JP5354206B2 - Antibacterial peptide - Google Patents

Abstract

Disclosed is a peptide having high safety and an excellent antibacterial activity or a salt of the peptide. Specifically disclosed is a peptide selected from the following peptides (1) and (2) or a salt of the peptide. (1) A peptide comprising an amino acid sequence represented by the general formula (I). (Pro Hyp Gly)<SUB>n</SUB>-[(Lys x Gly)<SUB>m</SUB>-(Pro Hyp Gly)<SUB>l</SUB>-(Lys X Gly)<SUB>p</SUB>]<SUB>a</SUB>- (Pro Hyp Gly)<SUB>r</SUB> (I) [wherein n represents an integer of 1 to 6; m represents an integer of 1 to 4; l represents an integer of 0 to 7; p represents an integer of 1 to 4; r represents an integer of 1 to 6; a represents an integer of 1 to 6; and X represents an amino acid residue selected from Ile, Leu and Hyp, provided that the (m+p) pieces of X's may be the same as or different from each other, n+m+l+p+r = 4 to 15, m+p=2 when l=0, and m=1, l=0 and p=1 when a=2.] (2) A peptide which comprises an amino acid sequence having the substitution, deletion or addition of 1 to 3 amino acid residues other than Lys, Gly and X in the amino acid sequence represented by the general formula (I), and which has an antibacterial activity.

Description

  The present invention relates to novel peptides having antibacterial activity and physiologically acceptable salts thereof. More particularly, the present invention relates to peptides and pharmaceutically acceptable salts thereof that are toxic to bacteria but not mammalian cells and are useful in the treatment of bacterial infections.

  Antibiotics play a major role in the treatment of bacterial infections, but most often lose their potency due to the emergence of resistant bacteria. Therefore, an antibacterial agent having a different mechanism of action from antibiotics is required. Such antibacterial agents include antibacterial peptides. In particular, a membrane-acting antibacterial peptide acts on the cell membrane / outer membrane of microorganisms and exhibits a bactericidal action by destroying its barrier ability, but is characterized in that resistant bacteria are not likely to appear in principle.

  There are many such antibacterial peptides in nature, for example, Melittin produced by bees. However, Meilittin acts on mammalian cells as well as bacterial membranes. On the other hand, it is considered that Magainin 2 secreted by Xenopus laevis selectively destroys the bacterial membrane and bears an innate biological defense action (for example, Non-Patent Document 1). Melittin and Maginin 2 are basic peptides containing Lys residues and Arg residues, and have been reported to form an α-helix and exhibit antibacterial activity. In addition, as antibacterial peptides exhibiting the same action, there are Cecropin A derived from silkworm, Defensin derived from human, insect, plant, etc., and electrostatic interaction and hydrophobic interaction are important for bacterial membrane selectivity. It has been reported (Non-Patent Document 2). For example, Patent Document 1 discloses an antibacterial peptide derived from human cornea and conjunctival secretory apoprotein that can be used for antibacterial agents, antibacterial drugs, and eye drops, and a polynucleotide encoding the same.

  As an artificially produced antibacterial peptide, for example, Patent Document 2 discloses a novel peptide based on CAP11, an antibacterial agent comprising this as an active ingredient, an inhibitor of LPS binding to cells, and bacterial infection Drugs such as therapeutic agents and endotoxin shock inhibitors have been reported. Patent Document 3 reports an antibacterial peptide represented by repeating hydrophilic amino acid residues-hydrophobic amino acid part-basic amino acid part-bridge part-basic amino acid part-hydrophobic amino acid part. Patent Document 4 reports an antibacterial peptide having 7 to 30 amino acid residues, including a portion where two or more basic amino acid residues are continuous at two positions that are not the ends of the peptide. Patent Document 5 reports an antibacterial peptide composition containing as an active ingredient peptides having an average molecular weight of 2 to 6 kDa obtained by decomposing lysozyme with trypsin. Patent Document 6 reports an antimicrobial peptide fractionated from a hydrolyzate of bovine lactoferrin. As a thing derived from a human, the protein or peptide which has the antibacterial activity which exists in the protein derived from a human is known (for example, refer patent document 7). Patent Document 8 reports a peptide derived from human and having very high LPS binding activity, antibacterial activity, and LPS neutralization activity.

  These antibacterial peptides have been proposed that peptides that bind to bacterial membranes and form α-helices take a channel-like structure within the bacterial membrane and form pores in the membrane. In general, the peptides that form the are susceptible to the external environment such as heat and salt concentration and are unstable.

On the other hand, the present inventors have reported a peptide that forms a stable triple helical structure based on the amino acid sequence of collagen (for example, Non-Patent Document 3 and Patent Document 9). The present inventors thought that a highly stable antibacterial peptide could be produced based on such knowledge.
JP2005-318829 JP 2006-45214 JP2005-120050 JP2004-175727 JP2003-267805 JP2000-319300 JP2000-217579 JP 11-335396 A JP2003-321500 Pharmaceutical Journal, Vol.117 (5), 253-264, 1997 Nature, 415, 389-395, 2002 Biopolymers, 79 (3), 163-172,2005

  However, the present invention provides a novel peptide having high stability and excellent antibacterial activity, and a physiologically acceptable salt thereof, as well as a peptide useful for the treatment of bacterial infections and the like and its pharmaceutical The main object is to provide an acceptable salt. Furthermore, an object of the present invention is to provide a method for preventing or treating antibacterial agents, pharmaceutical preparations, medical base materials, cosmetics, bacterial infection makeup or sepsis using the peptide or a salt thereof. is there.

  As a result of diligent studies, the present inventors have found a peptide having a specific amino acid sequence that can solve the above-mentioned problems. That is, a peptide having the amino acid sequence represented by the following general formula (I) can form a stable collagen-like triple helix structure, is hardly affected by heat, salt concentration, etc., and has excellent antibacterial properties. It has been found that the activity can be exerted. The present invention has been obtained as a result of further research based on such knowledge.

The present invention provides the following peptides or salts thereof having antibacterial activity, antibacterial agents containing these as active ingredients, pharmaceutical preparations, medical bases, cosmetics, methods for preventing or treating bacterial infections or sepsis. is there.
Item 1. A peptide represented by the following (1) or (2) or a salt thereof;
(1) Peptides having an amino acid sequence represented by the following general formula (I)
(Pro Hyp Gly) _n -{(Lys X Gly) _m- (Pro Hyp Gly) _l- (Lys X Gly) _p } _α- (Pro Hyp Gly) _r (I)
(Here, in formula (I), n is an integer of 1-6, m is an integer of 1-4, l is an integer of 0-7, p is an integer of 1-4, r is an integer of 1 to 6, and α is an integer of 1 to 6.
X is any amino acid residue selected from Ile, Leu and Hyp,
The m + p Xs may be the same or different from each other.
n + m + l + p + r = 4-15.
However, when l = 0, m + p ≧ 2 is satisfied,
When α ≧ 2, m = 1, l = 0, and p = 1 are satisfied. )
(2) The amino acid sequence represented by the general formula (I) is composed of an amino acid sequence in which 1 to 3 amino acid residues other than Lys, Gly and X are substituted, deleted or added, and has antibacterial activity. peptide.
Item 2. In said (1), n is an integer of 3-6, m is an integer of 1-4, l is an integer of 0-7, p is an integer of 1-4, r is 3- An integer of 6, α is 1,
X is Ile or Leu,
m + p Xs may be the same or different from each other,
n + m + l + p + r = 8-15,
Item 2. The peptide or salt thereof according to Item 1, which satisfies m + p ≧ 2 when l = 0.
Item 3. The peptide according to item 1 represented by the following (1-1) or (2-1) or a salt thereof:
(1-1) A peptide comprising an amino acid sequence represented by any one of (i) to (vi) below.
(I) (Pro Hyp Gly) _3- (Lys Ile Gly) _3- (Pro Hyp Gly) ₄ (SEQ ID NO: 1)
(Ii) (Pro Hyp Gly) ₃ -Lys Ile Gly Pro Hyp Gly Lys Ile Gly- (Pro Hyp Gly) ₄ (SEQ ID NO: 2)
(Iii) (Pro Hyp Gly) 4 - (Lys Ile Gly) 2 - (Pro Hyp Gly) 4 ( SEQ ID NO: 3)
(iv) (Pro Hyp Gly) _3- (Lys Hyp Gly Lys Leu Gly) _2- (Pro Hyp Gly) ₃ (SEQ ID NO: 4),
(v) (Pro Hyp Gly) _2- (Lys Leu Gly Lys Hyp Gly) _3- (Pro Hyp Gly) ₂ (SEQ ID NO: 5),
(vi) Pro Hyp Gly- (Lys Leu Gly Lys Hyp Gly) ₄ -Pro Hyp Gly (SEQ ID NO: 6)
(2-1) In the amino acid sequences (i) to (vi), 1 to 3 amino acids are substituted, deleted,
Alternatively, a peptide comprising an added amino acid sequence and having antibacterial activity, and salts thereof.
Item 4. Item 5. The peptide according to any one of Items 1 to 4, wherein the amino terminal hydrogen of the amino acid sequence is substituted with an acetyl group, a methoxycarbonyl group, a butoxycarbonyl group, or a peptide having 1 to 3 residues, and a pharmaceutically acceptable peptide thereof. Salt.
Item 5. Item 5. The peptide according to any one of Items 1 to 4, wherein the carboxyl group at the carboxy terminus of the amino acid sequence is substituted with an amide group, a methyl ester group, a butyl ester group, or a peptide having 1-3 residues, and a pharmaceutically acceptable peptide thereof. Salt.
Item 6. Item 6. The peptide according to any one of Items 1 to 5, comprising a collagen-like triple helix structure, and a pharmaceutically acceptable salt thereof.
Item 7. Item 7. An antibacterial agent comprising the peptide or salt thereof according to any one of items 1 to 6 as an active ingredient.
Item 8. Item 8. A pharmaceutical preparation comprising the peptide according to any one of Items 1 to 7 or a salt thereof, and a pharmaceutically acceptable carrier or substrate.
Item 9. Item 9. The pharmaceutical preparation according to Item 8, which is used for prevention or treatment of bacterial infection or sepsis.
Item 10. Item 7. A medical substrate in which the peptide according to any one of Items 1 to 6 or a salt thereof is supported on a pharmaceutically acceptable substrate.
Item 11. Item 11. The medical substrate according to Item 10, wherein the pharmaceutically acceptable substrate is at least one selected from the group consisting of alginate gel, collagen gel, lactic acid polymer, and cellulose.
Item 12. Cosmetics containing the peptide according to any one of claims 1 to 6 or a salt thereof.
Item 13. Item 7. A method for treating bacterial infection or sepsis, comprising administering an effective amount of the peptide or salt thereof according to any one of items 1 to 6 to a patient suffering from bacterial infection or sepsis.
Item 14. Item 12. A method for preventing or treating a bacterial infection, which comprises applying the medical substrate according to Item 10 or 11 to an affected area.
Item 15. Item 7. Use of the peptide according to any one of Items 1 to 6 or a salt thereof for producing an antibacterial agent.
Item 16. Item 7. Use of the peptide according to any one of Items 1 to 6 or a salt thereof for producing a pharmaceutical preparation for preventing or treating bacterial infection or sepsis.
Item 17. Item 7. Use of the peptide according to any one of Items 1 to 6 or a salt thereof for producing a medical substrate for preventing or treating bacterial infection.
Item 18.
A peptide represented by the following (1) or (2) or a salt thereof for use in the treatment of bacterial infection or sepsis;
A peptide represented by the following (1) or (2) or a salt thereof;
(1) Peptides having an amino acid sequence represented by the following general formula (I)
(Pro Hyp Gly) _n -{(Lys X Gly) _m- (Pro Hyp Gly) _l- (Lys X Gly) _p } _α- (Pro Hyp Gly) _r (I)
(Here, in formula (I), n is an integer of 1-6, m is an integer of 1-4, l is an integer of 0-7, p is an integer of 1-4, r is an integer of 1 to 6, and α is an integer of 1 to 6.
X is any amino acid residue selected from Ile, Leu and Hyp,
The m + p Xs may be the same or different from each other.
n + m + l + p + r = 4-15.
However, when l = 0, m + p ≧ 2 is satisfied,
When α ≧ 2, m = 1, l = 0, and p = 1 are satisfied. )
(2) The amino acid sequence represented by the general formula (I) is composed of an amino acid sequence in which 1 to 3 amino acid residues other than Lys, Gly and X are substituted, deleted or added, and has antibacterial activity. peptide.

  According to the peptide of the present invention, an antibacterial peptide that forms a stable triple helical structure can be provided. In addition, since the peptide of the present invention is not easily affected by external factors such as heat and salt concentration and can stably exhibit excellent antibacterial activity, it is prepared as a pharmaceutical preparation, such as bacterial infection, sepsis, etc. It can be suitably used for prevention or treatment.

  Furthermore, the polypeptide of the present invention can be used by binding to a medical substrate. For example, artificial skin produced by binding the peptide of the present invention to an artificial collagen gel or the like and using this as a raw material can prevent bacterial infection of the transplanted part.

  In addition, since the peptide of the present invention is hypoallergenic to humans and can maintain stable antibacterial properties, it can also be incorporated into cosmetics as an antibacterial agent (or preservative). Some of the conventional antibacterial agents and preservatives blended in cosmetics are annoying. However, the polypeptide of the present invention can be suitably used as an antibacterial agent blended in cosmetics without problems such as odor.

It is a graph which shows the result of antibacterial evaluation of the peptide of Examples 1-6 and Comparative Examples 1-3.

  In the present invention, various amino acid residues are described by the following abbreviations.

Ala: L-alanine residue
Arg: L-arginine residue
Asn: L-asparagine residue
Asp: L-aspartic acid residue
Cys: L-cysteine residue
Gln: L-glutamine residue
Glu: L-glutamic acid residue
Gly: Glycine residue
His: L-histidine residue
Hyp: L-hydroxyproline residue
Ile: L-isoleucine residue
Leu: L-leucine residue
Lys: L-lysine residue
Met: L-methionine residue
Phe: L-phenylalanine residue
Pro: L-proline residue
Ser: L-serine residue
Thr: L-threonine residue
Trp: L-tryptophan residue
Tyr: L-tyrosine residue
Val: L-valine residue In the present specification, the amino acid sequence of a peptide is arranged according to a conventional method so that the N-terminal amino acid residue is located on the left side and the C-terminal amino acid residue is located on the right side. Describe.

[Peptides having antibacterial activity and salts thereof]
Examples of the peptide having antibacterial activity of the present invention include peptides represented by the following (1) or (2).
(1) Peptide having an amino acid sequence represented by the following general formula (I)
(Pro Hyp Gly) _n -{(Lys X Gly) _m- (Pro Hyp Gly) _l- (Lys X Gly) _p } _α- (Pro Hyp Gly) _r (I)
In the formula (I), for example, (Lys X Gly) _m indicates that _m structural units (Lys X Gly) are linked.

Here, in formula (I), n, m, l, p, r, α, and X are as follows.
n is an integer of 1-6, preferably an integer of 1-5, more preferably an integer of 1-4.
m is an integer of 1-4, preferably an integer of 1-3, more preferably an integer of 1-2.
l is an integer of 0 to 7, preferably an integer of 0 to 5, more preferably an integer of 0 to 3.
p is an integer of 1-4, preferably an integer of 1-3, more preferably an integer of 1-2.
r is an integer of 1-6, preferably an integer of 1-5, more preferably an integer of 1-4.
α is an integer of 1 to 6, preferably an integer of 1 to 5, more preferably an integer of 1 to 4.

  X is the same or different and is at least one selected from Ile, Leu and Hyp.

In addition, m + p Xs contained in the general formula (I) may be the same or different from each other. That is, when m and p are each 2 or more, X may be the same or different in the repeating unit of (Lys X Gly). Specifically, the structural unit represented by (Lys X Gly) _m is an amino acid sequence composed only of a repeating unit (Lys Ile Gly), an amino acid sequence composed only of a repeating unit (Lys Lue Gly), and a repeating unit. It shows that the amino acid sequence is composed of only (Lys Hyp Gly), or is composed of amino acid sequences obtained by combining these repeating units in any order. In the general formula (I) of the present invention, X is preferably Ile or Hyp.

  In the said general formula (I), it is n + m + l + p + r = 4-15, Preferably it is 4-13, More preferably, it is 4-12. However, when l = 0, m + p ≧ 2 is satisfied, and when α ≧ 2, m = 1, l = 0, and n = 1 are satisfied.

  Furthermore, as for the amino acid sequence of the peptide provided by the above general formula (I), the following embodiments are preferable examples from the viewpoint of antibacterial activity.

  When l is 0, n is 1-6; m is 1-4, preferably 1-3; p is 1-4, preferably 1-3; r is 1-6; α = 1.

  When l is 1, n is 1-6; m is 1-4, preferably 1-3; p is 1-4, preferably 1-3; r is 1-6; α = 1.

  When l is 2, n is 1-6; m is 1-4, preferably 1-3; p is 1-4, preferably 1-3; r is 1-6; α = 1.

  When l is 3, n is 1-6; m is 1-4, preferably 1-3; p is 1-4, preferably 1-3; r is 1-6; α = 1.

  Further, when α ≧ 2, l is 0; n is 1 to 6, preferably 1 to 4; m is 1; p is 1; and r is 1 to 6, preferably 1 to 4.

  When l is 0, n is 3 to 6; m is 1 to 4; l is 0 to 7; p is 1 to 4; r is 3 to 6; α = 1; and X is Ile or Leu. Yes; m + p Xs may be the same or different; n + m + l + p + r = 8-15; m + p ≧ 2.

  The number of amino acids constituting the peptide represented by the general formula (I) is not particularly limited as long as the effect of the present invention is exhibited, but is usually 12 to 60, preferably 15 to 40, and more preferably 20 to 35.

Specific examples of the peptide represented by the general formula (I) include those having an amino acid sequence represented by the following (i) to (vi) (SEQ ID NOs: 1 to 6).
(i) (Pro Hyp Gly) _3- (Lys Ile Gly) _3- (Pro Hyp Gly) ₄ (SEQ ID NO: 1)
(ii) (Pro Hyp Gly) ₃ -Lys Ile Gly Pro Hyp Gly Lys Ile Gly- (Pro Hyp Gly) ₄ (SEQ ID NO: 2)
(iii) (Pro Hyp Gly) 4 - (Lys Ile Gly) 2 - (Pro Hyp Gly) 4 ( SEQ ID NO: 3)
(iv) (Pro Hyp Gly) _3- (Lys Hyp Gly Lys Leu Gly) _2- (Pro Hyp Gly) ₃ (SEQ ID NO: 4)
(v) (Pro Hyp Gly) _2- (Lys Leu Gly Lys Hyp Gly) _3- (Pro Hyp Gly) ₂ (SEQ ID NO: 5)
(vi) Pro Hyp Gly- (Lys Leu Gly Lys Hyp Gly) ₄ -Pro Hyp Gly (SEQ ID NO: 6)
(2) The present invention further comprises an amino acid sequence in which one or several arbitrary amino acid residues are substituted, deleted or added in the amino acid sequence represented by the general formula (I), and has antibacterial activity. Also included are peptides having

  In the peptide of (2) above, the number of amino acid residues to be substituted, deleted or added is not particularly limited as long as the modified peptide has antibacterial activity.

  For example, in the case of addition of amino acid residues, the number of amino acid residues to be added is 1 to 3, preferably 1 to 2, and more preferably 1.

  In the case of deletion of amino acid residues, the number of amino acid residues to be deleted is 1 to 3, preferably 1 to 2, and more preferably 1.

  Further, in the case of substitution of amino acid residues, the number of amino acid residues to be substituted is 1 to 3, preferably 1 to 2, and more preferably 1.

  In the peptide (2), the position of the amino acid residue to be substituted, deleted or added is not particularly limited as long as the modified peptide has antibacterial activity. However, amino acid substitution, deletion or addition is preferably performed on amino acid residues other than Lys, Gly and X in the above general formula (I).

  Furthermore, in the peptide of (2), the type of amino acid to be substituted or added is not particularly limited as long as the modified peptide has antibacterial activity. As long as the modified peptide has antibacterial activity, any amino acid can be selected regardless of whether it is a natural amino acid or an unnatural amino acid. However, for substitution, each amino acid residue is preferably a homologous substitution based on the properties of the side chain functional group. In the case of amino acid substitution, for example, Pro can be substituted with Hyp, Leu can be substituted with Ile or Val, and vice versa.

  The peptide of the present invention may be one in which the amino-terminal hydrogen is substituted with an acetyl group, a methoxycarbonyl group, a butoxycarbonyl group, a peptide having 1 to 3 residues, or the like. Further, the C-terminal carboxyl group may be substituted with an amide group, a methyl ester group, a butyl ester group, a peptide having 1 to 3 residues, or the like. Here, the case where the carboxyl group is substituted with the substituent means that —OH of the carboxyl group is substituted with an amino group, a methoxy group, or a butoxy group, respectively. Further, the case where the carboxyl group is substituted with a peptide means that dehydration condensation occurs between peptides bonded to the carboxyl group to form a peptide bond.

  The peptide of the present invention shown in the above (1) or (2) may form a pharmaceutically acceptable salt. Examples of the salt include hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaric acid, maleic acid. Salts with acids such as acid, fumaric acid, oxalic acid, malic acid, citric acid, oleic acid, palmitic acid; alkali metals or alkaline earth metals such as sodium, potassium, calcium, or aluminum hydroxide or carbonic acid Salts with salts; salts with triethylamine, benzylamine, diethanolamine, t-butylamine, dicyclohexylamine, arginine and the like.

  Such a peptide of the present invention is characterized by having a collagen-like triple helix structure. The collagen-like triple helix structure is a characteristic structure found in collagen molecules, and shows a rope-like super helical structure. Collagen contains a particularly large amount of proline, hydroxyproline and glycine, and glycine is present every 3 residues. Such characteristics are considered to be important for the formation of a stable triple helical structure. In the present invention, such a structure is considered to have a role of enhancing the stability of the peptide of the present invention.

  The peptide of the present invention can be obtained by an ordinary peptide synthesis method. For example, it is prepared by a solid phase synthesis method or a liquid phase synthesis method, but the solid phase synthesis method is simple in terms of operation. The solid-phase synthesis method is described in, for example, “Sequel Biochemistry Experiment Course 2 Protein Chemistry (2)” (published May 20, 1987, published by Tokyo Kagaku Dojin), pages 641-694. The method can be carried out with reference to the method.

  Preparation of the peptide of the present invention by a solid phase synthesis method includes, for example, an α-amino acid having an amino acid corresponding to the C-terminus of the target peptide in a polymer that is insoluble in a reaction solvent such as a styrene-divinylbenzene copolymer. An α-amino group other than the α-COOH group that the amino acid or peptide fragment has a corresponding amino acid or peptide fragment attached to the amino acid in the direction of the N-terminal of the target peptide, etc. The procedure of condensing and conjugating the functional group of the amino acid and the step of removing the protective group of the amino group that forms a peptide bond such as an α-amino group in the bound amino acid or peptide fragment is sequentially repeated. To elongate the peptide chain to form a peptide chain corresponding to the target peptide, and then the peptide chain Desorbed from the polymer, and obtain a peptide of interest by removing the protecting group from the functional group being protected, and then carried out by purification.

  Here, the elimination of the peptide chain from the polymer and the removal of the protecting group are preferably performed simultaneously using trifluoroacetic acid from the viewpoint of suppressing side reactions. Moreover, it is effective to purify the obtained peptide by reverse phase liquid chromatography or gel permeation chromatography.

  Moreover, the salt of the peptide of this invention is prepared by utilizing normal salt formation reaction.

  The peptide represented by the above (1) or (2) has excellent antibacterial activity. In the present invention, the peptide having antibacterial activity means a peptide having antibacterial action or bactericidal action against bacteria and / or fungi. The antibacterial activity of the peptide of the present invention (or a salt thereof) is usually measured by the following method. It is carried out by measuring the leakage amount (Biochim Biophys Acta, 1070, 259-264, 1991) of a fluorescent dye (carboxyfluorescein) encapsulated from a liposome containing DPPG (dipalmitoylphosphatidylglycerol) as a main constituent lipid. Examples of specific measurement methods include the following methods.

<Preparation of bacterial membrane model liposome>
A methylene chloride / methanol mixed solution containing 0.7 mg of dipalmitoylphosphatidylglycerol (DPPG: Sigma P9789) is placed in a brown glass vial, and nitrogen gas is blown to form a lipid film on the inner wall of the vial. Add 1 mL of 1 mM EDTA / TBS solution containing carboxyfluorescein (CF: Sigma C7153) at a concentration of 20 mM to a glass vial, and stir well with a vortex mixer until the lipid membrane peels from the inner wall of the vial. Next, ultrasonic treatment was performed at 60 ° C. for 20 minutes. Furthermore, 3-5 mL fractions are collected with a PD10 column (GE Healthcare Bioscience) to obtain a liposome solution.

<Production of mammalian membrane model liposome>
A liposome composed of phosphatidylcholine (PC: Sigma P2772 Type XI-E) is obtained in the same manner as described above except that sonication is performed at room temperature.

<Antimicrobial evaluation>
The peptide to be evaluated for antibacterial activity was added to 200 μL of the obtained DPPG liposome and PC liposome diluted 10-fold with PBS so that the final concentration would be 0.25 to 0.005 mM, and the mixture was allowed to stand at room temperature for 15 minutes. Leave at rest. Thereafter, the amount of CF in the supernatant obtained by centrifugation at 12,000 rpm for 5 minutes is measured at an excitation wavelength of 485 nm and a fluorescence wavelength of 535 nm. The amount of CF leakage due to each peptide is calculated assuming that the fluorescence when PBS is added instead of the peptide solution is 0% and the fluorescence when Triton X-100 at a final concentration of 0.1% is added is 100%.

  In the above test, the fluorescence due to CF leakage is 3% or more, preferably 5% when the fluorescence when PBS is added is 0% and the fluorescence when Triton X-100 is added at a final concentration of 0.1% is 100%. Above, more preferably 10% or more is referred to as a peptide having antibacterial activity in the present invention.

  It is also possible to evaluate antibacterial activity based on a method for measuring antibacterial activity against Staphylococcus aureus (Reference: Nature, 443, 867-869, 2006). At least one of the method for measuring the leakage amount of fluorescent dye (carboxyfluorescein) from the liposome and the method for measuring antibacterial activity against Staphylococcus aureus, preferably the antibacterial activity confirmed by both and To do.

  Although not wishing to be limited to the interpretation of the present invention, the peptide (or salt thereof) of the present invention selectively acts on the structure of the bacterial membrane and exhibits antibacterial activity by destroying the bacterial membrane structure. Therefore, it can act effectively against a wide variety of bacteria. Bacteria exhibiting excellent antibacterial activity of the peptide of the present invention are not particularly limited, such as Gram-negative bacteria, Gram-positive bacteria, etc. It is effective.

  Furthermore, the peptide of the present invention or a salt thereof can effectively act not only on the above general bacteria but also on fungi.

  Thus, since the polypeptide of the present invention or a salt thereof has excellent antibacterial activity against a wide range of bacteria and fungi, the peptide or salt can be used alone or in combination of two or more to obtain an antibacterial agent or antibacterial agent. It can be used as a fungicide. Moreover, based on the antibacterial action (antibacterial activity) of the polypeptide of the present invention or a salt thereof, it can also be used as a pharmaceutical preparation for the purpose of preventing infectious diseases. Furthermore, the peptide of the present invention or a salt thereof can be used by adding it to an additive such as a pharmaceutical preparation, a medical base, a cosmetic, a food, or a quasi-drug for the purpose of preserving. Hereinafter, it explains in full detail according to each usage form.

[Usage for the purpose of prevention of infectious diseases]
The peptide of the present invention or a salt thereof can exert a useful effect in the pharmaceutical field based on the antibacterial activity. Since the peptide of the present invention or a salt thereof has excellent antibacterial activity against bacteria and fungi such as Staphylococcus aureus and Pseudomonas aeruginosa, the peptide of the present invention or a salt thereof and a pharmaceutically acceptable substrate or It can be mixed with a carrier and prepared and used as a pharmaceutical preparation. When using the peptide of this invention or its salt as a pharmaceutical formulation, the said peptide or salt can also be used individually by 1 type or in combination of 2 or more types.

  The pharmaceutical preparation is useful for use based on the antibacterial activity, that is, for prevention or treatment of infectious diseases, sepsis and the like. In addition, since the peptide of the present invention or a salt thereof has excellent antibacterial activity against fungi, antifungal agents such as ringworm (foot ringworm (so-called athlete's foot), nail ringworm, hand ringworm, etc.), candidiasis, etc. It can also be prepared and used in the form of a pharmaceutical formulation for therapeutic purposes.

  The base material or carrier used in the pharmaceutical preparation is not particularly limited as long as it is pharmaceutically acceptable, and a wide variety of ingredients blended for pharmaceutical preparation can be used. The pharmaceutical preparation may contain additives and pharmacologically active ingredients in addition to the above ingredients.

  The dosage form of the pharmaceutical preparation is appropriately set according to the application form, but examples include solid preparations such as tablets, powders, powders, granules, capsules; and liquids, emulsions, suspensions, etc. Liquid preparations; semi-solid preparations such as ointments and creams. In addition, the pharmaceutical preparation may be prepared by liposomal formation of the peptide of the present invention or a salt thereof.

  Examples of the administration form of the pharmaceutical preparation include systemic administration such as intravenous administration, subcutaneous administration, intraperitoneal administration and oral administration; local administration such as intra-articular administration, etc. It can be set appropriately. Moreover, the form of transdermal administration which directly administers the peptide of this invention as an external preparation by application | coating or dispersion | spreading to a disease site etc. are mentioned.

  When administered systemically, a solution obtained by dissolving the peptide in a pharmaceutically acceptable solution such as 5% glucose solution or physiological saline is preferable. The solution may contain various pharmacologically acceptable additives. It is also possible to encapsulate or liposome peptides. In the case of topical administration, it can be mixed with a solution such as 5% glucose solution or physiological saline, or a commonly used ointment base or cream base.

  For example, the dosage when the pharmaceutical preparation of the present invention is intravenously administered can be appropriately set based on the sex, age, symptom, dosage form, expected effect and the like of the patient. As an example of the daily dose of the pharmaceutical preparation, it is usually 0.1 μg / kg to 3 g / kg (adult), preferably 1 μg / kg to the dose of the peptide of the present invention or a salt thereof. Examples include 500 mg / kg (adult), more preferably 1 μg / kg to 100 mg / kg (adult).

In addition, when the pharmaceutical preparation of the present invention is used in the form of an external preparation, an appropriate amount may be applied according to the size and symptoms of the affected part. For example, 0.01 μg in terms of peptide in the affected part of 1 to 10 cm ² What is necessary is just to apply so that it may become about -100 mg, Preferably it is 0.1 microgram-10 mg.

  In addition, the present invention provides a bacterial infection, sepsis, ringworm (eg, tinea pedis (so called athlete's foot), nail ringworm, hand ringworm, etc.) It is also possible to provide a method for preventing or treating diseases. The effective amount and administration method of the peptide of the present invention or a salt thereof in the treatment method can be appropriately set based on the above-mentioned dose and administration method.

  Furthermore, this invention can also provide the medical base material which carry | supported the peptide of this invention, or its salt on the pharmaceutically acceptable base material. As the base material that can be used in the present invention, biodegradable base materials such as alginic acid gel, collagen gel, lactic acid polymer, and cellulose are preferable. Such a substrate can be supported by physically or chemically bonding the peptide of the present invention or a salt thereof. Here, the collagen gel may be artificially produced, or may be naturally derived from animals such as cows and pigs, but it means to prevent animal-derived pathogen infection. Therefore, it is preferable to use artificial collagen.

  In addition, the polypeptide of the present invention or a salt thereof can be mixed with a biodegradable substrate to form a medical substrate. For example, the peptide of the present invention can be bound to an artificial collagen gel and used as a material for artificial skin.

  The present invention also provides a method for preventing or treating bacterial infection, characterized by applying such a medical substrate to an affected area. For example, the artificial skin carrying (or mixed with) the peptide of the present invention or a salt thereof may be used to cover a wound surface, a grafted portion by split-layer skin grafting, full-thickness skin grafting, or the like to prevent bacterial infection. it can. In the prevention or treatment method of bacterial infection using the medical base material, an appropriate application method can be selected according to the form of the medical base material.

The amount of the peptide of the present invention or a salt thereof carried or mixed on a medical substrate can be appropriately set based on the antibacterial activity, and is not particularly limited. For example, it is 0 per dry weight of the medical substrate. 0.01 to 10% by weight, preferably about 0.05 to 5% by weight, more preferably 0.1 to 2% by weight.
[Usage form to be added to the additive for the purpose of antiseptic]
Since the peptide of the present invention or a salt thereof has a wide range of antibacterial activity, it can be blended with additives such as pharmaceutical preparations, cosmetics, medical bases, foods and quasi drugs for the purpose of preserving.

  When the addition target is a pharmaceutical preparation, the form of the preparation to which the peptide of the present invention or a salt thereof is added is not particularly limited, and examples thereof include a solid preparation, a liquid preparation, and a semi-solid preparation.

  When the additive is a cosmetic, the form of the cosmetic is not particularly limited. For example, a cleansing agent, a skin cleanser, a massage agent, an ointment, a cream, a lotion, an oil, a pack, a face wash, A lotion, a milky lotion, a jelly, etc. are mentioned.

  Conventionally, parabens and the like have been widely used as antibacterial agents and preservatives, but there has been a problem such as annoying odors during use. When the peptide of the present invention is formulated as an antibacterial agent (or preservative), there is no such odor. In addition, the peptide of the present invention is hardly affected by heat, salt concentration, etc., and can maintain stable antibacterial activity for a long time even in cosmetics. Therefore, the peptide of the present invention or a salt thereof can be used in place of a conventional antibacterial agent or preservative.

  Further, when the object to be added is a medical base material, the peptide of the present invention or a salt thereof is bound to a biodegradable base material such as an alginate gel, an artificial collagen gel, a lactic acid polymer, cellulose, or the like. It can be used by mixing with a degradable substrate.

  In the case where the peptide of the present invention or a salt thereof is blended in the above-mentioned addition target for the purpose of preserving or the like, the amount to be added is 0.01 to 10% by weight, preferably 0.05 to 5% in the addition target in terms of peptide. %, More preferably 0.1 to 2% by weight. At this time, the peptide of this invention or its salt can be used individually by 1 type or in combination of 2 or more types. The peptide of this invention or its salt can be mix | blended with the said addition target according to a conventionally well-known method.

Hereinafter, the present invention will be described specifically by way of examples. In addition, this invention is not limited by these Examples.
[Example 1]
A peptide having the amino acid sequence represented by (Pro Hyp Gly) _3- (Lys Ile Gly) _3- (Pro Hyp Gly) ₄ (SEQ ID NO: 1) was synthesized by a solid phase synthesis method using an automatic peptide synthesizer. . That is, a styrene-divinylbenzene copolymer containing 4- (Nα-9- (fluorenylmethoxycarbonyl) -glycine) -oxymethyl-phenoxy-methyl group at a rate of 0.65 mmol / g (resin) [styrene and divinyl Amino acid corresponding sequentially from the carboxyl terminus to the amino terminus of the target peptide using 0.1 millimole of granular resin (HMP glycine, manufactured by Applied Biosystems, USA) consisting of 99: 1 benzene molar ratio Were combined.

In binding reaction, as the amino acid, Applied Biosystems (USA) for N ^{alpha-9-(fluorenyl} methoxy carbonyl) -L- proline [Fmoc-proline], N ^{alpha-9-(fluorenyl} methoxy carbonyl) -N ^epsilon - butoxycarbonyl -L- lysine [Fmoc ^lysine], N α -9- (fluorenyl methoxy carbonyl) -L- isoleucine [Fmoc ^isoleucine], N α -9- (fluorenyl methoxy carbonyl) - glycine [Fmoc-glycine], BACHEM Inc. of Germany made N ^{alpha-9-(fluorenyl} methoxy carbonyl) -O- butoxycarbonyl -L- hydroxyproline [Fmoc hydroxyproline], using one mmole respectively for each coupling step .

  The obtained peptide resin was treated with 10 ml of trifluoroacetic acid containing 5% water for 3 hours. The resulting solution was added to diethyl ether, and the resulting precipitate was further washed several times with diethyl ether to deprotect the peptide and desorb from the resin. The crude product was purified with a PD10 column (GE Healthcare Bioscience) to obtain a peptide. The purified peptide thus obtained was subjected to HPLC [AKTA explorer10XT system manufactured by GE Healthcare Biosciences Co., Ltd., column: Novapack C18 manufactured by Millipore Waters Co., Ltd., 3.9 mmφ × 150 mm, mobile phase: acetonitrile containing 0.05% by volume of trifluoroacetic acid. And a mixed solvent of acetonitrile and water (acetonitrile concentration was linearly changed from 5 vol% to 50 vol% over 30 minutes), flow rate 1.0 ml / min], a single peak was shown at 12.96 min. It was done.

  The obtained peptide is gel permeation chromatography (GPC; manufactured by GE Healthcare Biosciences, AKTApurifier system, column: Supedex peptide 10 / 30GL, flow rate: 0.5 mL / min, eluent: 150 mM NaCl When subjected to 10 mM phosphate buffer (pH 7.4)), a main peak was observed at an elution volume of 10.18 mL, and a minor peak was observed near 9 mL. Further, when the circular dichroism spectrum (CD) of the obtained peptide was measured, a positive cotton effect was observed at 223 nm and a negative cotton effect was observed at 199 nm, confirming that a triple helical structure was included. .

[Examples 2 and 3]
(Pro Hyp Gly) ₃ -Lys Ile Gly Pro Hyp Gly Lys Ile Gly- (Pro Hyp Gly) ₄ (Example 2), (Pro Hyp Gly) ₄ A peptide (Example 3) having an amino acid sequence represented by-(Lys Ile Gly) _2- (Pro Hyp Gly) ₄ (SEQ ID NO: 3) was synthesized by a solid phase synthesis method using an automatic peptide synthesizer.

That is, in Example 2, styrene-divinylbenzene copolymer containing 4- (Nα-9- (fluorenylmethoxycarbonyl) -glycine) -oxymethyl-phenoxy-methyl group at a rate of 0.65 mmol / g (resin). 4- (2 ′, 4′-dimethoxyphenyl-fluorenyl) instead of a granular resin (HMP glycine, manufactured by Applied Biosystems, USA) composed of a polymer [constitutive molar ratio of styrene and divinylbenzene: 99: 1] Granular resin comprising a styrene-divinylbenzene copolymer having a methoxycarbonyl) -aminomethyl) -phenoxyacetamido-ethyl group at a ratio of 0.62 mmol / g (resin) [constitutive molar ratio of styrene and divinylbenzene: 99 to 1] Synthesis was performed in the same manner as in Example 1 except that [Fmoc amide resin, manufactured by Applied Biosystems, USA] was used. Desorption from deprotection and resin peptide was subjected to purification. Also in Example 3, the peptide was prepared according to the same method as in Example 1.

In HPLC, Example 2 showed a single peak at 11.73 min. In Example 3, a single peak was shown at 12.19 min.

  In GPC, in Example 2, a major peak was observed at 10.22 mL and a minor peak was observed at 8.81 mL. In Example 3, a main peak was observed at 10.16 mL and a minor peak was observed at 8.74 mL.

  In CD, in Example 2, a positive cotton effect was observed at 225 nm and a negative cotton effect was observed at 199 nm. In Example 3, a positive cotton effect was observed at 225 nm and a negative cotton effect was observed at 199 nm. From this result, it was confirmed that the peptides of Examples 2 and 3 contain a triple helical structure.

[Comparative Examples 1 and 2]
A peptide consisting of the amino acid sequence represented by (Pro Hyp Gly) ₄ -Lys Ile Gly- (Pro Hyp Gly) ₅ (SEQ ID NO: 7) (Comparative Example 1) was synthesized in the same manner as in Example 1, and Deprotection, desorption from the resin, and purification were performed.

  The obtained peptide of Comparative Example 1 showed peaks at 11.03 min and 11.44 min on HPLC. In GPC, a major peak was observed at 10.61 mL and a minor peak was observed at 9.07 mL. Further, in CD, a positive cotton effect was observed at 224 nm and a negative cotton effect was observed at 199 nm, and it was confirmed that a triple helical structure was included.

A peptide consisting of the amino acid sequence represented by (Pro Hyp Gly) ₁₀ (SEQ ID NO: 8) (Comparative Example 2) was purchased from Peptide Institute.

  In GPC, a major peak was observed at 8.81 mL and a minor peak at 10.14 mL. In CD, a positive cotton effect was observed at 225 nm and a negative cotton effect was observed at 197 nm, confirming the inclusion of a triple helical structure.

[Test Example 1]
<Preparation of bacterial membrane model liposome>
A methylene chloride / methanol mixed solution containing 0.7 mg of dipalmitoyl phosphatidylglycerol (DPPG: Sigma P9789) was placed in a brown glass vial, and nitrogen gas was blown to form a lipid film on the inner wall of the vial. 1 mL of a 1 mM EDTA / TBS solution containing carboxyfluorescein (CF: Sigma C7153) at a concentration of 20 mM was added to a glass vial, and stirred well with a vortex mixer until the lipid membrane was peeled off from the inner wall of the vial. Next, ultrasonic treatment was performed at 60 ° C. for 20 minutes. Further, a 3 to 5 mL (preferably 3 to 4 mL) fraction was fractionated with a PD10 column (GE Healthcare Bioscience) to obtain a liposome solution.

<Production of mammalian membrane model liposome>
A liposome composed of phosphatidylcholine (PC: Sigma P2772 Type XI-E) was obtained in the same manner as above except that the ultrasonic treatment was performed at room temperature.

<Antimicrobial evaluation>
The peptides of Examples 1-3 and Comparative Examples 1 and 2 were added to 200 μL of the obtained DPPG liposome and PC liposome diluted 10-fold with PBS so that the final concentration would be 0.25 to 0.005 mM. And left at room temperature for 15 minutes. Thereafter, the amount of CF in the supernatant obtained by centrifugation at 12,000 rpm for 5 minutes was measured at an excitation wavelength of 485 nm and a fluorescence wavelength of 535 nm. The amount of CF leakage due to each peptide was calculated assuming that the fluorescence when PBS was added instead of the peptide solution was 0% and the fluorescence when Triton X-100 having a final concentration of 0.1% was added was 100%.

  For the bacterial membrane model DPPG liposome, the peptide of Example 1 shows 20% CF leakage at 50 μM, the peptide of Example 2 shows 11% at 125 μM, and Example 3 shows 3% CF leakage at 25 μM. It was. No leakage of CF was observed at any concentration against PC liposomes of mammalian cell membrane model, indicating that bacteria-selective antibacterial activity was expressed (see FIG. 1).

  In contrast, in the peptides of Comparative Examples 1 and 2, no CF leakage was observed in any concentration range for any liposome.

[Example 4]
A peptide having the amino acid sequence represented by (Pro Hyp Gly) _3- (Lys Hyp Gly Lys Leu Gly) _2- (Pro Hyp Gly) ₃ (SEQ ID NO: 4), wherein the carboxyl group at the carboxy terminus is substituted with an amide group That is, (Pro Hyp Gly) _3- (Lys Hyp Gly Lys Leu Gly) _2- (Pro Hyp Gly) ₃ -NH ₂ was synthesized by a solid phase synthesis method using an automatic peptide synthesizer.

That is, styrene-divinylbenzene copolymer containing 4- (2 ′, 4′-dimethoxyphenyl-fluorenylmethoxycarbonyl) -aminomethyl) -phenoxyacetamido-ethyl group at a rate of 0.62 mmol / g (resin) Using 0.1 mmol of a granular resin (Fmoc amide resin, manufactured by Applied Biosystems, USA) composed of a polymer (constituent molar ratio of styrene and divinylbenzene: 99 to 1) from the carboxyl terminal to the amino terminal of the target peptide. The corresponding amino acids were combined sequentially. In the binding reaction, N ^α -9- (fluorenylmethoxycarbonyl) -L-proline [Fmoc proline], N ^α -9- (fluorenylmethoxycarbonyl) -N manufactured by Applied Biosystems, Inc. ^epsilon - butoxycarbonyl -L- lysine [Fmoc ^lysine], N α -9- (fluorenyl methoxy carbonyl) -L- leucine ^{[Fmoc-leucine],} N α -9- (fluorenyl methoxy carbonyl) - glycine [Fmoc glycine], a Germany BACHEM Inc. N ^{alpha-9-(fluorenyl} methoxy carbonyl) -O- butoxycarbonyl -L- hydroxyproline [Fmoc hydroxyproline], using one mmole respectively for each coupling step.

  The obtained peptide resin was treated with 10 ml of trifluoroacetic acid containing 5% water for 3 hours. The resulting solution was added to diethyl ether, and the resulting precipitate was further washed several times with diethyl ether to deprotect the peptide and desorb from the resin. The crude product was purified with a PD10 column (GE Healthcare Bioscience) to obtain a peptide. The purified peptide thus obtained was HPLC [AKTA explorer 10XT system manufactured by GE Healthcare Biosciences Co., Ltd., column: Novopak C18 3.9 mmφ × 150 mm manufactured by Millipore Waters Co., Ltd., mobile phase: acetonitrile containing 0.05% by volume of trifluoroacetic acid. And a mixed solvent of acetonitrile and water (acetonitrile concentration was linearly changed from 5 vol% to 50 vol% over 30 minutes), flow rate of 1.0 ml / min], a single peak was observed at 10.7 min. Indicated.

  The obtained peptide is gel permeation chromatography (GPC; manufactured by GE Healthcare Biosciences, AKTApurifier system, column: Supedex peptide 10/30 GL, flow rate: 0.5 mL / min, eluent: 150 mM NaCl When subjected to 10 mM phosphate buffer (pH 7.4), a main peak was observed at an elution volume of 10.1 mL, and a minor peak was observed near 7.5 mL. Further, when the circular dichroism spectrum (CD) of the obtained peptide was measured, a positive cotton effect was observed at 223 nm and a negative cotton effect was observed at 200 nm, and it was confirmed that a triple helical structure was included.

[Examples 5 and 6]
(Pro Hyp Gly) _2- (Lys Leu Gly Lys Hyp Gly) _3- (Pro Hyp Gly) ₂ (SEQ ID NO: 5) A peptide in which the carboxyl group at the carboxy terminus is substituted with an amide group (Pro Hyp Gly) _2- (Lys Leu Gly Lys Hyp Gly) _3- (Pro Hyp Gly) ₂ -NH ₂ (Example 5),
Pro Hyp Gly- (Lys Leu Gly Lys Hyp Gly) ₄ -Pro Hyp Gly (SEQ ID NO: 6), a peptide in which the carboxyl group at the carboxy terminus is substituted with an amide group, ie, Pro Hyp Gly- (Lys Leu Gly Lys Hyp Gly) _4- Pro Hyp Gly-NH ₂ (Example 6) was synthesized in the same manner as in Example 4 by solid phase synthesis using an automatic peptide synthesizer, and peptide deprotection and resin were synthesized. From and purified.

  In HPLC, Example 5 showed a single peak at 10.2 min and Example 6 showed a single peak at 10.1 min. In GPC, in Example 5, a main peak was observed at 10.1 mL and a minor peak was observed at 7.5 mL, and in Example 6, a major peak was observed at 10.1 mL and a minor peak was observed at 7.5 mL. In CD, a positive cotton effect was observed at 222 nm in Example 5, a negative cotton effect at 198 nm, a positive cotton effect at 220 nm, and a negative cotton effect at 197 nm in Example 6. It was confirmed to contain a heavy helical structure.

[Comparative Example 3]
(Pro Hyp Gly) ₃ -Arg Leu Gly Pro Hyp Gly Arg Leu Gly- (Pro Hyp Gly) ₄ (SEQ ID NO: 9) peptide having a carboxy-terminal carboxyl group substituted with an amide group That is, (Pro Hyp Gly) ₃ -Arg Leu Gly Pro Hyp Gly Arg Leu Gly- (Pro Hyp Gly) ₄ -NH ₂ (Comparative Example 3) was synthesized in the same manner as in Example 1, peptide deprotection and resin From and purified. However, in the synthesis step, made in the US Applied Biosystems N ^{alpha-9-(fluorenyl} methoxy carbonyl) -N ^G - (2,2,5,7,8-pentamethyl chroman-6-sulfonyl) - 1 mmol of L-arginine [Fmoc arginine] was used for each binding step.

  A peak was shown at 12.2 min on HPLC. GPC showed a main peak at 10.6 mL and a minor peak at 7.5 mL. In CD, a positive cotton effect was observed at 222 nm and a negative cotton effect was observed at 199 nm, confirming that a triple helical structure was included.

  The antimicrobial activity of the peptides of Examples 4, 5, 6 and Comparative Example 3 was evaluated according to the method shown in Test Example 1 above.

  For the bacterial membrane model DPPG liposome, the peptide of Example 4 shows 10% CF leakage at 18 μM, the peptide of Example 5 shows 17% CF at 3.5 μM, and Example 6 shows 18% CF leakage at 7 μM. It was. No leakage of CF was observed at any concentration against PC liposomes of mammalian cell membrane model, indicating that bacteria-selective antibacterial activity was expressed (see FIG. 1).

  In contrast, with the peptide of Comparative Example 3, no CF leakage was observed in any concentration range for any liposome.

  As is clear from the above results, the novel peptide provided by the present invention exhibits selective antibacterial activity against bacterial cell membranes, and is useful for the treatment of bacterial infections, the suppression of the propagation of germs, and the like.

[Prescription example]
Although the formulation example of the cosmetics using the peptide of this invention or its salt is shown below, this invention is not limited to this.
Formulation Example 1
The following components were mixed to obtain a moisturizing liquid.
Artificial collagen aqueous solution (0.3%) 99.5 parts 0.5% peptide 1% aqueous solution of SEQ ID NO: 1

  The peptide of the present invention is not easily affected by external factors such as heat and salt concentration, and can stably exhibit excellent antibacterial activity. Therefore, it is prepared as a pharmaceutical preparation to prevent bacterial infection, sepsis, etc. Or it can be used for treatment.

  The polypeptide of the present invention can also be used by binding to a medical substrate. For example, artificial skin produced by binding the peptide of the present invention to an artificial collagen gel or the like and using this as a raw material can prevent bacterial infection of the transplanted part.

  Furthermore, since the peptide of the present invention is hypoallergenic to humans and can maintain stable antibacterial properties, it can also be formulated as an antibacterial agent in cosmetics. Some of the conventional antibacterial agents blended in cosmetics are annoying. However, the polypeptide of the present invention can be suitably used as an antibacterial agent blended in cosmetics without problems such as odor.

SEQ ID NO: 1 is an artificial peptide obtained in Example 1.
SEQ ID NO: 2 is an artificial peptide obtained in Example 2.
SEQ ID NO: 3 is an artificial peptide obtained in Example 3.
SEQ ID NO: 4 is an artificial peptide obtained in Example 4.
SEQ ID NO: 5 is an artificial peptide obtained in Example 5.
SEQ ID NO: 6 is an artificial peptide obtained in Example 6.
SEQ ID NO: 7 is an artificial peptide obtained in Comparative Example 1.
SEQ ID NO: 8 is the artificial peptide of Comparative Example 2.
SEQ ID NO: 9 is an artificial peptide obtained in Comparative Example 3.

Claims (16)

A peptide having antibacterial activity represented by the following (1) or (2) or a salt thereof;
(1) A peptide consisting of an amino acid sequence represented by the following general formula (I):
(Pro Hyp Gly) _n -{(Lys X Gly) _m- (Pro Hyp Gly) _l- (Lys X Gly) _p } _α- (Pro Hyp Gly) _r (I)
(Here, in Formula (I), n is an integer of 1-6, m is an integer of 1-4, l is an integer of 0-7, p is an integer of 1-4, r is an integer of 1 to 6, and α is an integer of 1 to 6.
X is any amino acid residue selected from Ile, Leu and Hyp,
The m + p Xs may be the same or different from each other.
n + m + l + p + r = 4-15.
However, when l = 0, m + p ≧ 2 is satisfied,
When α ≧ 2, m = 1, l = 0, and p = 1 are satisfied. ),
(2) The amino acid sequence represented by the general formula (I) consists of an amino acid sequence in which 1 to 3 amino acid residues other than Lys, Gly and X are substituted,
The substitution is
Pro to Hyp replacement, or
Hyp to Pro replacement,
A peptide which is at least one selected from. In said (1), n is an integer of 3-6, m is an integer of 1-4, l is an integer of 0-7, p is an integer of 1-4, r is 3- An integer of 6, α is 1,
X is Ile or Leu,
m + p Xs may be the same or different from each other,
n + m + l + p + r = 8-15,
When l = 0, m + p ≧ 2 is satisfied.
The peptide according to claim 1 or a salt thereof. The peptide or salt thereof according to claim 1, which is represented by the following (1-1) or (2-1);
(1-1) A peptide comprising an amino acid sequence represented by any one of (i) to (vi) below:
(i) (Pro Hyp Gly) _3- (Lys Ile Gly) _3- (Pro Hyp Gly) ₄ (SEQ ID NO: 1)
(ii) (Pro Hyp Gly) ₃ -Lys Ile Gly Pro Hyp Gly Lys Ile Gly- (Pro Hyp Gly) ₄ (SEQ ID NO: 2)
(iii) (Pro Hyp Gly) 4 - (Lys Ile Gly) 2 - (Pro Hyp Gly) 4 ( SEQ ID NO: 3)
(iv) (Pro Hyp Gly) _3- (Lys Hyp Gly Lys Leu Gly) _2- (Pro Hyp Gly) ₃ (SEQ ID NO: 4),
(v) (Pro Hyp Gly) _2- (Lys Leu Gly Lys Hyp Gly) _3- (Pro Hyp Gly) ₂ (SEQ ID NO: 5),
(vi) Pro Hyp Gly- (Lys Leu Gly Lys Hyp Gly) ₄ -Pro Hyp Gly (SEQ ID NO: 6), (2-1) 1-3 amino acids in the amino acid sequences (i) to (vi) A peptide comprising a substituted amino acid sequence,
The substitution is
Replacement of Pro to Hyp,
Hyp to Pro replacement,
Substitution of Leu to Ile or Val, and
At least one substitution selected from the group consisting of substitution of Ile or Val to Leu,
Peptides having antibacterial activity and salts thereof. The peptide according to any one of claims 1 to 3, wherein the amino terminal hydrogen of the amino acid sequence is substituted with an acetyl group, a methoxycarbonyl group, a butoxycarbonyl group, or a peptide having 1-3 residues, and a pharmaceutically acceptable peptide thereof. Salt. The peptide according to any one of claims 1 to 4, wherein the carboxyl group at the carboxy terminus of the amino acid sequence is substituted with an amide group, a methyl ester group, a butyl ester group, or a peptide having 1 to 3 residues, and pharmaceutically Acceptable salt. The peptide according to any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof, comprising a collagen-like triple helix structure. The antibacterial agent which uses the peptide in any one of Claims 1-6, or its salt as an active ingredient. A pharmaceutical preparation comprising the peptide according to any one of claims 1 to 6 or a salt thereof, and a pharmaceutically acceptable carrier or base material. The pharmaceutical preparation according to claim 8, which is used for prevention or treatment of bacterial infection, fungal infection or sepsis. The medical base material with which the peptide in any one of Claims 1-6, or its salt is carry | supported by the pharmaceutically acceptable base material. The medical base material according to claim 10, wherein the pharmaceutically acceptable base material is at least one selected from the group consisting of alginate gel, collagen gel, lactic acid polymer, and cellulose. Cosmetics containing the peptide according to any one of claims 1 to 6 or a salt thereof. The medical base material for preventing or treating bacterial infection according to claim 10 or 11, which is applied to an affected area. Use of the peptide according to any one of claims 1 to 6 or a salt thereof for producing an antibacterial agent. Use of the peptide according to any one of claims 1 to 6 for producing a pharmaceutical preparation for preventing or treating bacterial infection, fungal infection or sepsis. Use of the peptide according to any one of claims 1 to 6 or a salt thereof for producing a medical substrate for preventing or treating bacterial infection.

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