JPH08165248A - Endotoxin-derived inflammation inhibitor - Google Patents

Abstract

PURPOSE: To provide a new inflammation inhibitor capable of inhibiting endotoxin-inducible cytokine release, containing, as active ingredient, a lactoferrin-derived specific peptide. CONSTITUTION: This inflammation inhibitor contains, as active ingredient, a peptide <=10000 dalton in molecular weight derived from the N-terminal domain of lactoferrin. The peptide, which presents action to inhibit endotoxin-inducible cytokine release from immunocytes (for example, to inhibit interleukin-6 release from human monocytes due to endotoxin stimulation), has effect at concentrations of as low as 0.5-50ppm. Therefore, this inhibitor is useful for preventing and treating harmful effects due to endotoxin such as acute inflammation and septicemia, mediated by cytokine, due to Gram-negative bacteria. The peptide is obtained by hydrolysis of lactoferrin or by conventional peptide synthetic process, and purified. This inhibitor can be used in the form of oral agent, injection, eye drop or spray, or quasi-drug (e.g. mouthwash), cosmetic, food (e.g. chewing gum), etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an endotoxin-induced inflammation suppressant containing a lactoferrin-derived peptide as an active ingredient. More specifically, the invention relates to the release of endotoxin-induced cytokines from cells of the immune system (eg, interleukin-6 from monocytes.
Release) of the endotoxin, which can prevent the physiological effects of endotoxin such as acute inflammation mediated by cytokines, and is effective for the prevention and treatment of human and animal sepsis caused by Gram-negative bacteria. It relates to a new anti-inflammatory agent.

[0002]

2. Description of the Related Art Endotoxin (lipopolysaccharide), which is a constituent of the cell wall of all Gram-negative bacteria, is one of the major components that interact with the living body in Gram-negative bacterial infections. The human and animal immune systems are very sensitive to endotoxin and many pathological phenomena that are triggered as a biological response to endotoxin, for example, one of the immune system cells, monocytes, is sensitive to endotoxin. Is known to play an important role in mediating the biological reactions of the [advances in immunology
(Advances in Immunology), Volume 53, 267-28
Page 9, 1993].

[0003] Monocytes respond to picomolar concentrations (or higher) of endotoxin by inflammatory mediators [eg interleukin (interleukin-1, interleukin-1,
Interleukin-6, Interleukin-8), TN
F, etc.] is released. These inflammatory mediators play a central role in the regulation of biological functions in the inflammatory response and in the biological defense against infection [Annual Reviews of Biochemistry, No. 59.
Vol. 783-836, 1990].

Among the inflammatory mediators released from monocytes in response to endotoxin, interleukin-6
Influences the proliferation, differentiation and activity of various cells of the immune system along with other cytokines [Advances in Immunology, No. 54
, Pp. 1-78, 1993] also acts as a constituent of a complex network that controls the inflammatory response and the body's defense against bacterial infection.

Interleukin-6 is a component of the endogenous regulatory mechanism that inhibits cytokine-dependent acute inflammation induced by endotoxin. Antibodies to interleukin-6 are known to reduce mortality in mice infected with lethal doses of E. coli and mice given lethal doses of TNF [Journal of Immunology] , Volume 145,
4185-4191, 1990]. This result suggests that suppression of the activity of interleukin-6 can prevent acute inflammation caused by endotoxin.

[0006] Sepsis is a serious and fatal infection complication which is a major cause of death such as irreversible hypotension and multiple organ dysfunction [The Lancet, No. 338.
Vol. 732-736, 1991], endotoxin is widely known as a major mediator of sepsis in Gram-negative bacterial infections. The symptom of sepsis is caused as a biological reaction to endotoxin, and can be reproduced in an animal model injected with purified lipopolysaccharide. It is also known that antibodies against endotoxin can reduce the mortality rate of septic patients due to Gram-negative bacteria [New England Journal of Medicine.
Medicine), vol. 324, pp. 429-435, 1
991], the use of such an antibody is not always an effective means for endotoxin shock, and its prevention and treatment remain an important clinical problem.

On the other hand, lactoferrin has a molecular weight of about 8 present in various body fluids of mammals, milk, saliva and mucous secretions.
It is an iron-binding glycoprotein of 10,000 Daltons, which is released from neutrophils activated in an inflammatory response, but is known to directly bind to endotoxin [Infection and Immunit
y), vol. 62, pages 2628-2632, 1994.
Year]. Mice infected with a lethal dose of Escherichia coli have a reduced mortality rate after intravenous injection of bovine lactoferrin [British Journal of Experimental
Pathology (British Journal of Experimental Patholo
gy), vol. 70, pp. 697-704, 1989.
Year]. In this experimental model of Gram-negative sepsis, the activity of lactoferrin to reduce mortality is believed to be due to its ability to modulate immune cell responses to endotoxin.

Lactoferrin is an interleukin-
1. It is known to inhibit the release of cytokines such as TNF from human monocytes in vivo by endotoxin [Blood, Vol. 80, 235-235.
240 pages, 1992]. Endotoxin injection 2
As a result of intravenous injection of bovine lactoferrin into mice 4 hours before, it was reported that the increase in serum levels of TNF and interleukin-6 induced by endotoxin was inhibited [International Journal of Experimental・ Pasology (International J
ournal of Experimental Pathology), Vol. 74, No. 4
33-439, 1993]. Although this mechanism has not been fully elucidated, it is considered that lactoferrin directly binds to endotoxin to inhibit the systemic reaction. It is also known to use lactoferrin (particularly metal-bound lactoferrin) for the treatment of the toxic effects of endotoxins (Japanese Patent Laid-Open Publication No. 5 (1999) -135242).
No. 501,416).

[0009]

Gram-negative sepsis has become a serious clinical problem due to its high mortality rate due to endotoxin shock, although there are some preventive and therapeutic measures to date. ing. Therefore, there is a long-felt need for effective new means of protecting patients from the severe systemic reactions in Gram-negative sepsis. Moreover, such a situation is the same for other inflammatory diseases caused by the release of endotoxin-induced cytokines from monocytes.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a new anti-inflammatory agent that inhibits the release of endotoxin-inducible cytokines from human monocytes.

[0011]

SUMMARY OF THE INVENTION The inventors of the present invention have
We first discovered the effect of lactoferrin-derived peptides on endotoxin-induced cytokine release from immune cells. Surprisingly, the inventors of the present invention have found that a particular peptide derived from the N-terminal region of lactoferrin [Biochimica et biophysica Actor (Biochim
ica et Biophysica Acta), Volume 1121, Volume 130-
136, 1992] has an excellent activity of blocking the release of endotoxin-induced cytokines from human monocytes (eg, release of interleukins), which causes harmful effects such as endotoxin-induced acute inflammation. They have found that they are useful in protecting biological reactions and completed the present invention. Although such peptides are known to bind to lipopolysaccharides, [Infection and Immunity (I
nfection and Immunity), Volume 61, 719-728
Page, 1992], it has not previously been known to inhibit the release of cytokines by endotoxins.

[0012] In other words, the present invention provides, as a solution to the above problems, an agent for suppressing inflammation caused by endotoxin, which comprises a peptide derived from lactoferrin and having a molecular weight of 10,000 daltons or less as an active ingredient. Also,
In this invention, the inhibition of endotoxin-induced inflammation is at least inhibition of endotoxin-induced interleukin-6 release from monocytes, the peptide is a peptide derived from the N-terminal region of lactoferrin, and It is also a desirable embodiment that this peptide has the amino acid sequence set forth in SEQ ID NO: 1.

The present invention will be described in detail below.
The peptide derived from lactoferrin having a molecular weight of 10,000 daltons or less, which is used for the inflammation suppressant of the present invention, is produced and purified by hydrolysis of lactoferrin or a conventional peptide synthesis method. For example, JP-A-5-9299
The method disclosed in JP-A No. 4 or JP-A-5-238948 can be recommended as a desirable method. Furthermore, since it is known that lactoferrin of various mammals has an amino acid sequence with high homology, it has substantially the amino acid sequence of this peptide, and a slight substitution of amino acid residues that do not lose the endotoxin inhibitory activity, Obviously, other added and / or deleted peptides can also be used in the present invention, and they can also be produced by hydrolysis of lactoferrin in animals such as humans, buffalo, sheep and goats. Specifically, a peptide having the amino acid sequence of SEQ ID NO: 1 and a molecular weight of 3,100 daltons can be exemplified, and the lower limit of the molecular weight is about 50.
Peptides up to 0 Dalton can be shown as suitable examples.

In addition, another peptide having substantially the amino acid sequence of this peptide and having a slight substitution, addition, deletion, and / or a slight chemical modification of an amino acid residue which does not lose the endotoxin-inhibiting activity can be obtained. It is also obvious that it can be produced by a conventional peptide synthesis method. Further, pharmaceutically acceptable salts of the peptides (eg, hydrochloride,
Acid addition salts such as phosphates, sulfates, citrates, lactates, tartrates, etc.) and derivatives (eg, amidation of carboxyl groups, acetylation of amino groups, etc.) are also effective for the anti-inflammatory agent of the present invention. It can be used as an ingredient.

The peptide used for the inflammation suppressor of the present invention has an endotoxin inhibitory activity, and at least an endotoxin-induced interleukin-6 release inhibitory activity from monocytes. The useful activity of this peptide can be easily verified, for example, by the method described later in this specification (see Test Examples 1 to 3). By blocking the release of endotoxin-induced cytokines such as interleukin-6 from cells of the immune system, this peptide reduces the inflammatory response and adverse body reactions by endotoxin, such as during infection of human and animal Gram-negative bacteria. Acute inflammation in can be prevented. Such activity is particularly useful in protecting against and treating septic shock during Gram-negative bacterial infections in humans and animals. Further, as shown in Test Example 4, this peptide has extremely low toxicity and is safe.

As is apparent from Test Examples 1 to 3, the anti-inflammatory agent of the present invention contains at least 0.5 ppm, preferably 5 to 50 pp, of the peptide which is the active ingredient thereof.
It is contained at a concentration of m. The anti-inflammatory agent of the present invention is a known pharmacologically acceptable suitable solvent (for example, water, ethanol, glycerol, propylene glycol, liquid polyethylene glycol, etc.), fragrance, sweetener,
It can also be used in combination with a binder, an isotonic substance, a coating agent, a surfactant, an absorption delaying agent and the like. Furthermore, components effective in suppressing inflammation caused by other endotoxins [eg, corticosteroids (methylprednisolone, dexamethasone, etc.), antibiotics (ampicillin, methicillin, vancomycin, etc.), antibodies (anti-endotoxin antibody, anti-interleukin-6 antibody). , Anti-TNF antibody, etc.)]] can also be added to the inflammation inhibitor of the present invention. Obviously the substances used must, of course, be non-toxic at the concentrations used.

The anti-inflammatory agent of the present invention is applied to humans and animals, for example, to the skin, eyes, ears, nose, anus and vagina as powders, solutions, suspensions, creams, ointments, sprays and parenterally. It can be administered intraperitoneally or intramuscularly as a sterile injection solution. The anti-inflammatory agent of the present invention can also be orally administered, for example, as a powder, solution, capsule, tablet, syrup, tincture, or direct food.

The anti-inflammatory agent of the present invention is, for example, a drug (eye drops, mastitis drug, etc.), quasi drug (mouthwash, etc.), cosmetic (skin lotion, etc.), food (chewing gum, etc.).
Intake, mixing with products that require endotoxin inhibitory activity (surgical clothes, bandages, etc.), spraying, adhesion,
It is used for application, injection, and treatment of any product that requires endotoxin inhibiting activity.

The present invention will be described in more detail with reference to test examples. In the following description, percentages are expressed by weight unless otherwise specified. Test Example 1 This test examines the endotoxin-induced interleukin-6 release-inhibitory activity from human monocytes when a peptide used as an anti-inflammatory agent of the present invention is added before stimulation of cells with lipopolysaccharide. I went for. 1) Sample preparation E. E. coli 0127 purified endotoxin (lipopolysaccharide; manufactured by Sigma Chemical Co.), human lactoferrin (manufactured by Sigma Co.), bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) and a peptide manufactured by the same method as in Reference Example 1 were used as pyrogens. It was dissolved in free water, mixed with endotoxin absorption gel (manufactured by Taiyo Fisheries Co., Katz Clean-D), incubated overnight, and the gel was removed by centrifugation. After this treatment, the amount of endotoxin of these substances was measured by a Limulus assay (manufactured by Chromogenics). Human lactoferrin is 12
~ 36 EU / mg (about 1-3 ng of lipopolysaccharide)
/ Mg), bovine lactoferrin is 72 EU /
mg and peptide were 2.4 EU / mg or less. In addition, 1 EU is the amount of endotoxin having an activity corresponding to 1 unit of the standard substance of the United States Pharmacopeia in the Limulus assay. 2) Test method Human monocyte cell line, THP-1 cell (American
Type Culture Collection No. ATCC-T
IB202) is cultured in RPMI-1640 medium (manufactured by Sigma) supplemented with 10% heat-treated fetal bovine serum and β-mercaptoethanol, and the cells in the logarithmic growth phase are 1500 rp.
m, centrifuged for 10 minutes, and separated. Stimulation with lipopolysaccharide was carried out on a 24-well plate (Nunc) using fresh complete medium (RPMI-1640, 5% heat-treated fetal bovine serum, 1% gentamicin) for 20-24.
For 37 hours at 37 ° C.

In order to adjust the cell density to 1 × 10 ⁶ cells / ml and enhance the sensitivity to endotoxin stimulation,
It was pretreated with 200 U / ml of γ-interferon (units will be described later) 16 hours before the start of the experiment. Human lactoferrin, bovine lactoferrin, or peptide were administered with E. coli at a concentration of 5 μg / ml each. It was added 30 minutes before stimulation with lipopolysaccharide from E. coli 0127, the medium was centrifuged at 400 G, 4 ° C. for 10 minutes, and the resulting supernatant was stored at −20 ° C. until cytokine level assay.

Interleukin-6 bioassay was performed as follows. Using a sub-clone of cell line B13.29 that undergoes interleukin-dependent growth,
Cells are harvested from the tissue culture flask and 500 per well
The cells were seeded on a microtiter plate (Nunc) at a concentration of 0 cells. Samples or interleukin-6 standards diluted 1:50 or 1: 250 were added to the cells and incubated for 68 hours. ³ H 4 hours before harvesting cells
-Thymidine was added and the dose-dependent interleukin-6 release of lipopolysaccharide was quantified using the human interleukin-6 ELISA kit (manufactured by Hysite). The test was repeated at least 5 times for each sample, and the average value and standard deviation were calculated.

It should be noted that γ-interferon 1U was used to measure the Sindbis virus-induced cytopathic effect in FC cells using a reference substance of the World Health Organization as a standard.
% The amount of γ-interferon that protects. 3) Test result The result of this test is shown in FIG. Figure 1
The relationship between the dose of lipopolysaccharide and the interleukin-6 release is shown, and the vertical axis and the horizontal axis show the interleukin-6 concentration and the lipopolysaccharide concentration, respectively. In the figure, ○, ●, ▽, and ▼ represent no addition (control), human lactoferrin, bovine lactoferrin, and peptide, respectively, and bars extending above and below each symbol represent standard deviation.

As is apparent from FIG. 1, lipopolysaccharide-induced interleukin-6 release from THP-1 cells resulted in lipopolysaccharide doses of 10 and 1.
At 00 ng / ml, about 1200 for control respectively
And about 3000 U / ml, compared to about 500 and about 1600 U / ml for human lactoferrin, respectively.
ml and bovine lactoferrin were about 800 and about 500 U / ml, respectively.

On the other hand, the peptides used in the present invention were about 200 and 400 U / ml at the lipopolysaccharide doses of 10 and 100 ng / ml, respectively, and the release of interleukin-6 was increased by the increase of the lipopolysaccharide dose. And the release of interleukin-6 at a dose of 100 ng / ml of lipopolysaccharide was about 1/8 of that of the control, about 1/4 of human lactoferrin and bovine lactoferrin. It was found that the saccharide-induced release of interleukin-6 was significantly blocked. It should be noted that, although the type of peptide was changed and tested, almost the same results were obtained. Test Example 2 This test was carried out to examine the activity of inhibiting endotoxin-induced interleukin-6 release from human monocytes when the peptide used in the present invention was added after stimulation with lipopolysaccharide. 1) Preparation of sample The sample was prepared in the same manner as in Test Example 1. 2) Test method Human lactoferrin, bovine lactoferrin, and the same peptide as in Test Example 1 were used for lipopolysaccharide-stimulated 3
The test was performed in the same manner as in Test Example 1 except that 50 μg / ml was added after 0 minutes. 3) Test results The results of this test are shown in FIG. Figure 2
The relationship between the dose of lipopolysaccharide and the interleukin-6 release is shown, and the vertical axis and the horizontal axis show the interleukin-6 concentration and the lipopolysaccharide concentration, respectively. In the figure, ○, ●, ▽, and ▼ represent no addition (control), human lactoferrin, bovine lactoferrin, and peptide, respectively, and bars extending above and below each symbol represent standard deviation.

As is apparent from FIG. 2, the lipopolysaccharide-induced interleukin-6 release from THP-1 cells resulted in lipopolysaccharide doses of 10 and 1.
Approximately 1800 each for the control at 00 ng / ml
And about 3400 U / ml, compared to about 600 and about 2000 U / ml for human lactoferrin, respectively.
ml and bovine lactoferrin were about 400 and about 1800 U / ml, respectively.

On the other hand, the peptides used in the present invention were both about 600 U / ml at the doses of lipopolysaccharide of 10 and 100 ng / ml, and the release of interleukin-6 was increased by the increase of the dose of lipopolysaccharide. Not observed and interleukin-6 at a dose of 100 ng / ml lipopolysaccharide
Was about 1/6 of that of the control and about 1/3 of that of human lactoferrin and bovine lactoferrin, and it was found that the lipopolysaccharide-induced release of interleukin-6 was significantly blocked. It should be noted that, although the type of peptide was changed and tested, almost the same results were obtained. Test Example 3 This test was conducted to examine the activity of the peptide used in the present invention to prevent endotoxin-induced interleukin-6 release from human monocytes stimulated by bacterial cell wall fragments, bacterial cells and purified lipopolysaccharide. went. 1) Preparation of sample A sample was prepared in the same manner as in Test 1 except for the following. Preparation of Purified Lipopolysaccharide E. coli 018K1 was added to a Nutrient broth agar medium (manufactured by Difco) containing 1% glucose to 37
After culturing at ℃, collecting the cells, washing, and culturing the cells obtained by the hot phenol-water method [Methods in Carbohydrate
Chemistry (Methods in Carbohydrate Chemistry),
Vol. 5, p. 83, 1965], the purified lipopolysaccharide was extracted. Preparation of bacterial cell wall fragments E. E. coli 018K1 bacteria was cultivated in a minimal basic medium,
After centrifugation, the supernatant was filtered through a 0.2 μm Medicup filter (manufactured by Microgon), concentrated by ultrafiltration using an Ultraset Omega 100K membrane filter (manufactured by Filtron), and sterile pyrogen-free. It was dialyzed against water, and the concentrated supernatant was freeze-dried to obtain a bacterial cell wall fragment. Preparation of bacterial cells The bacterial cells (live cells) obtained by the same method as above were washed twice before being used for the test. 2) Test method 0.5 μg / ml 30 minutes before stimulating cells with bacterial cell wall fragments, bacterial cells and purified lipopolysaccharide.
The same method as in Test Example 1 except that (0.5 ppm) peptide was added, and 50 μg / ml (50 ppm) peptide 30 minutes after stimulating the cells with bacterial cell wall fragments, bacterial cells and purified lipopolysaccharide. Was tested in the same manner as in Test Example 2 except that was added.
At the same time, a similar test was conducted without adding the peptide as a control. The concentration of each endotoxin-containing preparation was adjusted to a lipopolysaccharide amount of 2 ng / ml. Further, each sample was tested at least 5 times and the average value thereof was calculated. 3) Test results The results of this test are shown in FIG. FIG.
The relationship with the interleukin-6 release by the addition of the peptide before and after stimulation of bacterial cell wall fragments, bacterial cells and purified lipopolysaccharide is shown, and the vertical axis and the horizontal axis show the interleukin-6 concentration and the test group, respectively. In the figure, the first column, the second column, and the third column of each test group show no addition (control), pre-stimulation addition, and post-stimulation addition, respectively.

As is clear from FIG. 3, in the group stimulated with bacterial cell wall fragments and purified lipopolysaccharide, the release of interleukin-6 was found to be significantly suppressed by the peptide as compared with the control. . In particular, the effect was remarkable when the peptide was added after stimulation. On the other hand, in the group stimulated with bacterial cells, the release of interleukin-6 was increased when the peptide was added before stimulation as compared with the control, but when the peptide was added after stimulation, it was significantly suppressed by the peptide. It was recognized that it was done. It is not clear why the interleukin-6 release was increased when the peptide was added before the stimulation, but it was confirmed that the peptide effectively acts even in the stimulation of live cells closer to the actual infection. .

From the results of this test and Test Example 1, it was found that the effective amount of the peptide was at least 0.5 ppm, preferably 5 to 50 ppm. It should be noted that, although the type of peptide was changed and tested, almost the same results were obtained. Test Example 4 This test was conducted to examine the acute toxicity of the peptide used as the inflammation suppressant of the present invention. 1) Test animals Eight groups (1 group) of 6-week-old CD (SD) rats of both sexes (purchased from Charles River Japan) were randomly selected.
It was divided into 5 groups). 2) Test method The peptide of Reference Example 1 was dissolved in water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), and 1000, 2000 and 400 per 1 kg of body weight.
A single oral gavage was administered using a needle with a metal ball at a rate of 0 mg to test acute toxicity. 3) Test results As a result of this test, no death was observed in any of the patients administered with the peptide. Therefore, the LD ₅₀ of the peptide is 4
It was found to be 000 mg / kg or more. It should be noted that, although the type of peptide was changed and tested, almost the same results were obtained. Reference Example 1 50 mg of commercially available bovine lactoferrin (manufactured by Sigma) was dissolved in 0.9 ml of purified water, pH was adjusted to 2.5 with 0.1 N hydrochloric acid, and then 1 mg of commercially available porcine pepsin (manufactured by Sigma). Was added and hydrolyzed at 37 ° C. for 6 hours. Then, adjust the pH to 7.0 with 0.1 N sodium hydroxide, heat at 80 ° C. for 10 minutes to inactivate the enzyme, cool to room temperature, and centrifuge at 15,000 rpm for 30 minutes,
A clear supernatant was obtained. 100 μl of this supernatant was added to TSK gel O
It was subjected to high performance liquid chromatography using DS-120T (manufactured by Tosoh Corp.), and after injecting the sample at a flow rate of 0.8 ml / min, elution was performed with 20% acetonitrile containing 0.05% TFA (trifluoroacetic acid) for 10 minutes, and then. 0.0 for 30 minutes
Elution was carried out with a gradient of 20 to 60% acetonitrile containing 5% TFA, and the fractions eluted during 24 to 25 minutes were collected and dried under vacuum. This dried product was dissolved in purified water at a concentration of 2% (W / V), and the TSK gel ODS-120T was dissolved again.
(Manufactured by Tosoh Corporation) and subjected to high performance liquid chromatography at a flow rate of 0.8 ml / min for 10 minutes after injection of the sample.
Elute with 24% acetonitrile containing 05% TFA, then elute with a gradient of 24-32% acetonitrile containing 0.05% TFA for 30 minutes, 33.5-35.
Fractions eluting during 5 minutes were collected. The above operation was repeated 25 times and dried in vacuum to obtain about 1.5 mg of the peptide.

The above peptide was hydrolyzed with 6N hydrochloric acid,
The amino acid composition was analyzed by an ordinary method using an amino acid analyzer. Identical sample is a gas phase sequencer (Applied
Edman degradation was performed 25 times using Biosystems) to determine the sequence of 25 amino acid residues. In addition, a disulfide bond analysis method using DTNB [5,5-dithio-bis (2-nitrobenzoic acid)] [Analytical B chemistry (Analytical B
iochemistry), vol. 67, p. 493, 1975]
Confirmed that a disulfide bond was present.

As a result, this peptide consisted of 25 amino acid residues, the 3rd and 20th cysteine residues were disulfide-bonded, and the 3rd cysteine residue was replaced with N.
-Confirmed to have the amino acid sequence of SEQ ID NO: 1 in which 2 amino acid residues at the terminal side and 5 amino acids at the C-terminal side from the 20th cysteine residue are bound respectively. Was done. Reference Example 2 Using a peptide automatic synthesizer (Pharmacia LKB Biotechnology Co., LKBBiolynx4170), a solid-phase peptide synthesis method by Shepard or the like [Journal of Chemical Society Perkin I (Jo
urnal of Chemical Society Perkin I), No. 538
Page, 1981], and the peptide having the amino acid sequence of SEQ ID NO: 1 was synthesized as follows.

An amino acid whose amine functional group is protected by a 9-fluorenylmethoxycarbonyl group [hereinafter sometimes referred to as Fmoc-amino acid or Fmoc-specific amino acid (for example, Fmoc-asparagine)] has N, N- Dicyclohexylcarbodiimide was added to produce the anhydride of the desired amino acid and this Fmoc-amino acid anhydride was used in the synthesis. In order to produce a peptide chain, Fmoc-phenylalanine anhydride corresponding to the C-terminal phenylalanine residue was used as a catalyst for ultrocin A resin (Pharmacia LK) using dimethylaminopyridine as a catalyst through its carboxyl group.
B Biotechnology). The resin was then washed with dimethylformamide containing piperidine,
The protecting group of the amine function of the C-terminal amino acid is removed.
After that, Fmoc corresponding to the second position from the C-terminal of the amino acid sequence.
-Alanine anhydride was coupled via the C-terminal amino acid residue to the deprotected amine functionality of phenylalanine anchored to the resin. Similarly in the following arginine, arginine, valine, cysteine, threonine, isoleucine, serine, proline, alanine, glycine,
Leucine, lysine, lysine, methionine, arginine,
Tryptophan, glutamine, tryptophan, arginine, arginine, cysteine, lysine and phenylalanine were fixed. After the coupling of all the amino acids is completed and the peptide chain of the desired amino acid sequence is formed, the removal of protecting groups other than acetamidomethyl and removal of the peptide with a solvent consisting of 94% TFA, 5% phenol, and 1% ethanediol. The peptide was purified by high performance liquid chromatography, and the solution was concentrated and dried to obtain a peptide powder.

The amino acid composition of the above peptide was analyzed by an ordinary method using an amino acid analyzer, and it was confirmed that it had the amino acid sequence shown in SEQ ID NO: 1.

[0033]

The present invention will be described in more detail and specifically with reference to the following examples, but the present invention is not limited to the following examples. Example 1 20 mg of the peptide obtained by the same method as in Reference Example 10 was used.
It was dissolved in 00 ml of purified water to obtain a solution-like agent for suppressing inflammation caused by endotoxin. Example 2 50 mg of the peptide obtained by the same method as in Reference Example 2 and 5 g of methylcellulose (manufactured by Wako Pure Chemical Industries, Ltd.) were added to 1000 m.
It was dissolved in 1 l of purified water to obtain a solution-like agent for suppressing inflammation caused by endotoxin. Example 3 30 mg of the peptide obtained in the same manner as in Reference Example 1 and 200 ml of ethyl alcohol were dissolved in 800 ml of purified water to obtain a solution-like agent for suppressing inflammation caused by endotoxin. Example 4 An eye drop having the following composition per 100 ml was produced by a conventional method.

Boric acid 1.9 g Methylcellulose (manufactured by Wako Pure Chemical Industries, Ltd.) 0.5 g Peptide obtained by the same method as Reference Example 1.0 mg Purified water 97.6 ml Example 5 100 g For the skin having the following composition The spray was manufactured in a conventional manner.

Propylene glycol (manufactured by Wako Pure Chemical Industries, Ltd.) 0.4 g Ethyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd.) 4.6 g Freon 11 (trademark. DuPont. Trichlorofluoromethane) 30 g Freon 12 (trademark. DuPont). Dichlorodifluoromethane) 48 g Diethyl ether (Wako Pure Chemical Industries, Ltd.) 17 g Peptide obtained by the same method as Reference Example 1 10 mg

[0036]

INDUSTRIAL APPLICABILITY As described in detail above, the present invention provides an inhibitor of inflammation caused by endotoxin, which comprises a peptide derived from lactoferrin and having a molecular weight of 10,000 daltons or less as an active ingredient. The present invention provides the following effects. To be 1) The peptide as an active ingredient of the present invention has an activity of suppressing the release of endotoxin-induced cytokines from cells of the immune system, for example, the release of interleukin-6 stimulated by endotoxin from human monocytes. It is effective in preventing 2) This peptide is effective at low concentrations in the range of 0.5 to 50 ppm. 3) This peptide prevents the harmful body effects of endotoxins such as cytokine-mediated acute inflammation and sepsis in humans and animals during Gram-negative bacterial infection,
It is effective in its treatment.

[Brief description of drawings]

1 shows the relationship between dose of lipopolysaccharide and interleukin-6 release.

FIG. 2 shows the relationship between lipopolysaccharide dose and interleukin-6 release.

FIG. 3 shows the relationship between bacterial cell wall fragments, bacterial cells, and interleukin-6 release by the addition of peptides before and after stimulation of purified lipopolysaccharide.

[Sequence list]

 SEQ ID NO: 1 Sequence length: 25 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro 1 5 10 15 Ser Ile Thr Cys Val Arg Arg Ala Phe 20 25

Claims (4)

[Claims] 1. Lactoferrin-derived molecular weight 1
An agent for suppressing inflammation caused by endotoxin, which comprises a peptide of 10,000 daltons or less as an active ingredient. 2. The inhibitor of inflammation by endotoxin according to claim 1, wherein the inhibition of inflammation by endotoxin is inhibition of endotoxin-induced interleukin-6 release from at least monocytes. 3. The agent for suppressing inflammation by endoxin according to claim 1, wherein the peptide is a peptide obtained from the N-terminal region of lactoferrin. 4. The inhibitor of inflammation by endotoxin according to claim 1, wherein the peptide has the amino acid sequence set forth in SEQ ID NO: 1.