JPH06172200A - Brain-protecting agent - Google Patents

Abstract

PURPOSE:To provide the subject medicine containing a substance such as a lactoferrin- hydrolyzed peptide having a specific amino acid sequence as an active ingredient, having a brain-protecting activity, useful for preventing ischemic cerebral diseases, etc., reduced in adverse reactions and good in its heat resistance and antigerminal property. CONSTITUTION:The brain-protecting agent comprising a peptide of the formula (Xaa is the residue of an arbitrary amino acid excluding Cys), etc., its derivative and salt, etc., or their mixture comprising two or more of the compounds is obtained by dissolving bovine lactoferrin in purified water, adjusting the pH of the solution to 2.5 with a 0.1N hydrochloric acid, hydrolyzing the bovine lactoferrin in the solution by the addition of swine pepsin at 37K!n for 6 hr, adjusting the hydrolyzed solution to a pH of 7.0 with a 0.1N sodium hydroxide aqueous solution, heating the solution at 80 deg.C for 10min for the inactivation of the enzyme, cooling the product, centrifuging the cooled solution, collecting the transparent supernatant, subjecting the supernatant to a high performance liquid chromatography using a 20% acetonitrile containing O.05% trifluoroacetic acid and subsequently using a 20-60% acetonitrile gradient containing 0.05% trifluoroacetic acid, collecting the eluted fractions and subsequently vacuum-drying the collected products.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a brain protective agent. More specifically, the present invention relates to a novel cerebral protective agent having no side effect which is effective for prevention of ischemic brain disease and the like.

[0002]

2. Description of the Related Art Heretofore, a search for substances having a brain protecting effect has been extensively carried out using experimental animals, and various effective substances for not only partial ischemia but also for complete ischemia due to decapitation and the like have been investigated. The substance is known. For example, it has been reported that glucose also has an effect [Pediatric Research.
(Pediatric Research), Vol. 8, p. 238, 19
1974]. However, most of the substances actually used for the prevention of ischemic brain diseases among those substances having a brain protective action are Y-9179 [Archives International Pharmacodynamics].
ternational Pharmacodynamie), Volume 233, Volume 136
Page, 1978], MCI-2016 (Japanese Journal of Pharmacology, Vol. 81, 421, 1983), Sloctide (Japanese Journal of Pharmacology, Vol. 82, page 37).
1983), Furnarizine [The Japanese Journal of Pharmacology
l of Pharmacology), Volume 38, Page 1, 1985
, Etc., and the side effects were a problem.

On the other hand, lactoferrin is
It is a natural iron-binding protein contained in tears, saliva, peripheral blood, milk, etc., and is known to show an antibacterial action against harmful microorganisms such as Escherichia coli, Candida and Clostridium [Journal] Of Pediatrics (Journa
l of Pediatrics), 94, 1 page: 1979
Year]. 0.5 against Staphylococcus and Enterococcus
It is known to have an antibacterial action at a concentration of -30 mg / ml [Journal of Dairy Science, Vol. 67, p. 606:
1984].

The applicant of the present application pays attention to the antibacterial activity of lactoferrin and focuses on mammalian lactoferrin, apolactoferrin, and / or metal-saturated lactoferrin (hereinafter sometimes referred to as lactoferrins) as an acid or an enzyme. It was found that the hydrolyzed hydrolyzate had no undesired side effects (eg, antigenicity, etc.) and had stronger heat resistance and antibacterial properties than undegraded lactoferrins, and a patent application has already been filed (patent pending). Japanese Patent Application No. 3-171736).

Further, the applicant of the present application isolated peptides having a strong antibacterial activity from decomposed products of lactoferrins, or synthesized peptides or peptide derivatives having the same amino acid sequence as those peptides to obtain lactoferrin-related antibacterial properties. A peptide has 2 amino acid residues
Antibacterial peptide having 0 residue (Japanese Patent Application No. 3-186260)
No.), an antimicrobial peptide having 11 amino acid residues (Japanese Patent Application No. 3-48196), an antimicrobial peptide having six amino acid residues (Japanese Patent Application No. 3-94492), amino acid residues An antimicrobial peptide having 5 residues (Japanese Patent Application No. 3-9
No. 4493), an antibacterial peptide having 3 to 6 amino acid residues (Japanese Patent Application No. 3-94494) was invented and a patent application has already been filed.

However, it is not known that peptides obtained by hydrolyzing lactoferrins have a protective effect on the brain, and neither is described in the literature.

[0007]

DISCLOSURE OF THE INVENTION As is apparent from the prior art relating to the above-mentioned brain-protective substances, despite the long-awaited need for a brain-protective agent with few side effects effective for the prevention of ischemic brain disease and the like, the present situation However, no substance that can be used as an active ingredient of such a drug has been found.

The present invention has been made in view of the above-mentioned circumstances, and as an alternative to the conventional brain protectant containing an organic compound as an active ingredient, there are few side effects and excellent effects can be obtained in a small amount. The aim is to provide new brain protectants.

[0009]

[Means for Solving the Problems] Generally, the test method using the opening reaction after complete cerebral ischemia due to decapitation as an index is widely used in the drug efficacy test of a brain protective agent [Pediatric Research (Pediatric Research) , Vol. 8, p. 238, 1974, Japanese Journal of Pharmacology, Vol. 81, Vol. 42.
1 page, 1983, Japanese Pharmacology Journal, Vol. 82, page 37, 1983, and The Japanese Journal of Pharmacology.
al of Pharmacology), Volume 38, Page 1, 198.
5 years], the inventors of the present invention, using this method,
As a result of screening a safe brain protective substance with few side effects, it was found that the above-mentioned lactoferrin-related antibacterial peptide obtained by hydrolyzing lactoferrin has a brain protective action, and completed the present invention.

That is, the present invention, as a solution to the above problems, is selected from the group consisting of the peptides listed in the sequence listing, pharmaceutically acceptable derivatives thereof, and pharmaceutically acceptable salts thereof. The present invention provides a brain protectant containing a substance or a mixture of two or more thereof as an active ingredient.
Next, the present invention will be described in detail.

Lactoferrins which may be used as a starting material in the present invention include commercially available lactoferrin, colostrum of mammals (eg, humans, cattle, sheep, goats, horses, etc.),
Lactoferrin separated from transitional milk, normal milk, end-stage milk, etc., or skim milk, whey, etc., which is a processed product of these milk, by a conventional method (for example, ion exchange chromatography), and those are removed with hydrochloric acid, citric acid, etc. Iron-containing apolactoferrin, metal-saturated or partially-saturated lactoferrin obtained by chelating apolactoferrin with a metal such as iron, copper, zinc, and manganese, and a commercially available product or a preparation produced by a known method can be used. .

The peptide used in the present invention is a peptide obtained by a separation means from a lactoferrin degradation product, a peptide having the same or a homologous amino acid sequence as the peptide obtained by a separation means from a lactoferrin degradation product, and these peptides. A compound selected from the group consisting of a derivative thereof, a pharmaceutically acceptable salt of these peptides and any mixture thereof. These peptides are disclosed, for example, in Japanese Patent Application No. 3-186260,
It can be obtained by the methods described in the inventions of Japanese Patent Application No. 3-48196, Japanese Patent Application No. 3-94492, Japanese Patent Application No. 3-944493, and Japanese Patent Application No. 3-94494.

Specifically, lactoferrins are hydrolyzed with an acid or an enzyme, and a fraction containing the desired peptide is obtained from the resulting peptide mixture by a separation means such as liquid chromatography, or as described above. The amino acid sequence of the obtained peptide is determined by a known method (for example, a method using a gas phase sequencer), and peptides having these amino acid sequences are known by a known method (for example, an automatic peptide synthesizer is used). Method) to obtain the desired peptide. The fraction containing the peptide separated from the degradation product of lactoferrin, or the peptide obtained by organic synthesis can be used as a solution as it is, in a concentrated liquid form, or in a powder form after concentration and drying.

The peptide obtained by such a method is a peptide having the following amino acid sequence or a derivative thereof: Peptides having the amino acid sequences of SEQ ID NOs: 1, 2, and 27, salts thereof or derivatives thereof (Japanese Patent Application No. Hei 10 (1999) -135242). 3-48196 invention); SEQ ID NOs: 3, 4, 5 and 6
Or a derivative thereof having the amino acid sequence of SEQ ID NO: 3 (the invention of Japanese Patent Application No. 3-94492); a peptide having the amino acid sequence of SEQ ID NOS: 7, 8, 9 and 31 or a salt thereof or a derivative thereof (the invention of Japanese Patent Application No. 3-94493). ); SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, 17,
Peptides having amino acid sequences of 18, 19, 20 and 21, salts thereof or derivatives thereof (Japanese Patent Application No. 3-944)
No. 94 invention); and SEQ ID NOS: 22, 23, 24, 2
Peptides having amino acid sequences of 5, 26, 28, 29 and 30; salts thereof or derivatives thereof (Japanese Patent Application No. 3-1
86260 invention).

Examples of the pharmaceutically acceptable salts of the above peptides include addition salts such as hydrochloride, phosphate, sulfate, citrate, lactate, tartrate, and the like. Examples include amidated and acylated derivatives of carboxyl groups. The brain protectant of the present invention contains a compound selected from the group consisting of the above-mentioned peptides, pharmacologically acceptable derivatives thereof, pharmaceutically acceptable salts thereof and any mixture thereof as an active ingredient. The effective dose of this active ingredient can be at least 10 mg for parenteral administration and at least 100 mg for oral administration. In addition, tablets, capsules, suppositories, troches, and
It can also be processed into a syrup, granule, powder, injection or the like, and can be made into a drug with a coating if necessary, for example, an enteric coating agent.

Next, the effects of the brain protectant of the present invention will be described in detail with reference to test examples. Test Example 1 This test was conducted to examine the brain protective effect of peptides. 1) Test animal Male ddY mice weighing 29 to 33 g were randomly
The animals were divided into 6 groups (7 to 8 animals per group). 2) Test method The peptide of SEQ ID NO: 26 produced by the same method as in Reference Example 1 was dissolved in physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) and administered at the doses and administration routes shown in Table 1 for 10 minutes and 30 minutes. After 90 minutes, the animals were decapitated, the duration of the opening reaction was measured by the method described in the above literature, and the average value and standard error of each group were calculated to evaluate the brain protective effect of the peptide. The statistical significance test was conducted using Mann-Whitne (Mann-Whitne
The U test of y) was applied. 3) Test results The results of this test are shown in Table 1. As is clear from Table 1, the duration of the opening reaction was prolonged depending on the dose of the peptide. In addition, the degree of extension is 10 mg / kg or more for intraperitoneal administration and 5 for subcutaneous administration.
It was statistically significant at a dose of 0 mg / kg.

Although the type of peptide was changed and tested, almost the same results were obtained.

[0018]

[Table 1]

Test Example 2 This test was conducted to examine the acute toxicity of peptides. 1) Test animal Both sexes of a 6-week-old CD (SD) strain rat were randomly divided into 4 groups (5 animals per group). 2) Test method Peptides produced by the same method as in Reference Example 1 were weighed 1 k
It was dissolved in water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) at a rate of 1000, 2000 and 4000 mg per gram, and a single forced oral administration was performed using a needle with a metal ball at a rate of 4 ml per 100 g of body weight to test acute toxicity. 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 the peptides at the rates of 1000 mg / kg and 2000 mg / kg. Therefore, the LD50 of this peptide was 2000 mg / kg or more. It should be noted that, although the type of peptide was changed and tested, almost the same results were obtained.

[0020]

[Table 2]

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 0.1N hydrochloric acid, and then commercially available porcine pepsin (manufactured by Sigma). ) 1 mg 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 antibacterial 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. In addition, a disulfide bond analysis method using DTNB (5,5-dithio-bis (2-nitrobenzoic acid)) [Analytical B chemistry (Analytical B
iochemistry), 67, 493, 1975.
Year] confirmed the existence of disulfide bonds.

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 to have the amino acid sequence of SEQ ID NO: 26 in which two amino acid residues were bound to the -terminal side and 5 amino acids were bound to the C-terminal side from the 20th cysteine residue, respectively. . Reference Example 2 Using an automatic peptide synthesizer (Pharmacia LKB Biotechnology Co., Ltd., LKBBiolynx4170), Shepard et al. [Journal of Chemical Society Parkin I (Journal of Chemical Societ
y Perkin I), p. 538, 1981], and antibacterial peptides were synthesized as follows.

N, N-dicyclohexylcarbodiimide was added to an amino acid whose amine functional group was protected by a 9-fluorenylmethoxycarbonyl group to produce the desired anhydride of the amino acid, and this Fmoc-amino acid anhydride was synthesized. Using. In order to produce a peptide chain, Fmoc-asparagine anhydride corresponding to the C-terminal asparagine residue is immobilized on Ultrosin A resin (Pharmacia LKB Biotechnology) via its carboxyl group using dimethylaminopyridine as a catalyst. To do. The resin is then washed with dimethylformamide containing piperidine to remove the amine functional group protecting group of the C-terminal amino acid. 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. 94% TF after coupling of all amino acids and formation of a peptide chain of the desired amino acid sequence
A, 5% phenol, and 1% ethanediol were used to remove protective groups other than acetamidomethyl and peptides to remove the peptide.
The peptide was purified by 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 of Reference Example 3 50 mg of commercially available human 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. 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 performed with a gradient of 20-60% acetonitrile containing 5% TFA, and the fractions eluted during 23-24 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.
Fractions were eluted with 24% acetonitrile containing 05% TFA, followed by a 30-minute gradient of 24-32% acetonitrile containing 0.05% TFA, and fractions eluting between 31-32 minutes. Repeat the above operation 25 times,
It was dried under vacuum to obtain about 2.0 mg of antibacterial 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
Biosystems) was used for Edman degradation in the same manner as in Reference Example 1 to determine the amino acid sequence. As a result, this peptide was found to contain two peptides linked by a disulfide bond. Then, the disulfide bond was reduced by a conventional method, the two peptides formed were separated by high performance liquid chromatography, and the amino acid sequences of the two peptides were analyzed. As a result, this peptide was found to be composed of two peptides of 36 and 11 amino acid residues. The peptide with 36 amino acid residues has cysteine residues at positions 9, 26, and 35 from the N-terminus,
SEQ ID NO: 9-cysteine residue and 26-cysteine residue are disulfide-bonded, and 35-cysteine residue is disulfide-bonded to the 10-cysteine residue from the N-terminus of a peptide with 11 amino acid residues It was confirmed to have 31 amino acid sequences. Reference Example 4 The peptide of SEQ ID NO: 14 in the sequence listing was synthesized by the same method as in Reference Example 2 except that the type of amino acid and the sequence were changed. Reference Example 5 The peptide of SEQ ID NO: 18 shown in the sequence listing was synthesized by the same method as in Reference Example 2 except that the type of amino acid and the sequence were changed. Reference Example 6 The peptide of SEQ ID NO: 24 shown in the Sequence Listing was synthesized by the same method as in Reference Example 2 except that the type of amino acid and the sequence were changed.

Next, the present invention will be described more specifically by showing examples, but the present invention is not limited to the following examples.

[0028]

【Example】

Example 1 The peptide powder of SEQ ID NO: 26 of Reference Example 1 and sodium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved in water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) at a ratio of 1 mg / ml and 9 mg / ml, respectively, and water was added. Adjust the pH to about 7 with sodium oxide (Wako Pure Chemical Industries, Ltd.) and hydrochloric acid (Wako Pure Chemical Industries, Ltd.), sterilize by filtration, and fill ampoules in 1 ml aliquots by the conventional method, and then use a brain protectant for injection. Was prepared. Example 2 A brain protectant for injection was prepared in the same manner as in Example 1 except that the peptide of SEQ ID NO: 10 of Reference Example 2 was used. Example 3 The peptide powder of SEQ ID NO: 26 of Reference Example 1 and D-mannite (manufactured by Wako Pure Chemical Industries, Ltd.) were added to water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) at 10 mg / ml and 49.5 mg / m, respectively.
Dissolve in a ratio of 1 and adjust the pH to about 7 with an aqueous solution of phosphate buffer powder (manufactured by Wako Pure Chemical Industries, Ltd.), sterilize by filtration, fill 1 ml vials in a conventional manner, and lyophilize. ,
A lyophilized brain protectant for injection was prepared. Example 4 A lyophilized brain protectant for injection was prepared in the same manner as in Example 3, except that the peptide of SEQ ID NO: 10 in Reference Example 2 was used. Example 5 A tablet brain protectant having the following composition per tablet was prepared.

Peptide of Reference Example 1 10.0 (mg) Lactose monohydrate (manufactured by Wako Pure Chemical Industries, Ltd.) 30.0 Corn starch (manufactured by Wako Pure Chemical Industries, Ltd.) 19.8 Crystalline cellulose (manufactured by Asahi Kasei Corporation) 28.0 Magnesium silicate pentahydrate (manufactured by Wako Pure Chemical Industries, Ltd.) 2.0 Magnesium stearate (manufactured by Wako Pure Chemical Industries, Ltd.) 0.2 Peptide of Reference Example 1, lactose monohydrate, corn starch and The mixture of crystalline cellulose is kneaded uniformly while appropriately adding sterile purified water, and dried at 50 ° C. for 3 hours,
Magnesium silicate pentahydrate and magnesium stearate were added to the obtained dried product and mixed, and the mixture was tableted by a tableting machine by a conventional method. Example 6 A brain protectant for tablets was prepared in the same manner as in Example 5, except that the peptide of SEQ ID NO: 10 of Reference Example 2 was used. Example 7 A brain protectant for injection was prepared in the same manner as in Example 1 except that the peptide of SEQ ID NO: 31 of Reference Example 3 was used. Example 8 A lyophilized brain protectant for injection was prepared in the same manner as in Example 3, except that the peptide of SEQ ID NO: 14 of Reference Example 4 was used. Example 9 A brain protectant for tablets was prepared in the same manner as in Example 5, except that the peptide of SEQ ID NO: 18 of Reference Example 5 was used. Example 10 A brain protectant for injection was prepared in the same manner as in Example 1 except that the peptide of SEQ ID NO: 24 of Reference Example 6 was used. Example 11 A tablet brain protectant was prepared in the same manner as in Example 5, except that the peptide of SEQ ID NO: 10 of Reference Example 2 and the peptide of SEQ ID NO: 31 of Reference Example 3 were used in the same amount. Example 12 A brain protectant for tablets was prepared in the same manner as in Example 5, except that the peptide of SEQ ID NO: 14 of Reference Example 4 and the peptide of SEQ ID NO: 18 of Reference Example 5 were used in the same amount. Example 13 A brain protectant for injection was prepared in the same manner as in Example 1, except that the peptide of SEQ ID NO: 26 of Reference Example 1 and the peptide of SEQ ID NO: 31 of Reference Example 3 were used in the same amount. Example 14 A freeze-dried brain protectant for injection was prepared in the same manner as in Example 3, except that the peptide of SEQ ID NO: 14 of Reference Example 4 and the peptide of SEQ ID NO: 24 of Reference Example 6 were used in the same amount. .

[0030]

As described in detail above, according to the present invention, there are few side effects, heat resistance, solubility in water and stability in an aqueous solution, and thus excellent stability as a drug, and the peptide has an antibacterial action. A brain protectant is provided that does not require the use of an antiseptic agent in its formulation in order to have.

[0031]

[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. Xaa in the sequence below
Indicates any amino acid residue except Cys Sequence: Lys Xaa Xaa Xaa Xaa Gln Xaa Xaa 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 peptides containing this peptide as a fragment. Xaa in the sequence below
Indicates any amino acid residue except Cys Sequence: Lys Xaa Xaa Xaa Xaa Gln Xaa Xaa 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 peptides containing this peptide as a fragment. Xaa in the sequence below
Indicates any amino acid residue except Cys Sequence: Arg Xaa Xaa Xaa Xaa 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. Xaa in the sequence below
Indicates any amino acid residue except Cys Sequence: Lys Xaa Xaa Xaa Xaa 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. Xaa in the sequence below
Indicates any amino acid residue except Cys Sequence: Lys Xaa Xaa Xaa Xaa 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. Xaa in the sequence below
Indicates any amino acid residue except Cys Sequence: Arg Xaa Xaa Xaa Xaa 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. Xaa in the sequence below
Indicates any amino acid residue except Cys Sequence: Arg Xaa Xaa Xaa 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. Xaa in the sequence below
Indicates any amino acid residue except Cys Sequence: Lys Xaa Xaa Xaa 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. Xaa in the sequence below
Indicates any amino acid residue except Cys Sequence: Arg Xaa Xaa Xaa 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 Polylogy: Linear Sequence type: Peptide Sequence characteristics: Peptide containing this peptide and this peptide as a fragment Sequence: Trp Gln Arg 1 SEQ ID NO: 14 Sequence length: 5 Sequence type: Amino acid Topology: Type of linear sequence: Peptide Sequence characteristics: Peptide containing this peptide and this peptide as a fragment Sequence: Arg Arg Trp Gln Trp 15 Sequence number: 15 Sequence length: 4 Sequence type: Amino acid Topology: Type of linear sequence: Peptide Sequence characteristics: 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 Type of sequence: Peptide Sequence characteristics: This peptide and peptides containing this peptide as a fragment : Trp Gln Trp Arg 1 SEQ ID NO: 17 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: Peptide containing this peptide and 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: Peptide containing this peptide and 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: Peptide containing this peptide and this peptide as a fragment Sequence: Leu Arg Trp Gln Asn 15 Sequence number: 20 Sequence length: 4 Sequence type: Amino acid Topology-: Linear sequence type: Peptide Sequence characteristics Character: 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 comprising this peptide as a fragment. In the sequence below, the 2nd Cys and the 19th Cys are disulfide-bonded: 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
Shows a cysteine with a thiol group chemically modified to prevent disulfide bond formation.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, the 2nd Cys and the 19th Cys are disulfide-bonded: 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
Shows a 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: Peptide containing this peptide and this peptide as a fragment of
Sequence in which Cys and No. 20 Cys are disulfide-bonded: 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: Peptide containing this peptide and 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: Peptide containing this peptide and this peptide as a fragment Sequence in which Cys and No. 33 Cys are disulfide-bonded: 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: Peptide containing this peptide and this peptide as a fragment In the following sequence, the 10th Cys and the 27th Cys are disulfide-bonded: 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 this peptide as fragments Containing peptides. In the following sequence, the 9th Cys and 26th Cys of the 36-length peptide are disulfide-bonded, and the 35th Cys and 10th Cys of the 11-length peptide are disulfide-bonded. 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 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 this peptide as fragments Containing peptides. Xaa in the sequence below
Indicates any amino acid residue except Cys Sequence: Lys Xaa Xaa Xaa Lys 15

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display area C07K 7/06 ZNA Z 8318-4H 7/08 8318-4H 7/10 8318-4H C07K 99:00

Claims (1)

[Claims] 1. A substance selected from the group consisting of the peptides listed in the sequence listing, pharmaceutically acceptable derivatives thereof, and pharmaceutically acceptable salts thereof, or 2 thereof.
A brain protectant containing the above mixture as an active ingredient.