JPH07291874A - Heparin neutralizing agent - Google Patents

Abstract

PURPOSE:To obtain a heparin neutralizing agent containing a peptide (derivative), etc., having a specific amino acid sequence as an active ingredient, having hardly any adverse effect, excellent in heat resistance, water soluble property, chemical safety and antimicrobial property and not necessary to use preservative in preparing. CONSTITUTION:This heparin neutralizing agent contains e.g. a peptide having an amino acid sequence of formulae I to V, their pharmaceutically permissible peptide salts or a mixture of these peptide derivatives as an active ingredient. Furthermore, the active ingredient is preferably parenterally administered at a dose of 10-15mg based on 1000 units of heparin.

Description

Detailed Description of the Invention

[0001]

This invention relates to a heparin neutralizing agent. More specifically, the present invention relates to a new heparin neutralizing agent which has a lactoferrin-derived peptide having a specific amino acid sequence as an active ingredient and has no side effect.

[0002]

2. Description of the Related Art Heparin, which is a sulfur-containing polysaccharide, is used for the treatment of generalized intravascular blood coagulation syndrome and hemodialysis, artificial cardiopulmonary,
In addition, it is widely used for the purpose of preventing blood clots when using an internal circulation device and when inserting a blood vessel catheter. On the other hand, when this heparin is used during surgery, heparin neutralizing agents such as protamine, which is a basic peptide, are used after surgery in order to suppress the bleeding tendency of the surgical site due to its strong anticoagulant effect. The Pharmacolological Basis of Therapeutics
ogical Basis of Therapeutics, 7th Edition, page 1338, Macmilan, Inc., 1985]. Also,
Protamine is also used to sustain the efficacy of insulin administration [The Pharmacologic Basis
The Pharmacological Basis
of Therapeutics, 7th Edition, page 1490, Macmilan, Inc., 1985], or when fixing heparin as an antithrombotic medical material (JP-A-58-180162).

However, protamine binds to proteins such as fibrinogen and platelets and has side effects such as inhibition of blood coagulation, and antigenicity has been a problem. Therefore, a heparin neutralizing agent that does not have these drawbacks and has no side effects has been desired. In addition to protamine, as substances that bind to heparin, in-vivo substances such as acidic fibroblast growth factor and basic fibroblast growth factor are known, and recently, 59 amino acid residues isolated from the brain. A peptide consisting of
It has been suggested that it has potential as a therapeutic agent for wounds and a therapeutic agent for bone diseases (JP-A-5-339287). In addition, fibronectin and antithrombin III also have a heparin binding site in the molecule, and bind those parts to other peptides,
Attempts have been made to change the function of the peptide (Japanese Unexamined Patent Publication Nos. 2-311498 and 4-7749).
No. 9).

On the other hand, many inventions have been disclosed regarding peptides having antibacterial activity against various microorganisms. For example, phosphonotripeptides effective against Gram-positive bacteria and Gram-negative bacteria (JP-A-57-106689) and phosphonodipeptide derivatives (JP-A-58-135).
94), cyclic peptide derivatives (JP-A-58-21)
3744), peptides exhibiting antibacterial and antiviral effects (JP-A-59-51247), polypeptides effective for yeast (JP-A-60-130599),
Glycopeptide derivatives effective against Gram-positive bacteria (JP-A-60-
172998, JP-A-61-251699 and JP-A-63-44598), oligopeptides effective for Gram-positive bacteria (JP-A-62-22798), peptide antibiotics (JP-A-62-22798). 62-51697 and JP-A-63-17897), other antibacterial peptides extracted from blood cells of horseshoe crab from North America (JP-A-2-53799), antibacterial peptide isolated from bee haemolymph ( There is an antimicrobial peptide 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]. Furthermore, a method for separating and purifying lactoferrin from milk by utilizing the property that lactoferrin binds to heparin (JP-A-63-255299) is also disclosed.

[0006] The inventors of the present invention focused on the antibacterial properties of lactoferrin and focused 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 a peptide having a strong antibacterial activity from a degradation product of lactoferrin, or synthesized a peptide having the same amino acid sequence as those peptides or a derivative 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.

[0008] Further, physiologically active peptides of milk include, in addition to growth hormone, cell differentiation and growth factor, calcium absorption promoting peptide (Food Chemical, Vol. 11, p. 33, 1988), opioid peptide [Hoppe Sailors Zeitschrift Fur Physiologische Chemie (Hoppe-Seyler's Zeitschrift furPh
ysiologische Chemie), 360, 1211, 1979 and The Journal of Biological Chemi.
stry), vol. 254, p. 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 a peptide having the same amino acid sequence as a 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.

However, it is not known that these lactoferrin-derived peptides have a heparin-neutralizing action equivalent to protamine in blood, and do not have a blood coagulation-inhibiting action like protamine. Is also not listed.

[0011]

As is clear from the above-mentioned prior art, a heparin neutralizing agent with few side effects has been long-awaited, but at present, no excellent substance has been known. The present invention has been made in view of the above circumstances, and an object thereof is to provide a heparin neutralizing agent that has few side effects and is effective in a small amount.

[0012]

SUMMARY OF THE INVENTION The inventors of the present invention have
We searched for a substance that neutralizes the anticoagulant action of heparin, but we found that the peptide obtained by hydrolyzing lactoferrin was safe and had few side effects, and completed the present invention. That is, the present invention, in order to solve the above-mentioned problems, comprises a peptide having the amino acid sequence of any of SEQ ID NO: 1 to SEQ ID NO: 31, pharmaceutically acceptable derivatives of these peptides, and pharmaceutically acceptable peptides. A heparin-neutralizing agent containing a salt of these peptides (hereinafter, these may be collectively referred to as peptides) or a mixture of two or more thereof as an active ingredient.

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 heparin neutralizing agent of the present invention, are produced from lactoferrins, the lactoferrins used as starting materials are commercially available lactoferrin and mammals (for example, humans, cows, sheep, goats, horses). From colostrum, transitional milk, normal milk, end-stage milk, etc., or skimmed milk, whey, etc., which are processed products of these milk, a conventional method (eg, ion exchange chromatography)
Lactoferrin separated by, hydrochloric acid, apolactoferrin deironed with citric acid or the like, iron, copper, zinc, metal saturated or partially saturated lactoferrin chelated with a metal such as manganese, commercially available or known methods It is also possible to use the preparations manufactured by

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-
No. 148297), SEQ ID NOS: 7, 8, 9 and 31
Having the amino acid sequence of SEQ ID NO: 10, a salt thereof or a derivative thereof (JP-A-5-1498296), a peptide having an amino acid sequence of SEQ ID NOS: 10 to 21, a salt or a derivative thereof (JP-A-5-148295), a sequence It is a peptide having the amino acid sequences of Nos. 22 to 26, 28, 29 and 30 and salts or derivatives thereof (Japanese Patent Laid-Open No. 5-19924).

Examples of the pharmaceutically acceptable salts of the above peptides include acid addition salts such as hydrochloride, phosphate, sulfate, citrate, lactate, tartrate, and the like. The derivative is a carboxyl group. Examples thereof include amidated or acylated derivatives. The therapeutic agent of the present invention can be processed into an injection or the like by a known method. The heparin neutralizing agent of the present invention varies depending on age, symptoms, etc.,
At least 10-15 mg per 1000 units of heparin
Can be administered parenterally.

Next, the present invention will be described in detail with reference to test examples. Test Example 1 This test was conducted to examine the neutralizing effect of peptides on the heparin anticoagulant effect. 1) Test animal Male ddY mice (purchased from Japan SLC) having a body weight of 29 to 33 g were randomly divided into 12 groups (10 to 12 animals per group) and used. 2) Test method Mice were anesthetized with chloroform (Wako Pure Chemical Industries, Ltd.),
The chest was incised, and blood was collected from the heart with a syringe (made by Terumo Corp.) prefilled with heparin (final concentration 5 U / ml) and the peptide of SEQ ID NO: 26 produced by the same method as in Reference Example 1. It was transferred to a test tube (Falcon) and left standing in a 37 ° C water bath. The time (coagulation time) from when blood was collected to when the test tube was tilted until it did not flow out (coagulation time) was measured, and the case where the coagulation time was 60 minutes or less was determined to be effective. As a control, the coagulation time was similarly measured for blood collected by a syringe filled with heparin and protamine. The peptide of SEQ ID NO: 26, protamine sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) and sodium heparin (manufactured by Kodama Co., Ltd.) were dissolved or diluted in physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) before use. 3) Test results The results of this test are shown in Table 1. In addition, E
D50 is a numerical value read from the log probability graph. As is clear from Table 1, the peptide of SEQ ID NO: 26 neutralized the anticoagulant action of heparin, and its efficacy was almost the same as that of protamine sulfate. Similar tests were conducted for other peptides, but almost the same results were obtained.

[0018]

[Table 1]

Test Example 2 This test was conducted to examine the neutralizing effect of peptides on the anticoagulant effect of sodium citrate. 1) Test animals Male ddY mice (purchased from Japan SLC) having a body weight of 29 to 33 g were randomly divided into 3 groups (8 mice per group) and used. 2) Test method A test was conducted in the same manner as in Test Example 1 except that a syringe prefilled with 10% sodium citrate solution (manufactured by Fuso Yakuhin Co., Ltd.) having a final concentration of 50 μl / ml was used instead of heparin. 3) Test results The results of this test are shown in Table 2. As is clear from Table 2, it was confirmed that the peptide of SEQ ID NO: 26 produced by the same method as in Reference Example 1 did not neutralize the anticoagulant action of sodium citrate. Similar tests were performed for other peptides, but almost the same results were obtained.

[0020]

[Table 2]

Test Example 3 This test was carried out to investigate the blood coagulation-inhibiting effect of the peptide. 1) Test animal Male ddY mice (purchased from Japan SLC) with a body weight of 29 to 33 g were used by randomly dividing them into 13 groups (10 to 20 animals per group). 2) Test method SEQ ID NO: 2 previously produced by the same method as in Reference Example 1
Test except that a syringe filled with the peptide of No. 6, protamine sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) or protamine base (manufactured by Sigma) was used, and that it was determined to be effective when the coagulation time exceeded 60 minutes Tested by the same method as in Example 1. 3) Test results The results of this test are shown in Table 3. As is clear from Table 3, the peptide of SEQ ID NO: 26 was found to have a weaker anticoagulant action than protamine sulfate and protamine base. Similar tests were performed for other peptides, but almost the same results were obtained.

[0022]

[Table 3]

Test Example 4 This test was conducted to examine the acute toxicity of peptides. 1) Animals used Six-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 The peptide of SEQ ID NO: 26 produced by the same method as in Reference Example 1 was used for 1000, 2000 or 400 per 1 kg of body weight.
It was dissolved in water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) at a rate of 0 mg, and a single forceful 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 4. As is clear from Table 4, 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 performed for other peptides, but almost the same results were obtained.

[0024]

[Table 4]

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). (Manufactured by Mitsui Chemicals Co., Ltd.) 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 consisted of 25 amino acid residues, the 3rd and 20th cysteine residues were disulfide-bonded, and the 3rd cysteine residue resulted in 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 a peptide automatic synthesizer (Pharmacia LKB Biotechnology Co., Ltd., LKBBiolynx4170), a solid-phase peptide synthesis method by Shepard or the like [Journal of Chemical Society Parkin I (Jo
urnal of Chemical Society Perkin I), No. 538
Page, 1981], and the peptide was synthesized as follows.

An amino acid having an amine functional group 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.

[0030]

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 1 mg of peptide powder of SEQ ID NO: 26 produced by the same method as in Reference Example 1 in 1 ml of water for injection (Otsuka Pharmaceutical Co., Ltd.)
And sodium chloride (manufactured by Wako Pure Chemical Industries) at a ratio of 9 mg, and the pH was adjusted to about 7 with sodium hydroxide (manufactured by Wako Pure Chemical Industries) and hydrochloric acid (manufactured by Wako Pure Chemical Industries) and sterilized by filtration. Then, 1 ml each was filled in an ampoule by a conventional method to prepare a heparin neutralizing agent for injection. Example 2 10 mg of the peptide powder of SEQ ID NO: 10 produced by the same method as in Reference Example 2 in 1 ml of water for injection (Otsuka Pharmaceutical Co., Ltd.)
And D-mannite (manufactured by Wako Pure Chemical Industries, Ltd.) 49.5 m
Dissolve at a rate of g, 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 by a conventional method, and lyophilize, A heparin neutralizer for injection was prepared.

[0031]

INDUSTRIAL APPLICABILITY As described above in detail, the present invention relates to a heparin neutralizing 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.

[0032]

[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 A 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 A 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 Arg 15 Sequence number: 4 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and this peptide Peptides included as fragments. 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 this peptide Peptides included as fragments. 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 this peptide Peptides included as fragments. 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 this peptide Peptides included as fragments. 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 this peptide fragment As a peptide. In the sequence below, R01
Indicates any amino acid residue except Cys.

Sequence: Lys R01 R01 R01 Arg 15 Sequence number: 9 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and this peptide are fragments As a peptide. 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 this peptide fragment As a peptide.

Sequence: Phe Gln Trp Gln Arg Asn 15 Sequence number: 11 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and this peptide Peptides included as fragments.

Sequence: Phe Gln Trp Gln Arg 15 Sequence number: 12 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and this peptide are fragments As a peptide.

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 this peptide as a fragment peptide.

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 15 Sequence number: 15 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and this peptide are fragments As a peptide.

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

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 this peptide as a fragment peptide.

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 this peptide Peptides included as fragments.

Sequence: Leu Arg Trp Gln Asn 15 Sequence number: 20 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and this peptide are fragments As a peptide.

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 this peptide as a fragment peptide.

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 Type of sequence: Peptide Sequence characteristics: This peptide and peptides 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 peptides 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 Type of sequence: Peptide Sequence characteristics: This peptide and peptides 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 peptides 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 A 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 of the 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 this peptide Peptides included as fragments. In the sequence below, R01
Indicates any amino acid residue except Cys.

Sequence: Lys R01 R01 R01 Lys 15

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area C07K 5/103 5/11 5/113 5/117 7/06 7/08 ZNA 14/47 (72 ) Inventor Mitsunori Takase 5-1-83 Higashihara, Zama-shi, Kanagawa Morinaga Milk Industry Co., Ltd. (72) Inventor Shuzo Orikasa 5-1-83 Higashihara, Zama-shi, Kanagawa 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. A heparin neutralizer containing a mixture of two or more species as an active ingredient.