JPH07309774A - Antifungal agent - Google Patents

Abstract

PURPOSE:To provide an antifungal agent having a specified amino acid sequence, derived from lactoferrin, excellent in antifungal activity, reduced in side effects, exhibiting heat-resistant properties and water solubility, stable in water, having antibacterial properties and not requiring addition of a preservative. CONSTITUTION:Bovine lactoferrin is dissolved in purified water and pH is adjusted to 2.5 by using 0.1 N hydrochloric acid. Swine pepsin is added thereto and the resultant mixture is hydrolyzed at 37 deg.C for 6 hr. pH us subsequently adjusted to 7.0 by using 0.1 N sodium hydroxide and the reaction solution is heated at 80 deg.C for 10min to deactivate the enzyme. The reaction solution is cooled to the room temperature and centrifuged at 15000rpm for 30min to obtain a transparent supernatant. The resultant supernatant is then fractionated according to the high-performance liquid chromatography method and the active fraction is dried under vacuum to obtain a peptide having, e.g. an amino acid sequence represented by formula I (RO1 is an arbitrary amino acid residue excluding Cys), formula II, etc. One or more kinds of the obtained peptides are used as the active components and prepared, thus affording the objective heat-resistant and water-soluble antifungal agent reduced in side effects, excellent in stability and not requiring addition of a preservative.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates to an antifungal agent. More specifically, the present invention relates to a novel antifungal agent which has lactoferrin-derived peptides having a specific amino acid sequence as an active ingredient and has no side effect.

[0002]

2. Description of the Related Art Antifungal agents containing peptides as active ingredients include ε-poly-lysine (JP-A-5-246883) and peptides derived from oleobasidium (JP-A-5-58).
No. 279384) is known. However, these peptides are distinct peptides that are quite different from the peptides of the invention.

On the other hand, many inventions have been disclosed regarding peptides having an antibacterial action against various microorganisms including not only fungi. For example, a phosphonotripeptide effective against Gram-positive and Gram-negative bacteria (Japanese Patent Laid-Open No. 57-106).
689), phosphonodipeptide derivatives (Japanese Patent Application Laid-Open No. Sho 5)
8-13594), cyclic peptide derivatives (JP-A-58-213744), peptides having antibacterial and anti-viral effects (JP-A-59-51247),
Polypeptides effective for yeast (JP-A-60-130599)
Gazette), glycopeptide derivatives effective against Gram-positive bacteria (JP-A-60-172998, JP-A-61-2516).
99 and JP-A-63-44598), oligopeptides effective against Gram-positive bacteria (JP-A-62-22).
798), peptide antibiotics (JP-A-62-5).
1697 and Japanese Patent Laid-Open No. 63-17897), other antibacterial peptides extracted from blood cells of horseshoe crab from North America (Japanese Patent Laid-Open No. 2-35799), antibacterial peptides isolated from hemolymph of bees (Special Table) Flat 2-500
No. 084), antibacterial peptides isolated from royal jelly (JP-A-2-268198), and the like.

Lactoferrin is used in milk and saliva, tears,
It is an iron-binding protein that is present in body fluids of mammals including humans such as mucous secretions, and is known to show antibacterial action against harmful microorganisms such as Escherichia coli, Candida, Clostridium [Journal of・ Pediatrics (Journal of Pediatrics), Volume 94, Page 1,
1979]. It is also known to have an antibacterial action against staphylococci and enterococci at a concentration of 0.5 to 30 mg / ml [Journal of Dairy Science, Vol. 67, Sixth
06, 1984].

The inventors of the present invention pay attention to the antibacterial property of lactoferrin, and focus on mammalian lactoferrin, apolactoferrin, and / or metal-saturated lactoferrin (hereinafter, these may be referred to as lactoferrins).
It was found that a substance obtained by hydrolyzing lactic acid with an acid or an enzyme has no undesired side effects (for example, antigenicity) and has stronger heat resistance and antibacterial property than undegraded lactoferrins, and already applied for a patent ( JP-A-5-3
20068).

Further, the inventors of the present invention isolated peptides having strong antibacterial activity from lactoferrin degradation products, or synthesized peptides having the same amino acid sequence as those peptides, or derivatives of these peptides, Antibacterial peptide consisting of 20 amino acid residues (JP-A-5-92994), antibacterial peptide consisting of 11 amino acid residues (JP-A-5-78392), consisting of 6 amino acid residues Antibacterial peptide (JP-A-5-148297), antibacterial peptide consisting of 5 amino acid residues (JP-A-5-1498296), antibacterial peptide consisting of 3 to 6 amino acid residues (JP-A-5-29187) Nos. 5 to 148295) have already been applied for patents.

[0007] In addition to physiologically active peptides of milk, in addition to growth hormone, cell differentiation and growth factor, etc., calcium absorption promoting peptide (Food Chemical, Vol. 11, p. 33, 1988), opioid peptide [Hoppe. Hope-Seyler's Zeitschrift furPhy
siologische Chemie), 360, 1211, 1979 and The Journal of Biological Chemis.
try), Volume 254, Page 2446, 1979.
Year], angiotensin converting enzyme inhibitory peptide (Food Chemical, Vol. 11, p. 39, 1988), peptide having gastric acid secretion inhibitory action (JP-A-5-2627).
No. 93) are known.

The inventors of the present invention have also found that the peptide having the same amino acid sequence as the substance obtained by hydrolyzing lactoferrins with an acid or an enzyme or a derivative of these peptides has a brain-protecting action (Japanese Patent Application No. 4-327738). , Lactoferrin hydrolyzate, promoting fibroblast growth by epidermal growth factor (JP-A-6-48955) and nerve growth factor production promoting effect (JP-A-5-23)
557), and already applied for a patent. Also disclosed is a method for separating and purifying lactoferrin from milk by utilizing the property of lactoferrin binding to heparin (JP-A-63-255299).

However, it is not known that these peptides derived from lactoferrin have an antifungal action, and neither is described in the literature.

[0010]

As is clear from the above-mentioned prior art, an antifungal agent with few side effects has been long-awaited, but the excellent substance is still unknown.

The inventors of the present invention have few side effects,
We searched for substances with safe antifungal activity, but we found that peptides obtained by hydrolyzing lactoferrin have this effect, and completed the present invention.

The present invention has been made in view of the above circumstances, and an object thereof is to provide an antifungal agent which has few side effects and is effective in a small amount.

[0013]

Means for Solving the Problems The present invention is provided to solve the above-mentioned problems by providing peptides having an amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 31 and pharmaceutically acceptable peptides thereof. Derivatives, pharmaceutically acceptable salts of these peptides (hereinafter, these may be collectively referred to as peptides), or these 2
An antifungal agent containing a mixture of at least one species as an active ingredient is provided.

The structure and preferred embodiments of the present invention will be described in detail below.

When the peptides, which are the active ingredients of the antifungal agent of the present invention, are produced from lactoferrins, the lactoferrins used as starting materials are commercially available lactoferrin, mammals (eg, human, bovine, sheep, goat, horse). ) Colostrum, transitional milk, normal milk, end-stage milk, etc., or non-fat milk that is a processed product of these milks, whey, etc.
Lactoferrin separated by ion exchange chromatography), apolactoferrin deironed with hydrochloric acid, citric acid, etc., metal saturated or partially saturated lactoferrin obtained by chelating apolactoferrin with a metal such as iron, copper, zinc, manganese, etc. It is also possible to use a product or a preparation produced by a known method.

The peptides used in the present invention are:
A peptide obtained by a separation means from a degradation product of lactoferrin, a peptide having the same amino acid sequence as this peptide, a peptide having a homologous amino acid sequence, a derivative of these peptides, a pharmaceutically acceptable salt of these peptides, or any of these And can be chemically synthesized by a known method. These peptides are
For example, JP-A-5-199294 and JP-A-5-19924.
78392, JP-A-5-148297, JP-A-5-1498296, and JP-A-5-148.
It can be obtained by the method described in each invention of Japanese Patent Publication No. 295.

As the peptide obtained by the above method, a peptide having the following amino acid sequence, its derivative or salt can be exemplified as a desirable embodiment. For example, peptides having the amino acid sequences of SEQ ID NOs: 1, 2 and 27, salts or derivatives thereof (JP-A-5-78392), peptides having the amino acid sequences of SEQ ID NOs: 3, 4, 5 and 6, salts thereof or Derivatives thereof (JP-A-5-
148297), peptides having the amino acid sequences of SEQ ID NOS: 7, 8, 9 and 31, salts or derivatives thereof (JP-A-5-1498296), SEQ ID NO: 1
A peptide having an amino acid sequence of 0 to 21, a salt thereof or a derivative thereof (JP-A-5-148295),
It is a peptide having the amino acid sequences of SEQ ID NOs: 22 to 26, 28, 29 and 30, a salt thereof or a derivative thereof (JP-A-5-92994).

Examples of pharmaceutically acceptable salts of the above peptides include acid addition salts such as hydrochloride, phosphate, sulfate, citrate, lactate and tartrate, and the derivative is a carboxyl group. Examples thereof include amidated or acylated amino group.

The antifungal agent of the present invention can be processed into an ointment, liquid, etc. by a known method.

The antifungal agent of the present invention can be externally administered to an affected area affected by mycosis by an administration method such as spraying, coating, or sticking.

The antifungal agent of the present invention varies depending on age, symptoms, etc., but is at least 0.64 m / cm ² of skin.
It can be administered topically at a rate of g / day.

Next, the present invention will be described in detail with reference to test examples.

Test Example 1 This test was conducted to examine the antifungal effect of peptides. 1) Test animal SPF Hartley guinea pig (female, 330 to 430 g:
(Purchased from Japan SLC, Inc.) were randomly divided into 4 groups (5 animals per group) and used. These guinea pigs were tested in a breeding room at a temperature of 24 ± 2 ° C. and a humidity of 55 ± 7% by freely ingesting solid feed (manufactured by Oriental Yeast Co., Ltd., RC-4) and water.

2) Strains used The inoculum used is Trichophyton mentagrophyte, which is a kind of fungus Trichophyton mentagrophyte.
s) TIMM1189 (obtained from Teikyo University Medical and Fungal Research Center)
It was used.

3) Preparation of inoculum solution 1/10 SDA (bactopeptone 0.2%, glucose 0.1%, KH ₂ PO ₄ 0.1%, MgSO ₄ 0.1%, agar 1.6%)
, PH5.2) After culturing in a slant medium at 27 ° C. for 2 weeks, the slant portion on which the fungus was grown was covered with a sterilized physiological saline containing 0.05% Tween80, and conidia were released by shaking. Sterilize this
The mycelium was removed by filtration with ze. The number of conidia in the obtained bacterial suspension was counted by a hemocytometer using an optical microscope and adjusted to a concentration of 2 × 10 ⁷ conidia / ml, which was used as an inoculum.

4) Agents Used As the ointment of the present invention, an external antifungal agent produced by the same method as in Example 1 (containing 10% of the peptide produced by the same method as Reference Example 1) was used. As a control base, the cream containing no peptide in Example 1 was used. As a control drug, 1% bifonazol (Bifo
nazole) cream (made by Bayer Yakuhin. Mycospo
RECREAM) was used.

5) Test method i) Infection method After cutting the hair on the back of the guinea pig with an electric clipper,
The operation of selecting a total of two sites on the left and right at appropriate intervals, applying a gum tape here, and peeling off vigorously was repeated 3 to 4 times. As a result, hair is removed from the local area,
The upper stratum corneum of the skin was peeled off. The remaining hair was completely removed by using a hair remover to obtain a hair removal site having a diameter of 2 cm. Bacterial inoculation was carried out by rubbing 0.05 ml of the inoculated bacterial solution on each of these hair removal sites.

Ii) Experimental treatment method Treatment was started at the time when redness was clearly observed on all infected sites (5 days after infection treatment), and 0.2 g of the drug was administered per site per day, and a spatula for ointment was applied. It was applied topically once a day and administered daily for 14 days. The conditions and contents of the experiment conducted by this method are as follows.

Twenty guinea pigs that had been treated with infection were used, and the experiment was carried out in four groups of a non-treatment control group, a control base group, the ointment group of the present invention, and a control drug group, with 5 animals in each administration group.

Iii) Culture test method Three days after the final treatment day, all guinea pigs were slaughtered under anesthesia with dry ice, and then the whole skin of each infected site was cut into a circle. Then wipe the skin with a disinfecting alcohol,
After soaking in 1% Osban (0.01% benzalkonium chloride) solution, the disinfectant was washed off with sterile water and the skin was minced into 10 pieces. Cycloheximide each small piece
ide) 500 μg / ml, chloramphenicol
nicol) 100 μg / ml, and sisomicin
PYG agar (Bactopeptone 1.0%, yeast extract 0.5%, glucose 4.0%, agar 1.5%) containing 50 μg / ml was placed on a plate and cultured at 27 ° C. for 10 days. Those in which a colony of Trichophyton mentaglophytes developed around the skin piece was determined to be culture positive.

6) Test Results The results of this test are shown in Table 1. As is apparent from Table 1, in the ointment group of the present invention containing the peptide produced by the same method as in Reference Example 1, 7 sites out of 10 infected sites were negative. On the other hand, the control drug group became negative only in 4 out of 10 infected sites. From this test result, it was confirmed that the peptide has an effect of healing ringworm. In addition, the positive rate per small piece obtained by further dividing the infected site into 10 equal parts revealed that the peptide was more effective than the control drug in negatively treating Trichophyton.

From the results of this test, it was confirmed that the peptide produced by the same method as in Reference Example 1 had an effect of curing mycosis. Also, by using the ointment of the present invention,
No adverse side effects such as skin allergic reaction were observed. Similar tests were conducted on other peptides, but almost the same results were obtained.

[0033]

[Table 1] Test Example 2 This test was conducted to examine the acute toxicity of peptides. 1) Animals used 6-week-old CD (SD) strain rats (purchased from Japan SLC) were used, and males and females were randomly divided into 4 groups (5 animals per group) and used.

2) Test method 1000, 2000 and 4000 mg / kg body weight
SEQ ID NO: 26 produced by the same method as in Reference Example 1
Was dissolved in water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), and 4 ml per 100 g of body weight was orally administered by force once using a needle with a metal ball, and acute toxicity was tested.

3) Test Results The results of this test are shown in Table 2. As is clear from Table 2, no deaths were observed in the groups administered with this peptide at the rates of 1000 mg / kg body weight and 2000 mg / kg body weight. Therefore, the L of this peptide
The D ₅₀ was 2000 mg / kg body weight or more, and the toxicity was found to be extremely low. Similar tests were conducted on other peptides, but almost the same results were obtained.

[0036]

[Table 2] Test Example 3 This test was conducted to investigate the effective amount of peptides. 1) Test Method The peptide dosage was changed by changing the peptide content of the ointment of the present invention (an external antifungal agent produced by the same method as in Example 1) used in Test Example 1 as follows. A test was conducted in the same manner as in Test Example 1 except that the effective dose showing an antifungal effect was determined.

2) Experimental group No treatment; no treatment control Ointment 1 of the present invention; 0.1 produced by the method of Example 1
% Peptide-containing external antifungal agent (the peptide dose is 0.064 mg / cm ² / day according to the present experimental system) Ointment 2 of the present invention; 1.0 produced by the method of Example 1
% Peptide-containing external antifungal agent (the peptide dose is 0.64 mg / cm ² / day according to the present experimental system) Ointment 3 of the present invention; 5.0 produced by the method of Example 1
% Anti-fungal agent containing peptide (According to this experimental system, the dose of peptide is 3.2 mg / cm ² / day.

3) Test Results The results of this test are shown in Table 3. As is clear from Table 3, in the group administered with the peptide produced by the same method as in Reference Example 1 at 0.64 mg / cm ² / day, 2 out of 10 infected sites were negative. In addition, 3.2 mg of the peptide
/ Cm ² / day administration group became negative in 4 out of 10 infected sites. From this test result, it was confirmed that the peptide has an effect of curing ringworm at a dose of 0.64 mg / cm ² / day or more. In addition, the peptide is 0.064 mg /
The group administered with cm ² / day, compared to the untreated control group,
In the positive rate per small piece with the infection site divided into 10 equal parts,
Although it had the effect of making some ringworm fungus negative, it did not cure the entire infected site. Similar tests were conducted on other peptides, but almost the same results were obtained.

[0039]

[Table 3] 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 Corporation) and eluted with 20% acetonitrile containing 0.05% TFA (trifluoroacetic acid) for 10 minutes after injecting the sample at a flow rate of 0.8 ml / min. , Then 30 minutes 0.0
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 vacuum drying was performed 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 vapor phase sequencer (Applied
Edman degradation was performed 25 times using Biosystems) to determine the sequence of 25 amino acid residues. Further, 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 consists of 25 amino acid residues, the 3rd and 20th cysteine residues are disulfide-bonded, and the 3rd cysteine residue leads to N
-It was confirmed that the two amino acid residues on the terminal side have the amino acid sequence set forth in SEQ ID NO: 26 in which 5 amino acids were bonded to the C-terminal side from the 20th cysteine residue, respectively. It was

Reference Example 2 Using an automatic peptide synthesizer (Pharmacia LKB Biotechnology, LKBBiolynx4170), a solid-phase peptide synthesis method [Journal of Chemical Society Parkin I] by Shepard or the like was used. (Jo
urnal of Chemical Society Perkin I), No. 538
Page, 1981], and the peptide 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 name (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. An Fmoc-asparagine anhydride corresponding to the C-terminal asparagine residue was prepared to produce a peptide chain,
Through the carboxyl group, it is immobilized on Ultrosin A resin (Pharmacia LKB Biotechnology) using dimethylaminopyridine as a catalyst. The resin is then washed with dimethylformamide containing piperidine, C-
The protecting group of the amine function of the terminal amino acid is removed. Thereafter, Fmoc-arginine anhydride, which corresponds to the second position from the C-terminal of the amino acid sequence, was coupled to the deprotected amine functional group of arginine fixed to the resin via the C-terminal amino acid residue. In the same manner, glutamine, tryptophan, glutamine, and phenylalanine were sequentially fixed in the same manner. After the coupling of all amino acids is completed and the peptide chain of the desired amino acid sequence is formed, 94% T
The protective group other than acetamidomethyl was removed and the peptide was eliminated with a solvent consisting of FA, 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-mentioned peptide was analyzed by an ordinary method using an amino acid analyzer, and SEQ ID NO: 10 was used.
It was confirmed to have the amino acid sequence described in 1.

[0045]

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 Ten peptides prepared by the same method as in Reference Example 1 were used.
g, 0.1 g of methyl paraoxybenzoate, 0.1 g of propyl paraoxybenzoate, 12 g of propylene glycol, and 27.8 g of purified water were dissolved with stirring while heating. Separately, in advance, 25 g of white petrolatum, 20 g of stearyl alcohol, polyoxyethylene hydrogenated castor oil 6
A solution obtained by stirring and mixing 4 g of 0 and 1 g of glyceryl monostearate with heating was added to the above solution and emulsified using a homomixer to obtain an O / W type cream. This was filled in an aluminum tube in an amount of 10 g by a conventional method to produce an antifungal agent for external use.

Example 2 40 g of white petrolatum, 10 g of cetanol, 5 g of thalassemi wax, 5 g of sorbitan sesquioleate, and 0.5 g of lauromacrogol were stirred and mixed while heating. Separately, 5 g of a peptide prepared in the same manner as in Reference Example 2 and 0.1 g of methyl paraoxybenzoate were prepared in advance.
g, 0.1 g of propyl paraoxybenzoate, and 34.3 g of purified water while stirring and dissolving were added to the above solution, and the mixture was emulsified using a homomixer to obtain a W / O type cream preparation. This was filled in an aluminum tube in an amount of 10 g by a conventional method to produce an antifungal agent for external use.

[0048]

INDUSTRIAL APPLICABILITY As described above in detail, the present invention relates to an antifungal agent containing peptides as an active ingredient, and the effects of the present invention are as follows. (1) There are few side effects. (2) It has heat resistance, is soluble in water, and is stable in an aqueous solution, so that it is stable as a drug. (3) Since the peptide has an antibacterial action, it is not necessary to use a preservative for formulation. (4) It does not show cytotoxicity to animal cells, but only to fungal cells. (5) Since it is a peptide, it is rapidly metabolized in the body.

[0049]

[Sequence list]

SEQ ID NO: 1 Sequence length: 11 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys. Sequence: Lys R01 R01 R01 R01 Gln R01 R01 Met Lys Lys 1 5 10

SEQ ID NO: 2 Sequence length: 11 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys. Sequence: Lys R01 R01 R01 R01 Gln R01 R01 Met Arg Lys 1 5 10

SEQ ID NO: 3 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys. Array: Arg R01 R01 R01 R01 Arg 1 5

SEQ ID NO: 4 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys. Sequence: Lys R01 R01 R01 R01 Arg 1 5

SEQ ID NO: 5 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys. Sequence: Lys R01 R01 R01 R01 Lys 1 5

SEQ ID NO: 6 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys. Sequence: Arg R01 R01 R01 R01 Lys 1 5

SEQ ID NO: 7 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys. Sequence: Arg R01 R01 R01 Arg 1 5

SEQ ID NO: 8 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys. Sequence: Lys R01 R01 R01 Arg 1 5

SEQ ID NO: 9 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys. Sequence: Arg R01 R01 R01 Lys 1 5

SEQ ID NO: 10 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Sequence: Phe Gln Trp Gln Arg Asn 1 5

SEQ ID NO: 11 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. Sequence: Phe Gln Trp Gln Arg 1 5

SEQ ID NO: 12 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Sequence: Gln Trp Gln Arg 1

SEQ ID NO: 13 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. Sequence: Trp Gln Arg 1

SEQ ID NO: 14 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. Sequence: Arg Arg Trp Gln Trp 1 5

SEQ ID NO: 15 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Sequence: Arg Arg Trp Gln 1

SEQ ID NO: 16 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Sequence: Trp Gln Trp Arg 1

SEQ ID NO: 17 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. Sequence: Gln Trp Arg 1

SEQ ID NO: 18 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Sequence: Leu Arg Trp Gln Asn Asp 1 5

SEQ ID NO: 19 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. Sequence: Leu Arg Trp Gln Asn 1 5

SEQ ID NO: 20 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. Sequence: Leu Arg Trp Gln 1

SEQ ID NO: 21 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. Sequence: Arg Trp Gln 1

SEQ ID NO: 22 Sequence length: 20 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the following sequence, Cys 2 and 19 are disulfide-bonded. Sequence: Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro 1 5 10 15 Ser Ile Thr Cys Val 20

SEQ ID NO: 23 Sequence length: 20 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Cys * in the sequence below
Indicates cysteine in which a thiol group is chemically modified to prevent the formation of a disulfide bond. Sequence: Lys Cys * Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro 1 5 10 15 Ser Ile Thr Cys * Val 20

SEQ ID NO: 24 Sequence length: 20 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the following sequence, Cys 2 and 19 are disulfide-bonded. Sequence: Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg Gly Pro 1 5 10 15 Pro Val Ser Cys Ile 20

SEQ ID NO: 25 Sequence length: 20 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. Cys * in the sequence below
Indicates cysteine in which a thiol group is chemically modified to prevent the formation of a disulfide bond. Sequence: Lys Cys * Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg Gly Pro 1 5 10 15 Pro Val Ser Cys * Ile 20

SEQ ID NO: 26 Sequence length: 25 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the sequence below, Cys 3 and 20 are disulfide-bonded. Sequence: Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala 1 5 10 15 Pro Ser Ile Thr Cys Val Arg Arg Ala Phe 20 25

SEQ ID NO: 27 Sequence length: 11 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Sequence: Lys Thr Arg Arg Trp Gln Trp Arg Met Lys Lys 1 5 10

SEQ ID NO: 28 Sequence length: 38 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. 16 in the sequence below
Cys No. 33 and Cys No. 33 have a disulfide bond. Sequence: Lys Asn Val Arg Trp Cys Thr Ile Ser Gln Pro Glu Trp Phe Lys 1 5 10 15 Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro Ser 20 25 30 Ile Thr Cys Val Arg Arg Ala Phe 35

SEQ ID NO: 29 Sequence length: 32 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. 10 in the sequence below
No. 27 Cys and No. 27 Cys are disulfide-bonded. Sequence: Thr Ile Ser Gln Pro Glu Trp Phe Lys Cys Arg Arg Trp Gln Trp 1 5 10 15 Arg Met Lys Lys Leu Gly Ala Pro Ser Ile Thr Cys Val Arg Arg 20 25 30 Ala Phe

SEQ ID NO: 30 Sequence length: 47 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. In the following sequence, in the peptide having a sequence length of 36 and having Cys at positions 9, 26, and 35, the 9th Cys and the 26th Cys form a disulfide bond, and 35 of peptides
No. 10 Cys has a sequence length of 11 and has a disulfide bond with No. 10 Cys of a peptide having Cys at No. 10. Sequence: Val Ser Gln Pro Glu Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn 1 5 10 15 Met Arg Lys Val Arg Gly Pro Pro Val Ser Cys Ile Lys Arg Asp 20 25 30 Ser Pro Ile Gln Cys Ile 35 Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala 1 5 10

SEQ ID NO: 31 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys. Sequence: Lys R01 R01 R01 Lys 1 5

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07K 7/06 ZNA 8318-4H 7/08 8318-4H 14/79 8318-4H // A61K 38 / 16 (72) Inventor Hiroyuki Wakabayashi 5-1-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Milk Industry Co., Ltd.

Claims (1)

[Claims] 1. A peptide having the amino acid sequence of any of SEQ ID NOs: 1 to 31, a pharmaceutically acceptable derivative of these peptides, a pharmaceutically acceptable salt of these peptides, or 2 thereof. An antifungal agent containing a mixture of two or more species as an active ingredient.