. 5-24972), an effect of promoting animal growth (Japanese Patent Application Laid-Open No. 5-260902), and the like. In addition, a peptide which is a degradation product of ovalbumin derived from egg white has an angiotensin converting enzyme inhibitory action (JP-A-5-331190, JP-A-10-36394, Japanese Nippon Kaiho,
63, 867-877, 1992), having an active oxygen production promoting action of neutrophils and macrophages (Japanese Patent Laid-Open No. 9-12470), having an antioxidant action (Journal of the Japanese Society of Agricultural Chemistry, 65,
1635-1641, 1991).

Neutrophils are cells that control cellular immunity together with monocytes, eosinophils, and macrophages, infiltrate inflammation sites, and exert phagocytosis on harmful microorganisms and tumor cells. Its specific bactericidal factors are considered to be active oxygen whose production is increased by stimulation of bacterial infection and the like, and bactericidal peptides (lactoferrin and the like) and hydrolytic enzymes (lysozyme and the like) contained in granule components (such as lysozyme) ( Leukocytes and host defense, Shigeki Mizukami, et al., Kodansha Scientific, 1990., p
211-219). Therefore, by promoting the production of active oxygen by neutrophils, an effect of enhancing host immunity and an effect of protecting the living body from bacterial or fungal infection can be expected.

[0004] On the other hand, when the production of reactive oxygen is excessive, such as when excessive inflammation occurs, the normal tissues of the living body may be damaged and the organ may be damaged (see, ibid., P.
221-225). For example, active oxygen produced by neutrophils attached to vascular endothelium may damage endothelial cells, resulting in thrombus formation, which may cause ischemic disease, shock, disseminated intravascular coagulation (DIC), etc. It is known. In addition, in addition to this, acute respiratory distress syndrome (ARDS) induced by endotoxin, erosions and ulcers of the stomach and duodenal mucosa, inflammatory skin diseases such as Behcet's disease and cutaneous vasculitis, and the development of glomerulonephritis, etc. It has been reported that active oxygen generated from neutrophils is involved (ibid., P225-23
0). Therefore, the effect of preventing, ameliorating, or treating the above-mentioned diseases and excessive inflammation by suppressing the production of neutrophil active oxygen is expected.

[0005]

Japanese Patent Application Laid-Open No. 9-12470 discloses that an octapeptide having an amino acid sequence of Val-Asn-Val-His-Ser-Ser-Leu-Arg is capable of producing active oxygen by neutrophils. It is disclosed that it has a promoting effect. This amino acid sequence is equivalent to the 77th to 84th amino acid sequence of ovalbumin contained in egg white, but which part in this octapeptide shows the above activity,
Not yet reported.

[0006]

Means for Solving the Problems The present inventors have made intensive studies based on these backgrounds, and as a result, produced a peptide in which amino acids were cleaved one by one from the above octapeptide.
Along with finding a peptide having a necessary and sufficient amino acid sequence to show neutrophil active oxygen production promoting action,
The present inventors have found a peptide having an inhibitory action and completed the present invention. That is, the present invention provides (1): Asn-Val-His-Ser-Ser-Le
a peptide represented by the amino acid sequence of u-Arg, (2): Val-His
A peptide represented by the amino acid sequence of -Ser-Ser-Leu-Arg,
(3): a neutrophil active oxygen production promoter containing the peptide of (1) and (2), an immunopotentiator and an infectious disease preventive, (4): His-Se
A peptide represented by the amino acid sequence of r-Ser-Leu-Arg, and an inhibitor of neutrophil active oxygen production containing the peptide of (5) :( 4) and pneumonia, acute respiratory distress syndrome, bronchitis, meningitis , Encephalitis, optic neuritis, conjunctivitis, choroiditis, otitis externa, tonsillitis, dermatitis, arthritis, vasculitis, polymyositis, endocarditis, pleurisy, peritonitis, pancreatitis, glomerulonephritis, esophagitis, gastritis, Ulcerative colitis, prostatitis, metritis; rheumatism, asthma, atopic dermatitis, psoriasis, lupus erythematosus and inflammation associated with infections; sepsis, shock, erosion of gastrointestinal mucosa.
Ulcer, multiple organ failure, disseminated intravascular coagulation
It is a preventive and therapeutic agent for minated intravascular coagulation (DIC) and sarcoidosis.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The peptide according to the present invention can be obtained by decomposing and purifying ovalbumin with an enzyme such as chymotrypsin or pepsin, and by sequentially binding constituent amino acids by a known method. Can also. The present invention is not limited by the method for producing the peptide. The amino acids in the present invention are all L-forms.

[0008] The peptide according to the present invention can be used as an active oxygen production promoter or inhibitor, an immunopotentiator, an infectious disease preventive, an inflammation inhibitor, and also pneumonia, acute respiratory distress syndrome, bronchitis, pancreatitis, meninges. It can be used as an agent for preventing or treating various inflammatory diseases such as inflammation, encephalitis, ulcerative colitis, dermatitis, nephritis, arthritis, vasculitis, endocarditis, pleurisy, peritonitis, conjunctivitis, choroiditis and the like. Further, as an agent for preventing or treating rheumatism, asthma, atopic dermatitis, psoriasis, puerperium, lupus erythematosus, glomerulonephritis and inflammation associated with infectious diseases, sepsis,
It can be used as an agent for preventing or treating shock, ulcer, multiple organ failure, disseminated intravascular blood coagulation and sarcoidosis.

[0009] In the present invention, the term "immune enhancement" means to enhance the immune function of humans or animals, and as a result, the effect of preventing or treating various diseases such as infectious diseases can be expected. Such infections are not particularly limited,
For example, human salmonellosis (Sa1mone11a enteritidis, Sd
ub1in etc.), Vibrio parahaemolyticu
s), pathogenic Escherichia coli, tuberculosis (Myc
bacterium tuberculosis), Shigella dyse
nteriae, S. flexneri etc.), influenza (Human in
fluenzavirus), immunodeficiency syndrome (Human immunodeficien)
cy syndrome virus), rubella (Rubellavirus), epidemic parotitis (Mumps virus), etc., bovine mastitis (Staphylococcus aur
eus, Klebsiella pneumoniae, Streptcoccus agalactia
e, Actinomyces pyogenes etc.), Brucella disease
abortus), Campylobacter fetu
s), anthrax (Bacillus anthracis), cowpox (Cowpox virus),
Akabane virus, transport fever (Pasteurella mult)
ocida, Bovine parainfluenza virus), horse rhinitis (Bor
detella mallei), equine contagious metritis (Taylorella equigen)
italis), Equine infectious anemia vir
us), equine arteritis (Equinearteritis virus), Borna disease (Bor
navirus), and atrophic rhinitis in pigs (Bordetella bronchisepti)
ca), erysipelas erysipelas (Erysipelothrix rhusiopathiae), Glaser disease (Haemophilus parasuis), porcine infectious gastroenteritis (Porci
ne transmissible gastroenteritis virus), Porcine reproductive and respiratory sy
ndrome virus), Aujeszky's disease (Pseudorabies viru)
s), swine epidemic pneumonia (Mycoplasma hyopneumoniae), chicken white leukemia (Salmonella pullorum), infectious coryza (Haem
ophilus paragallinarum), infectious bursal disease
(Infectious bursal disease virus), Newcastle disease virus (Newcastle disease virus), fowlpox (Fowlpox virus),
Leptospira canine, tetanus (Clostr) in dogs such as Marek's disease virus, infectious laryngotracheitis virus, and infectious bronchitis in chickens.
idium tetani), rabies (Rabies virus), distemper
(Canine distemper virus), infectious hepatitis (Canine adenov
irus 1), parvovirus infection (Canine parvovirus)
Cat feline leukemia virus, feline immunodeficiency syndrome (Feline immnodeficiency virus), feline infectious peritonitis virus
Fish streptococcal disease (Enterococcus seriolicida), nodose disease (Pasteurella piscicida), vibrio disease (Vivrio a
nguillarum), Iridovirus infection (Flouder virus),
Infectious haemotopoietic necro
sis virus), Infectious pancreatic
necrosis virus).

When the peptide according to the present invention is used, it may be administered as it is, or it may be made into a solid preparation, a liquid preparation or an external preparation by a known method depending on a commonly used pharmaceutical carrier. They can also be used by mixing them with food, feed, drinking water and the like.

[0011] When preparing an oral solid preparation, an excipient, a binder, a binder, a disintegrant, a lubricant, a colorant,
After adding a flavoring agent, an antioxidant, a solubilizing agent, and the like, tablets, coated tablets, granules, powders, capsules, and the like are produced in a conventional manner.

The excipients include starch, corn starch, dextrin, flour, wheat middling, bran, rice bran, rice bran oil cake, soybean meal, soybean meal, soybean oil cake, kinako, glucose, lactose, sucrose, maltose, vegetable oil, Animal oil, hydrogenated oil, higher saturated fatty acids, fatty acids, yeast, mannitol, sorbitol, crystalline cellulose, silicon dioxide, anhydrous silicon, calcium silicate, silicic acid, calcium monohydrogen phosphate, dibasic calcium phosphate, tricalcium phosphate, calcium phosphate, phosphoric acid Calcium dihydrogen or the like is used.

Examples of the binder include polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, sodium alginate, sodium caseinate, sodium carboxymethyl cellulose, propylene glycol, Sodium polyacrylate, block polymer, meglumine and the like are used.

As disintegrants, starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate, calcium citrate, dextrin, pectin,
Carboxymethylcellulose and calcium can be used.

As the lubricant, magnesium stearate, talc, polyethylene glycol, silica, hydrogenated vegetable oil and the like can be used. The coloring agent and flavoring agent are not particularly limited as long as they are permitted to be added to pharmaceuticals and feeds. As the flavoring agent, cocoa powder, peppermint brain, aromasan, peppermint oil, dragon brain, cinnamon powder and the like are used. As the antioxidant, any substance which is permitted to be added to pharmaceuticals and feeds, such as ascorbic acid, α-tocopherol, ethoxyquin, dibutylhydroxytoluene and butylhydroxyanisole, may be used. Tablets and granules may be coated as necessary.

In preparing an injection preparation, a pH adjusting agent, a buffering agent, a suspending agent, a solubilizing agent, a stabilizing agent, an isotonic agent, an antioxidant, a preservative, etc., if necessary. And then can be produced by a conventional method. At this time, if necessary, a freeze-dried agent can be used. This injection is subcutaneous,
It can be administered intradermally, intraperitoneally, intramuscularly, and the like.

Examples of the suspending agent include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, tragacanth powder, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate and the like. As the dissolution aid, polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, and the like are used. As a preservative, methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid and the like are used.

When the external preparation is produced, the method is not particularly limited, and it can be produced by a known method. As a base material used in formulating, various raw materials usually used for pharmaceuticals, quasi-drugs, cosmetics, and the like can be used.

Examples of base materials include animal and vegetable oils, mineral oils, ester oils, waxes, higher alcohols, fatty acids, silicone oils, surfactants, phospholipids, alcohols, polyhydric alcohols, and water-soluble Raw materials such as polymers, clay minerals, and purified water are mentioned.
A pH adjuster, an antioxidant, a chelating agent, a preservative / antifungal agent, a coloring agent, a fragrance, and the like can be added, but are not limited thereto. Further, if necessary, components having a differentiation-inducing action, components such as a blood flow promoter, a bactericide, an anti-inflammatory agent, a cell activator, vitamins, amino acids, a humectant, and a keratolytic agent may be added. Note that the amount of the base material added is an amount that usually results in a concentration set in the production of an external preparation.

The method of administering the peptide according to the present invention may be appropriately selected depending on the purpose of administration, and is not particularly limited.
For example, oral, intramuscular, intrathecal, intradermal, subcutaneous, transdermal,
Instillation, intraperitoneal administration and the like can be mentioned, but oral administration is preferred. The dosage varies depending on the purpose of administration, but when administered orally, 1 to 1000 mg / kg, preferably 10 to 500 m
g / kg, more preferably 100-250 mg / kg. Also,
When administered by injection, 0.01 to 100 mg / kg, preferably 0.1 to 100 mg / kg.
It is 1 to 10 mg / kg.

[0021]

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. Example 1 Preparation of Ovalbumin Fragments Six types of ovalbumin fragments shown below were
It was prepared using an automatic peptide synthesizer (PSSM-8) manufactured by Shimadzu Corporation. 1) Val-Asn-Val-His-Ser-Ser-Leu-Arg (hereinafter, octapeptide Arg) 2) Asn-Val-His-Ser-Ser-Leu-Arg (hereinafter, heptapeptide)
Arg) 3) Val-His-Ser-Ser-Leu-Arg (hereinafter, hexapeptide Arg) 4) His-Ser-Ser-Leu-Arg (hereinafter, pentapeptide Arg) 5) Val-Asn-Val-His- Ser-Ser-Leu (hereinafter heptapeptide)
Leu) 6) Val-Asn-Val-His-Ser-Ser (hereinafter, hexapeptide Ser) These peptides are Resource RPC (trade name, Pharmacia
FPLC (Fast protein liquid using Biotech)
It was purified by chromatography.

Example 2 Neutrophils Promote Active Oxygen Production
(1) Octapeptide Arg, heptapeptide Arg, hexapeptide Arg and pentapeptide prepared by the method of Example 1
Regarding Arg, the action of neutrophils to promote the production of active oxygen was examined. First, rat peritoneal exudate neutrophils were collected according to the method of Kohn et al. (Cohn, ZA and Morse, SI, J.
Exp. Med., 110, 419-429, 1959). Rats were injected intraperitoneally with phosphate buffered saline containing 0.2% oyster glycogen at 1/20 of body weight. Sixteen hours later, 50 ml of ice-cold phosphate buffered saline was injected into the peritoneal cavity, and ascites was collected. This operation was performed twice, and the collected ascites was centrifuged at 150 × g for 5 minutes to collect peritoneal exudate neutrophils. When erythrocytes were mixed in the cells, hemolysis was performed by adding a 0.2% sodium chloride solution. Each of the above peptides was added thereto, and the mixture was incubated for 6 minutes. Then, phorbol ester (Phorbol 12-Myristate 13-Acetate, below
PMA) or N-formyl-methionyl-leucyl-phenylalanine (hereinafter fMLP) was added, and the amount of active oxygen produced from neutrophils was measured by a chemiluminescence method.

As a result, the amount of active oxygen produced per minute by neutrophils stimulated with PMA gradually increased immediately after the stimulation, and reached a maximum after about 3 minutes (FIG. 1). The amount of active oxygen production at the peak was about twice as high for the octapeptide Arg (A), heptapeptide Arg (B), and hexapeptide Arg (C) as compared to the peptide-free control (E). . On the other hand, the pentapeptide Arg (D) decreased to about 1/2 of the control without peptide. Even when stimulated with fMLP, the amount of ROS produced per minute by neutrophils rapidly increased after stimulation (FIG. 2). The amount of active oxygen production at the peak was about 10% lower for the octapeptide Arg (A) than for the control (E) containing no peptide.
It increased about 2.3 times, about 2 times for heptapeptide Arg (B) and about 2 times for hexapeptide Arg (C). In contrast, the pentapeptide Arg (D) was reduced to about 2/3. PMA stimulation and fMLA
In stimulation, although there are some differences in the magnification of the amount of active oxygen produced by the addition of peptides, octapeptide Arg, heptapeptide Arg, and hexapeptide Arg all have a priming effect that enhances the neutrophil's ability to produce active oxygen. It was suggested to have. On the other hand, the pentapeptide Arg had an effect of inhibiting neutrophil production of active oxygen.

Example 3 Neutrophils Promote Active Oxygen Production
(2) The effect of octapeptide Arg, heptapeptide Leu and hexapeptide Ser produced by the method of Example 1 on neutrophil active oxygen production was examined. The method for measuring the amount of active oxygen produced from neutrophils was the same as in Example 2. As a result, in the PMA stimulation, the amount of active oxygen produced when the heptapeptide Leu (F) was added was almost the same as that in the control without peptide added (H), and no priming effect was observed. In addition, hexapeptide Ser (G) was about 1.2 when no peptide was added.
Although active oxygen was produced twice as much, it did not have the same priming activity as octapeptide Arg. In fMLP stimulation,
Both the hexapeptide Ser (G) and the heptapeptide Leu (F) exhibited the same active oxygen production behavior as the control without the peptide, and no production promoting effect was observed (FIG. 4).

[0025]

The heptapeptide of the present invention (Asn-Val-H
is-Ser-Ser-Leu-Arg) and hexapeptide (Val-His-Se
r-Ser-Leu-Arg) has an action of promoting the production of neutrophils' active oxygen, and is useful as a immunity enhancer for living organisms and a protective agent for infection. The pentapeptide (His-Ser-Ser-Leu-Ar
g) has an action of inhibiting neutrophil production of active oxygen, and can suppress inflammation associated with inflammatory diseases, autoimmune diseases, infectious diseases and the like.

[0026]

[Brief description of the drawings]

FIG. 1 is a graph showing the amount of active oxygen produced during PMA stimulation.

FIG. 2 is a graph showing the amount of active oxygen produced during fMLP stimulation.

FIG. 3 is a graph showing the amount of active oxygen produced during PMA stimulation.

FIG. 4 is a graph showing the amount of active oxygen produced during fMLP stimulation.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) A61P 9/00 A61P 9/14 9/14 11/00 11/00 11/06 11/06 13/12 13 / 12 17/00 17/00 17/06 17/06 19/02 19/02 25/00 25/00 29/00 29/00 101 101 31/00 31/00 37/04 37/04 43/00 105 43/00 105 A61K 37/02 (72) Inventor Seiichi Araki 1-35 Eikunidai, Tsuchiura City, Ibaraki Prefecture (72) Inventor Mitsunobu Matsuura 2-10-1-501 Miyazaki, Miyamae-ku, Kawasaki City, Kanagawa Prefecture (72) Inventor Tohru Suzuki 3-3-7 Shimoyoya, Hoya-shi, Tokyo

Claims (10)

[Claims] 1. Asn-Val-His-Ser-Ser
-A peptide represented by the amino acid sequence of Leu-Arg. 2. Val-His-Ser-Ser-Leu
A peptide represented by the amino acid sequence of Arg; 3. The peptide according to claim 1, which has an activity of promoting neutrophil production of active oxygen. 4. An agent for promoting neutrophil active oxygen production comprising the peptide according to claim 1 or 2. (5) An immunopotentiator comprising the peptide according to (1) or (2). [6] An agent for preventing infectious diseases comprising the peptide according to [1] or [2]. 7. His-Ser-Ser-Leu-Arg
A peptide represented by the amino acid sequence of 8. The peptide according to claim 7, which has a neutrophil active oxygen production inhibitory effect. 9. A neutrophil active oxygen production inhibitor comprising the peptide according to claim 7. 10. Pneumonia, acute respiratory distress syndrome, bronchitis, pancreatitis, meningitis, encephalitis, ulcerative colitis, dermatitis, nephritis, arthritis, vasculitis, endocarditis containing the peptide according to claim 7. , Pleurisy, peritonitis, conjunctivitis, choroiditis; rheumatism, asthma, atopic dermatitis, psoriasis, inflammation associated with lupus erythematosus, glomerulonephritis and infections; sepsis, shock, ulcers, multiple organ failure, disseminated intravascular blood An agent for preventing and treating coagulation syndrome and sarcoidosis.