JP6693728B2 - Novel functional peptide - Google Patents

Description

  The present invention relates to a novel functional peptide or derivative thereof. The present invention also relates to a pharmaceutical or cosmetic product containing the functional peptide.

  Glucosaminoglycan is a polysaccharide having an amino sugar in its molecule, and has a repeating structure of a disaccharide composed of uronic acid and glucosamine or galactosamine as a basic skeleton. Glucosaminoglycan can be divided into hyaluronic acid, chondroitin sulfate, deltaman sulfate, heparan sulfate, etc.For example, hyaluronic acid is widely present in the extracellular matrix in vivo such as joints, vitreous body, skin and brain, Chondroitin sulfate is known to exist in all tissues including the extracellular matrix of cartilage and connective tissues such as skin.

  These glycosaminoglycans interact with various cell growth factors and extracellular matrix components to control cell activities such as cell adhesion, migration, proliferation, differentiation, morphogenesis, and diseases such as inflammation and metastasis of cancer cells. Since it is known that it is also involved in biological phenomena, various studies have been conducted on the effect of glucosaminoglycan on the living body and its mechanism. Glucosaminoglycan is known to exhibit extremely high water retention capacity and viscoelasticity, and by utilizing this property, it is possible to treat various diseases in various fields including ophthalmology and dermatology. It is clinically applied as a medicine and preventive drug.

  For example, in the field of ophthalmology, the amount of tear secretion decreases, or the composition of tears changes even if the amount is sufficient, due to the recent increase in the use of contact lenses and the increased chances of watching monitors such as personal computers and games. By doing so, an increasing number of people have the symptoms of dry eyes, in which the power to moisturize the surface of the eyes is reduced (Non-Patent Document 1). An eye drop containing sodium hyaluronate, which is a kind of glucosaminoglycan, as an active ingredient is known as a drug for relieving such dry eyes. Sodium hyaluronate as an ethical drug is an intrinsic disease such as Sjogren's syndrome, Stevens-Johnson syndrome, and dry eye syndrome (dry eye), and extrinsic diseases caused by postoperative, drug-induced, trauma, contact lens wearing, etc. The indications are keratoconjunctival epithelial disorders associated with any of the above diseases. As described above, the sodium hyaluronate preparation has a wide range of application and is a useful medicine. However, since the instilled medicine runs off the cornea due to tears, it is necessary to inject it 5 to 6 times a day. Is left.

  Further, in the dermatological field, for example, external preparations containing a heparin-like substance, which is a kind of glucosaminoglycan, as an active ingredient are known, and sebum deficiency, progressive palmar keratoderma or atopic dermatitis, etc. Used in the treatment of. Also in the field of cosmetics, lotions containing hyaluronic acid, which is a kind of glucosaminoglycan, are commercially available. Also in these external preparations and cosmetics, if the active ingredient stays in the skin for a long time, the effect lasts for a long time.

Further, as described above, since glucosaminoglycans are widely present in connective tissues such as extracellular matrix and skin in the living body, not only as a therapeutic drug, but also glucosaminoglycine which already exists in the living body. It is also expected to be applied to the treatment of diseases using glycans. One example of such a disease is retinal detachment. Retinal detachment refers to a state in which the neural retina is detached from the retinal pigment epithelium. As a treatment method, there is a laser photocoagulation method. If only a retinal tear occurs, a laser beam is emitted from the pupil to the periphery of the retinal tear to strengthen the retinal adhesion around the rupture of the retinal aperture, thereby preventing retinal detachment that may occur in the future. If retinal detachment has already occurred, a surgical instrument is inserted into the vitreous body, and vitreous surgery is performed to reposition the retina from within the eye. However, in refractory cases, the retina re-exfoliates even after vitreous surgery. In this case, prevention of re-peeling can be expected by improving the adhesiveness between the retina and the retinal pigment epithelium. The extracellular matrix existing in the space between the neural retina and the retinal pigment epithelium, especially hyaluronic acid, which is a glucosaminoglycan, is abundant. It is considered that if this extracellular matrix is used to promote the adhesion of the retina, it will contribute to the increase in the success rate of vitreous surgery.

On the other hand, hyaluronic acid (HA) is a simple structure composed of repeating disaccharides of N-acetylglucosamine and glucuronic acid. Although many kinds of proteins (HABP: hyaluronan-binding protein) that bind to hyaluronan in vivo have been reported, the mode of binding to hyaluronan can be roughly divided into two. That is, among HABP, aggrecan (aggrecan), versican (also called PG-M), neurocan (neurocan), brevican (also called brevican, also called BEHAB), and other HABPs are about 100 amino acids in the N-terminal region. The spherical three-dimensional structure (G1 domain) has hyaluronic acid binding activity. On the other hand, HABPs such as SPACR (Sialoprotein Associated with Cones and Rods) and SPACRCAN (Sialoproteoglycan Associated with Rods) are nine amino acids in which B (X ₇ ) B is an amino acid composed of B (X ₇ ) B acid. (Non-patent document 2). In recent years, it was reported that a peptide created based on the G1 domain of versican assists the binding between hyaluronic acid and cornea and can be used as a new medical material (Patent Document 1), but B (X ₇ ) B There is no such report for the peptide created on the basis.

JP, 2004-154038, A

Ministry of Health, Labor and Welfare, "A Report on the Investigation of Candidate Ingredients Considered to be Possible to be Used as OTC Drugs among Active Ingredients", 2011, pp87-95 Masahiko Yoneda et al., Cell engineering, 2001, Volume 20, Issue 8, pp1114-1121

  An object of the present invention is to provide a functional peptide or a derivative thereof that promotes adhesion between living tissues or promotes adhesion between glucosaminoglycan administered as an active ingredient and living tissues.

As a result of intensive studies to solve such a problem, the present inventors have found that a peptide consisting of the following amino acids has a glucosaminoglycan-binding ability far exceeding that of a biologically-derived peptide, particularly hyaluronic acid-binding ability, hyaluronic acid. The present invention has been completed by finding that it exhibits a coadjunctive action between the cornea and between the retina and the retinal epithelium.
That is, the present invention relates to the following inventions.

[1] A peptide represented by the following general formula (1) or general formula (2), a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof (provided that they are represented by SEQ ID NOS: 3 to 6). Excluding peptides consisting of amino acid sequences).
^{Y 1 -X 1 -X 2 -X 3} -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -Y 2 ( SEQ ID NO: 1)
(1)
^{Y 1 - (X 1 -X 2} -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9) 2 -Y 2 ( SEQ ID NO: 2)
(2)
[In the formula,
X ¹ independently represents a basic amino acid, an acidic amino acid or a branched amino acid,
X ² independently represents an amide-containing amino acid or a basic amino acid,
X ³ and X ⁶ each independently represent a branched amino acid or a hydroxy-containing amino acid,
X ⁴ independently represents a branched amino acid, an aliphatic amino acid, an amide-containing amino acid or an acidic amino acid,
X ⁵ independently represents a branched amino acid,
X ⁷ independently represents an aliphatic amino acid or an amide-containing amino acid,
X ⁸ independently represents an aromatic amino acid,
X ⁹ independently represents a basic amino acid,
Y ¹ and Y ² do not exist at the same time, or simultaneously represent a sulfur-containing amino acid, or
Y ¹ and Y ² simultaneously represent a sulfur-containing amino acid, and the amino acids Y ¹ and Y ² form an intramolecular bond]
[2] A peptide represented by the general formula (1) or the general formula (2) according to the above [1], a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof, which is basic. The amino acid is Arg, His or Lys, the acidic amino acid is Asp or Glu, the branched amino acid is Leu, Ile or Val, the amido-containing amino acid is Asn or Gln, and the hydroxy-containing amino acid is Ser. Or Thr, the aliphatic amino acid is Ala or Gly, the aromatic amino acid is Phe, Trp or Tyr, and the sulfur-containing amino acid is Cys or Met. An acceptable salt or a solvate thereof.
[3] A peptide represented by the general formula (1) according to the above [1] or [2], a derivative thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof,
In the general formula (1),
X ¹ and X ⁹ each independently represent Arg or Lys,
X ² and X ⁷ independently of each other represent Asn or Gln,
X ³ , X ⁴ , X ⁵ and X ⁶ independently of each other represent Leu, Ile or Val,
X ⁸ represents Phe, Trp or Tyr,
Y ¹ and Y ² simultaneously represent Cys, and the amino acid forms a disulfide bond,
The peptide, its derivative, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[4] A peptide represented by the general formula (2) according to the above [1] or [2], a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof,
In the general formula (2),
X ¹ and X ⁹ each independently represent Arg or Lys,
X ² and X ⁷ independently of each other represent Asn or Gln,
X ³ , X ⁴ , X ⁵ and X ⁶ independently of each other represent Leu, Ile or Val,
X ⁸ represents Phe, Trp or Tyr,
Y ¹ and Y ² do not exist at the same time, or simultaneously represent Cys, or
Y ¹ and Y ² simultaneously represent Cys, and the amino acid forms a disulfide bond,
The peptide, its derivative, a pharmaceutically acceptable salt thereof, or a solvate thereof.
[5] The peptide according to [1] or [2] above, whose amino acid sequence is represented by SEQ ID NOs: 7 to 38, a derivative thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof.
[6] The above [1] or [2], wherein the amino acid sequence is represented by SEQ ID NO: 15, 27 or 28.
The peptide according to 1 above, a derivative thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[7] A medicament comprising as an active ingredient a peptide represented by the following general formula (1) or general formula (2), a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof.
^{Y 1 -X 1 -X 2 -X 3} -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -Y 2 ( SEQ ID NO: 1)
(1)
^{Y 1 - (X 1 -X 2} -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9) 2 -Y 2 ( SEQ ID NO: 2)
(2)
[In the formula,
X ¹ independently represents a basic amino acid, an acidic amino acid or a branched amino acid,
X ² independently represents an amide-containing amino acid or a basic amino acid,
X ³ and X ⁶ each independently represent a branched amino acid or a hydroxy-containing amino acid,
X ⁴ independently represents a branched amino acid, an aliphatic amino acid, an amide-containing amino acid or an acidic amino acid,
X ⁵ independently represents a branched amino acid,
X ⁷ independently represents an aliphatic amino acid or an amide-containing amino acid,
X ⁸ independently represents an aromatic amino acid,
X ⁹ independently represents a basic amino acid,
Y ¹ and Y ² do not exist at the same time, or simultaneously represent a sulfur-containing amino acid, or
Y ¹ and Y ² simultaneously represent a sulfur-containing amino acid, and the amino acids Y ¹ and Y ² form an intramolecular bond]
[8] The pharmaceutical agent according to [7] above, wherein the basic amino acid is Arg, His or Lys, the acidic amino acid is Asp or Glu, and the branched amino acid is Leu, Ile or Val, The amide-containing amino acid is Asn or Gln, the hydroxy-containing amino acid is Ser or Thr, the aliphatic amino acid is Ala or Gly, the aromatic amino acid is Phe, Trp or Tyr, and the sulfur-containing amino acid. Is Cys or Met.
[9] A medicament comprising the peptide represented by the general formula (1) according to [7] or [8] above, a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient. There
In the general formula (1),
X ¹ and X ⁹ each independently represent Arg or Lys,
X ² and X ⁷ independently of each other represent Asn or Gln,
X ³ , X ⁴ , X ⁵ and X ⁶ independently of each other represent Leu, Ile or Val,
X ⁸ represents Phe, Trp or Tyr,
Y ¹ and Y ² simultaneously represent Cys, and the amino acid forms a disulfide bond,
The medicine according to the above [7] or [8].
[10] A medicament comprising the peptide represented by the general formula (2) according to the above [7] or [8], a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient. There
In the general formula (2),
X ¹ and X ⁹ each independently represent Arg or Lys,
X ² and X ⁷ independently of each other represent Asn or Gln,
X ³ , X ⁴ , X ⁵ and X ⁶ independently of each other represent Leu, Ile or Val,
X ⁸ represents Phe, Trp or Tyr,
Y ¹ and Y ² do not exist at the same time, or simultaneously represent Cys, or
Y ¹ and Y ² simultaneously represent Cys, and the amino acid forms a disulfide bond,
The medicine according to the above [7] or [8].

[11] The above-mentioned [7] or [8], which comprises a peptide consisting of the amino acid sequences represented by SEQ ID NOs: 3 to 38, a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient. The medicine according to.
[12] The above [7] or [containing the peptide comprising the amino acid sequence represented by SEQ ID NO: 15, 27 or 28, a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient. 8] The medicine according to [8].
[13] The medicament according to any of [7] to [12] above, which is used for prevention and / or treatment of eye diseases.
[14] The medicine according to the above [13], wherein the eye disease is retinal detachment.
[15] The medicine according to [14] above, wherein the retinal disease is retinal detachment, diabetic retinopathy or macular degeneration.

[16] Administration to a patient in need of a therapeutically effective amount of a peptide represented by the following general formula (1) or general formula (2), a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof. A method for preventing and / or treating an eye disease, which comprises the step of:
^{Y 1 -X 1 -X 2 -X 3} -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -Y 2 ( SEQ ID NO: 1)
(1)
^{Y 1 - (X 1 -X 2} -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9) 2 -Y 2 ( SEQ ID NO: 2)
(2)
[In the formula,
X ¹ independently represents a basic amino acid, an acidic amino acid or a branched amino acid,
X ² independently represents an amide-containing amino acid or a basic amino acid,
X ³ and X ⁶ each independently represent a branched amino acid or a hydroxy-containing amino acid,
X ⁴ independently represents a branched amino acid, an aliphatic amino acid, an amide-containing amino acid or an acidic amino acid,
X ⁵ independently represents a branched amino acid,
X ⁷ independently represents an aliphatic amino acid or an amide-containing amino acid,
X ⁸ independently represents an aromatic amino acid,
X ⁹ independently represents a basic amino acid,
Y ¹ and Y ² do not exist at the same time, or simultaneously represent a sulfur-containing amino acid, or
Y ¹ and Y ² simultaneously represent a sulfur-containing amino acid, and the amino acids Y ¹ and Y ² form an intramolecular bond]
[17] A peptide represented by the following general formula (1) or general formula (2), a derivative thereof, or a pharmaceutically acceptable thereof, for producing a medicament for preventing and / or treating an eye disease. Use of salts or solvates thereof.
^{Y 1 -X 1 -X 2 -X 3} -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -Y 2 ( SEQ ID NO: 1)
(1)
^{Y 1 - (X 1 -X 2} -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9) 2 -Y 2 ( SEQ ID NO: 2)
(2)
[In the formula,
X ¹ independently represents a basic amino acid, an acidic amino acid or a branched amino acid,
X ² independently represents an amide-containing amino acid or a basic amino acid,
X ³ and X ⁶ each independently represent a branched amino acid or a hydroxy-containing amino acid,
X ⁴ independently represents a branched amino acid, an aliphatic amino acid, an amide-containing amino acid or an acidic amino acid,
X ⁵ independently represents a branched amino acid,
X ⁷ independently represents an aliphatic amino acid or an amide-containing amino acid,
X ⁸ independently represents an aromatic amino acid,
X ⁹ independently represents a basic amino acid,
Y ¹ and Y ² do not exist at the same time, or simultaneously represent a sulfur-containing amino acid, or
Y ¹ and Y ² simultaneously represent a sulfur-containing amino acid, and the amino acids Y ¹ and Y ² form an intramolecular bond]
[18] (A) A peptide represented by the following general formula (1) or general formula (2), a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof,
(B) A medicine containing glucosaminoglycan or a salt thereof.
^{Y 1 -X 1 -X 2 -X 3} -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -Y 2 ( SEQ ID NO: 1)
(1)
^{Y 1 - (X 1 -X 2} -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9) 2 -Y 2 ( SEQ ID NO: 2)
(2)
[In the formula,
X ¹ independently represents a basic amino acid, an acidic amino acid or a branched amino acid,
X ² independently represents an amide-containing amino acid or a basic amino acid,
X ³ and X ⁶ each independently represent a branched amino acid or a hydroxy-containing amino acid,
X ⁴ independently represents a branched amino acid, an aliphatic amino acid, an amide-containing amino acid or an acidic amino acid,
X ⁵ independently represents a branched amino acid,
X ⁷ independently represents an aliphatic amino acid or an amide-containing amino acid,
X ⁸ independently represents an aromatic amino acid,
X ⁹ independently represents a basic amino acid,
Y ¹ and Y ² do not exist at the same time, or simultaneously represent a sulfur-containing amino acid, or
Y ¹ and Y ² simultaneously represent a sulfur-containing amino acid, and the amino acids Y ¹ and Y ² form an intramolecular bond]
[19] The drug according to [18] above, wherein the basic amino acid is Arg, His or Lys, the acidic amino acid is Asp or Glu, and the branched amino acid is Leu, Ile or Val, The amide-containing amino acid is Asn or Gln, the hydroxy-containing amino acid is Ser or Thr, the aliphatic amino acid is Ala or Gly, the aromatic amino acid is Phe, Trp or Tyr, and the sulfur-containing amino acid. Is Cys or Met.

[20] The component (A) is the peptide represented by the general formula (1) described in [18] or [19], a derivative thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof. hand,
In the general formula (1),
X ¹ and X ⁹ each independently represent Arg or Lys,
X ² and X ⁷ independently of each other represent Asn or Gln,
X ³ , X ⁴ , X ⁵ and X ⁶ independently of each other represent Leu, Ile or Val,
X ⁸ represents Phe, Trp or Tyr,
Y ¹ and Y ² simultaneously represent Cys, and the amino acid forms a disulfide bond,
The medicament according to [18] or [19] above.

[21] The component (A) is a peptide represented by the general formula (2) described in [18] or [19], a derivative thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof. ,
In the general formula (2),
X ¹ and X ⁹ each independently represent Arg or Lys,
X ² and X ⁷ independently of each other represent Asn or Gln,
X ³ , X ⁴ , X ⁵ and X ⁶ independently of each other represent Leu, Ile or Val,
X ⁸ represents Phe, Trp or Tyr,
Y ¹ and Y ² do not exist at the same time, or simultaneously represent Cys, or
Y ¹ and Y ² simultaneously represent Cys, and the amino acid forms a disulfide bond,
The medicament according to [18] or [19] above.

[22] The above [18] or [19], wherein the component (A) is a peptide consisting of the amino acid sequences represented by SEQ ID NOS: 3 to 38, a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof. ] The pharmaceutical as described in.
[23] The above-mentioned [18], wherein the component (A) is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 15, 27 or 28, a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof. The medicine according to [19].
[24] The medicine according to any of [18] to [23] above, wherein the component (B) is hyaluronic acid or a salt thereof.
[25] The medicament according to any of [18] to [24] above, which is used for prevention and / or treatment of eye diseases or skin diseases.

[26] The medicine according to the above [25], wherein the eye disease is keratoconjunctival epithelial disorder.
[27] The medicine according to the above [26], wherein the keratoconjunctival epithelial disorder is dry eye, keratoconjunctivitis sicca, punctate superficial keratopathy, corneal erosion or corneal ulcer.
[28] The drug according to the above [25], wherein the skin disease is sebum deficiency, progressive palmar keratoderma or atopic dermatitis.
[29] The medicament according to any of [18] to [28] above, which is in the form of a pharmaceutical composition containing the component (A) and the component (B) in the same preparation.
[30] The medicament according to any one of [18] to [28] above, which is in the form of a kit formulation containing the component (A) and the component (B) in separate formulations.

[31] (A) A therapeutically effective amount of a peptide represented by the following general formula (1) or general formula (2), a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof,
(B) A method for preventing and / or treating an eye disease or a skin disease, which comprises the step of administering a therapeutically effective amount of glucosaminoglycan or a salt thereof to a patient in need thereof at the same time or separately at different times.
^{Y 1 -X 1 -X 2 -X 3} -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -Y 2 ( SEQ ID NO: 1)
(1)
^{Y 1 - (X 1 -X 2} -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9) 2 -Y 2 ( SEQ ID NO: 2)
(2)
[In the formula,
X ¹ independently represents a basic amino acid, an acidic amino acid or a branched amino acid,
X ² independently represents an amide-containing amino acid or a basic amino acid,
X ³ and X ⁶ each independently represent a branched amino acid or a hydroxy-containing amino acid,
X ⁴ independently represents a branched amino acid, an aliphatic amino acid, an amide-containing amino acid or an acidic amino acid,
X ⁵ independently represents a branched amino acid,
X ⁷ independently represents an aliphatic amino acid or an amide-containing amino acid,
X ⁸ independently represents an aromatic amino acid,
X ⁹ independently represents a basic amino acid,
Y ¹ and Y ² do not exist at the same time, or simultaneously represent a sulfur-containing amino acid, or
Y ¹ and Y ² simultaneously represent a sulfur-containing amino acid, and the amino acids Y ¹ and Y ² form an intramolecular bond]
[32] A peptide represented by the following general formula (1) or general formula (2), a derivative thereof, for producing a medicament for the prevention and / or treatment of an eye disease or a skin disease: Use of the combination of the pharmaceutically acceptable salt or the solvate thereof of (B) Glucosaminoglycan or a salt thereof.
^{Y 1 -X 1 -X 2 -X 3} -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -Y 2 ( SEQ ID NO: 1)
(1)
^{Y 1 - (X 1 -X 2} -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9) 2 -Y 2 ( SEQ ID NO: 2)
(2)
[In the formula,
X ¹ independently represents a basic amino acid, an acidic amino acid or a branched amino acid,
X ² independently represents an amide-containing amino acid or a basic amino acid,
X ³ and X ⁶ each independently represent a branched amino acid or a hydroxy-containing amino acid,
X ⁴ independently represents a branched amino acid, an aliphatic amino acid, an amide-containing amino acid or an acidic amino acid,
X ⁵ independently represents a branched amino acid,
X ⁷ independently represents an aliphatic amino acid or an amide-containing amino acid,
X ⁸ independently represents an aromatic amino acid,
X ⁹ independently represents a basic amino acid,
Y ¹ and Y ² do not exist at the same time, or simultaneously represent a sulfur-containing amino acid, or
Y ¹ and Y ² simultaneously represent a sulfur-containing amino acid, and the amino acids Y ¹ and Y ² form an intramolecular bond]

[33] (A) A peptide represented by the following general formula (1) or general formula (2), a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof,
(B) A cosmetic containing glucosaminoglycan or a salt thereof.
^{Y 1 -X 1 -X 2 -X 3} -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -Y 2 ( SEQ ID NO: 1)
(1)
^{Y 1 - (X 1 -X 2} -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9) 2 -Y 2 ( SEQ ID NO: 2)
(2)
[In the formula,
X ¹ independently represents a basic amino acid, an acidic amino acid or a branched amino acid,
X ² independently represents an amide-containing amino acid or a basic amino acid,
X ³ and X ⁶ each independently represent a branched amino acid or a hydroxy-containing amino acid,
X ⁴ independently represents a branched amino acid, an aliphatic amino acid, an amide-containing amino acid or an acidic amino acid,
X ⁵ independently represents a branched amino acid,
X ⁷ independently represents an aliphatic amino acid or an amide-containing amino acid,
X ⁸ independently represents an aromatic amino acid,
X ⁹ independently represents a basic amino acid,
Y ¹ and Y ² do not exist at the same time, or simultaneously represent a sulfur-containing amino acid, or
Y ¹ and Y ² simultaneously represent a sulfur-containing amino acid, and the amino acids Y ¹ and Y ² form an intramolecular bond]
[34] The cosmetic product according to [33], wherein the basic amino acid is Arg, His or Lys, the acidic amino acid is Asp or Glu, and the branched amino acid is Leu, Ile or Val, The amide-containing amino acid is Asn or Gln, the hydroxy-containing amino acid is Ser or Thr, the aliphatic amino acid is Ala or Gly, the aromatic amino acid is Phe, Trp or Tyr, and the sulfur-containing amino acid. Is Cys or Met.

[35] The component (A) is the peptide represented by the general formula (1) described in [33] or [34], a derivative thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof. hand,
In the general formula (1),
X ¹ and X ⁹ each independently represent Arg or Lys,
X ² and X ⁷ independently of each other represent Asn or Gln,
X ³ , X ⁴ , X ⁵ and X ⁶ independently of each other represent Leu, Ile or Val,
X ⁸ represents Phe, Trp or Tyr,
Y ¹ and Y ² simultaneously represent Cys, and the amino acid forms a disulfide bond,
The cosmetic product according to [33] or [34].

[36] The component (A) is the peptide represented by the general formula (2) described in [33] or [34], a derivative thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof. ,
In the general formula (2),
X ¹ and X ⁹ each independently represent Arg or Lys,
X ² and X ⁷ independently of each other represent Asn or Gln,
X ³ , X ⁴ , X ⁵ and X ⁶ independently of each other represent Leu, Ile or Val,
X ⁸ represents Phe, Trp or Tyr,
Y ¹ and Y ² do not exist at the same time, or simultaneously represent Cys, or
Y ¹ and Y ² simultaneously represent Cys, and the amino acid forms a disulfide bond,
The cosmetic product according to [33] or [34].

[37] The above [33] or [34], wherein the component (A) is a peptide consisting of the amino acid sequences represented by SEQ ID NOs: 3 to 38, a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof. ] The cosmetics described in.
[38] The component [A] is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 15, 27 or 28, a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof, [33] to The cosmetic product according to any one of [37].
[39] The cosmetic product according to the above [33] or [34], wherein the component (B) is hyaluronic acid or a salt thereof.
[40] The cosmetic product according to any of [33] to [39] above, which is in the form of a composition containing the component (A) and the component (B) in the same preparation.
[41] The cosmetic product according to any of [33] to [39] above, which is in the form of a kit containing the component (A) and the component (B) in separate formulations.

  The peptide of the present invention, a derivative thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof has a binding ability to glucosaminoglycan far superior to that of a peptide derived from a living body. Glucosaminoglycan is present in all connective tissues of the living body, and thus the peptide of the present invention can be used for treatment or prevention of diseases such as retinal detachment that can be treated by improving the connectivity between living tissues. Can be used.

  Further, when the peptide of the present invention and glucosaminoglycan are administered in combination, glucosaminoglycan, which is an active ingredient, is strong at the administration site as glucosaminoglycan existing in the living tissue via the peptide of the present invention. Join. Therefore, the glucosaminoglycan administered as an active ingredient remains at the application site for a long period of time, and as a result, the pharmacological effect of the glucosaminoglycan is significantly longer than that of the conventional one. In particular, the peptide of the present invention, a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof can suppress the outflow of hyaluronic acid from the cornea, and further can assist the binding between hyaluronic acid and cornea. Therefore, a highly durable drug can be provided.

Further, the peptide of the present invention is a glycosaminoglycan other than hyaluronic acid, for example, chondroitin (chn), chondroitin sulfate A (CSA), chondroitin sulfate C (CSC), dermatan sulfate (DS), heparan sulfate (HS), The peptide of the present invention is not limited to those containing hyaluronic acid as an active ingredient, since it has a much higher binding ability to heparin (hep) and the like than a peptide derived from a living body. It is also possible to apply to a drug containing noglycan as an active ingredient.

  Furthermore, the peptide of the present invention binds via glucosaminoglycan present in living tissues, and therefore the application of the peptide of the present invention is not limited to the retina and cornea, and for example, skin surface, glucosaminoglycan is It can be applied to all living tissues.

FIG. 1 is a graph showing the results of Test 1 (comparison test of binding activity between biotinylated hyaluronic acid and each peptide). FIG. 2 is a graph showing the results of Test 2 (comparison test of binding activity between biotinylated heparan sulfate and each peptide). FIG. 3 is a graph showing the results of Test 3 (comparison test of binding activity between biotinylated chondroitin sulfate A and each peptide). FIG. 4 is a graph showing the results of Test 4 (comparison test of binding activity between biotinylated chondroitin sulfate C and each peptide). FIG. 5 is a photograph showing the appearance of the porcine corneal surface after Test 5 (application of peptide + hyaluronic acid to the cornea). FIG. 6 is a photograph showing the appearance of the porcine corneal surface after Test 6 (application of peptide + chondroitin sulfate to the cornea). FIG. 7 is a graph showing the results of Test 7 (peeling retinal adhesion of the peptide of the present invention).

The terms used in the present specification have the meanings commonly used in the art, unless otherwise specified. The present invention will be described in more detail below.
The “basic amino acid” means an amino acid having a basic side chain and includes arginine (Arg), histidine (His), lysine (Lys) and the like.
The “acidic amino acid” means an amino acid having an acidic side chain, and examples thereof include aspartic acid (Asp) and glutamic acid (Glu).
The “branched amino acid” means an amino acid having a branched alkyl side chain, and examples thereof include leucine (Leu), isoleucine (Ile), valine (Val) and the like.
The “amide-containing amino acid” means an amino acid having an amide or carbamoyl group in its side chain, and examples thereof include asparagine (Asn) and glutamine (Gln).
“Hydroxyl-containing amino acid” means an amino acid having a hydroxyl group in its side chain, and examples thereof include serine (Ser) and threonine (Thr).
Examples of the “aliphatic amino acid” include alanine (Ala) and glycine (Gly).
Examples of the “aromatic amino acid” include phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr) and the like.
The “sulfur-containing amino acid” means an amino acid having a sulfur atom in its side chain, and examples thereof include cysteine (Cys) and methionine (Met).
"Glucosaminoglycans" include hyaluronic acid (HA), chondroitin (chn), chondroitin sulfate A (CSA), chondroitin sulfate C (CSC), dermatan sulfate (DS), heparan sulfate (HS), heparin (hep). , Heparin-like substances and the like.

By "derivative of a peptide" is meant a polypeptide that has substantially the same biological function or activity as the polypeptide of the invention. A modified polypeptide in which the N-terminal of the derivative of the polypeptide is modified with an acyl group such as an acetyl group or another modifying group, and a C-terminal carboxyl group of the derivative of the polypeptide is converted to a carboxylate, amide or ester. Polypeptides are also included in the invention. Further, the present invention also includes a polypeptide PEGylated for the purpose of extending the half-life in blood and a polypeptide to which a sugar chain is added.

  Examples of the "pharmaceutically acceptable salt" include physiologically acceptable salts with acids or bases, and as the pharmaceutically acceptable salt of the peptide, acid addition salts are particularly preferable. Such salts include, for example, hydrochloric acid, phosphoric acid, hydrobromic acid, salts with inorganic acids such as sulfuric acid, or acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, Salts with organic acids such as malic acid, oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid are used. Examples of the glucosaminoglycan salt include sodium salt and calcium salt.

The “solvate” may be coordinated with any number of solvates with respect to the peptide of the present invention. A hydrate is preferable.
The “pharmaceutical composition” may include at least the peptide of the present invention, a pharmaceutically acceptable salt thereof, or a solvate thereof.
Examples of the “eye disease” include, but are not limited to, retinal disease, keratoconjunctival epithelial disorder and the like. Retinal diseases include retinal detachment, diabetic retinopathy, macular degeneration and the like. Examples of keratoconjunctival epithelial disorders include dry eye, keratoconjunctivitis sicca, punctate corneal keratopathy, corneal erosion, and corneal ulcer.
Examples of the “skin disease” include sebum deficiency, progressive keratoderma palmaris, atopic dermatitis and the like.

Arginine (Arg) and lysine (Lys) are preferable as the basic amino acid at X ¹ , X ² and X ⁹ of the peptide of the present invention.
Glutamic acid (Glu) is preferable as the acidic amino acid in X ¹ and X ⁴ of the peptide of the present invention.
Leucine (Leu), isoleucine (Ile) and valine (Val) are preferable as the branched amino acids at X ¹ , X ³ , X ⁴ and X ⁵ of the peptide of the present invention.
As amide-containing amino acids in X ² , X ⁴ and X ⁷ of the peptide of the present invention, asparagine (Asn) and glutamine (Gln) are preferable.
Serine (Ser) and threonine (Thr) are preferred as the hydroxy-containing amino acid in X ³ and X ⁶ of the peptide of the present invention.
Alanine (Ala) and glycine (Gly) are preferable as the aliphatic amino acid at X ⁴ and X ⁷ of the peptide of the present invention.

Phenylalanine (Phe) is preferred as the aromatic amino acid at X ⁸ of the peptide of the present invention.
Cysteine (Cys) is preferred as the sulfur-containing amino acid in Y ¹ and Y ² of the peptide of the present invention.
Examples of the intramolecular bond in Y ¹ and Y ² of the peptide of the present invention include a peptide bond and a disulfide bond, and a disulfide bond is preferable.
As X ¹ of the peptide of the present invention, arginine (Arg), lysine (Lys), glutamic acid (Glu) and leucine (Leu) are preferable, and arginine (Arg) and lysine (Lys) are particularly preferable.
As X ² of the peptide of the present invention, arginine (Arg), asparagine (Asn) and glutamine (Gln) are preferable, and arginine (Arg) and glutamine (Gln) are particularly preferable.

As X ³ of the peptide of the present invention, leucine (Leu), isoleucine (Ile), valine (Val) and serine (Ser) are preferable, and leucine (Leu) and serine (Ser) are particularly preferable.
X ⁴ of the peptide of the present invention is preferably glutamic acid (Glu), leucine (Leu), isoleucine (Ile), valine (Val), asparagine (Asn), alanine (Ala), and particularly glutamic acid (Glu) and valine. (Val), asparagine (Asn), and alanine (Ala) are preferable.
As X ⁵ of the peptide of the present invention, leucine (Leu), isoleucine (Ile) and valine (Val) are preferable, and leucine (Leu) and isoleucine (Ile) are particularly preferable.
As X ⁶ of the peptide of the present invention, leucine (Leu), isoleucine (Ile), valine (Val) and threonine (Thr) are preferable, and leucine (Leu) and threonine (Thr) are particularly preferable.
As X ⁷ of the peptide of the present invention, asparagine (Asn), glutamine (Gln) and glycine (Gly) are preferable, and asparagine (Asn) and glycine (Gly) are particularly preferable.
As X ⁸ of the peptide of the present invention, phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr) are preferable, and phenylalanine (Phe) is particularly preferable.
As X ⁹ of the peptide of the present invention, arginine (Arg) and lysine (Lys) are preferable.

  Peptides preferably used in the present invention are peptides consisting of the amino acid sequences represented by SEQ ID NOs: 3 to 38, derivatives thereof, pharmaceutically acceptable salts thereof, or solvates thereof, and specifically, the following: It has the structure shown in Table 1.

  The method for producing the peptide of the present invention is not particularly limited, and it can be produced according to a known peptide synthesis method based on the amino acid sequence. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. For example, in the solid-phase synthesis method, a peptide or amino acid that can form a peptide compound such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method) is condensed with the remaining portion to produce a product. When has a protecting group, the target peptide compound can be produced by removing the protecting group. For solid-phase synthesis, APEX396 (manufactured by Advanced Chemtech), 433A (manufactured by Applied Biosystems), PS3 (manufactured by Protein Technologies), 9050 (manufactured by Perceptive), PSSM-8 (manufactured by Shimadzu), etc. Peptide synthesizers are commercially available. The resin used in the solid phase synthesis method is not particularly limited, and for example, Rink amide AM Resin, Fmoc-AA-Wang Resin, AA-2-Cl-Trt Resin can be used. Further, in the case of the liquid phase synthesis method, the peptide compound can be produced by a method in which the N-protected amino acid derivative is condensed step by step for each residue. A dicyclohexylcarbodiimide (DCC) method, an active ester method, a mixed acid anhydride method or the like can be used depending on the presence or absence of a protecting group. Further, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSC · HCl), (1H-benzotriazol-1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate (BOP) reagent, diisopropyl The target peptide compound can also be produced by a method using a condensing agent such as carbodiimide (DIPCDI).

  The method for producing the peptide derivative of the present invention is not particularly limited, and the peptide derivative of the present invention can be produced by a method usually used in the art. For example, the derivative can be produced by an acylation reaction with acetic anhydride or acetyl chloride for the amino group of the peptide, an amidation reaction with ammonia for the carboxyl group of the peptide, or the like. In addition, a cyclized peptide having a disulfide bond can also be produced by removing a protecting group of cysteine, which is a constituent amino acid of a polypeptide, and then using a method such as air oxidation or iodine oxidation. When the protecting group for the thiol group contained in cysteine is an acetamidomethyl group, deprotection and formation of a disulfide bond can be simultaneously performed by using iodine.

  Alternatively, the peptide of the present invention can be produced by producing a polynucleotide (DNA or RNA) corresponding to the amino acid sequence of the peptide of the present invention and performing a genetic engineering technique using the polynucleotide.

The produced peptide of the present invention can be purified by protein isolation and purification methods generally known in the field of protein chemistry. Specifically, for example, extraction, recrystallization, salting out with ammonium sulfate or sodium sulfate, centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reverse Treatment operations such as phase chromatography, gel filtration method, gel permeation chromatography, affinity chromatography, electrophoresis method, countercurrent distribution, and combinations thereof are included.

  The peptide of the present invention has a strong binding ability to glucosaminoglycans such as hyaluronic acid, heparan sulfate, and chondroitin sulfate, as confirmed in the experimental examples described below. Therefore, the peptide of the present invention can be used as an active ingredient in the prevention or treatment of diseases treatable by improving the connectivity between living tissues, such as retinal detachment. In addition, the peptide of the present invention can be used as an auxiliary agent for improving the durability of glucosaminoglycan in the prevention or treatment of diseases that have been conventionally treated with glucosaminoglycan. Such diseases include, as described above, keratoconjunctival epithelial disorders such as dry eye, keratoconjunctivitis sicca, punctate corneal erosion, corneal erosions, and corneal ulcers, and skin diseases such as sebum deficiency and progressive disease. Palm keratoderma, atopic dermatitis, etc. are mentioned.

  When the peptide of the present invention is used as an active ingredient, the drug used for the prevention or treatment of various diseases includes the peptide of the present invention, a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof. It should be. For example, the above-mentioned medicament may be composed only of the polypeptide of the present invention, or a composition comprising the peptide of the present invention, a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof, and a pharmaceutical carrier. It may be in the form of a product.

  In addition, when the peptide of the present invention is used as an auxiliary agent, as a medicine, (A) the peptide of the present invention, a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof and (B) gluco are used. It suffices that the aminoglycan or its salt is contained. For example, the above-mentioned medicament may be composed only of the polypeptide of the present invention and glucosaminoglycan, and also the peptide of the present invention, a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof, gluco. It may be in the form of a composition comprising a aminoglycan or a salt thereof and a pharmaceutical carrier. Further, the above-mentioned medicament may be in the form of a kit preparation containing the peptide of the present invention and a glycosaminoglycan in separate preparations.

  The pharmaceutical carrier that can be used in the above-mentioned medicament of the present invention is not particularly limited, and is a pharmaceutically acceptable excipient, binder, lubricant, colorant, disintegrating agent, buffering agent, isotonicity agent, dissolving agent. Auxiliary agents, chelating agents, stabilizers, preservatives, antioxidants, bases, emulsifiers and the like can be used.

Preferable examples of excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, gum arabic, pullulan, soft Examples thereof include anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminometasilicate, xylitol, sorbitol, and erythritol.
Preferable examples of the binder include pregelatinized starch, sucrose, gelatin, gum arabic, methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropyl cellulose, hydroxy. Examples include propylmethyl cellulose and polyvinylpyrrolidone.

Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica, polyethylene glycol 6000 and the like.
Preferable examples of the colorant are water-soluble colored tar dyes such as food reds 2 and 3, food yellows 4 and 5, food blues 1 and 2, and aluminum salts thereof. Insoluble lake pigments, natural pigments such as β-carotene, chlorophyll, red iron oxide and the like can be mentioned.

Preferable examples of the disintegrant include lactose, sucrose, starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, soft anhydrous silicic acid, calcium carbonate and the like. ..
Preferable examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate and the like.
Preferable examples of the isotonicity agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, xylitol, fructose and the like.
Preferable examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate. And so on.

Suitable examples of the chelating agent include edetate salts such as edetate disodium, edetate calcium disodium, edetate trisodium, edetate tetrasodium, and edetate calcium, ethylenediaminetetraacetic acid salt, nitrilotriacetic acid or a salt thereof. , Sodium hexametaphosphate, citric acid and the like.
Suitable examples of the stabilizer include sodium bisulfite and the like.
Suitable examples of preservatives include sorbic acid, potassium sorbate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, paraoxybenzoic acid esters such as butyl paraoxybenzoate, chlorhexidine gluconate, and benzalkonium chloride. , Benzethonium chloride, quaternary ammonium salts such as cetylpyridinium chloride, alkylpolyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, chlorhexidine and the like.

Preferable examples of the antioxidant include sodium hydrogen sulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherols and the like.
Preferable examples of the solubilizing agent include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol and the like.
Suitable examples of bases include petroleum jelly, liquid paraffin, hydrocarbons such as squalane, beeswax, jojoba oil, esters such as carnauba wax, triglycerides other than oleic acid triglyceride, olive oil, fats and oils such as beef tallow, Examples include higher alcohols such as cetanol, stearyl alcohol and behenyl alcohol, higher fatty acids such as stearic acid, oleic acid and behenic acid, lower alcohols such as ethanol and propanol, and water.
Preferable examples of emulsifiers include fatty acid monoglyceride, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, nonionic surfactant such as polyoxyethylene hydrogenated castor oil, higher fatty acid salt, sodium lauryl sulfate, anion such as alkylsulfosuccinic acid ester. Examples thereof include surfactants, cation surfactants such as quaternary alkylamine salts, and amphoteric surfactants such as alkyl betaines.

It is desirable to use the most effective route for treatment, and it is possible to administer an oral preparation, an injection, a transdermal preparation or an eye drop, but in particular, a transdermal preparation or an eye drop may be used. preferable. Moreover, the peptide of the present invention can be directly applied to a living tissue to be treated. As such an embodiment, for example, direct application to the retina during vitreous surgery for retinal detachment can be mentioned. Oral preparations include tablets (including sublingual tablets and orally disintegrating agents), capsules (including soft capsules and microcapsules), powders, granules, troches, syrups, emulsions, suspensions, etc. .. Examples of the injection include intradermal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrathecal injection, epidural injection, local injection, intraocular injection and the like. Examples of transdermal preparations include patches, ointments, creams, lotions and dusting agents. These formulations may be controlled release formulations (including sustained release microcapsules) such as immediate release formulations or sustained release formulations. The eye drops may be any of aqueous eye drops, non-aqueous eye drops, suspension eye drops, emulsion eye drops, eye ointments and the like. In addition, examples of the formulation for direct application to living tissue include a liquid formulation having the same composition as the aqueous eye drop.

  In the case of producing an oral preparation, it is produced by adding, for example, an excipient, a disintegrating agent, a binder, a lubricant or the like to the peptide of the present invention and compression-forming. Further, for the purpose of taste masking, enteric coating or sustained release, an oral preparation may be coated by a method known per se. In the case of producing an injectable preparation, it is produced by dissolving, suspending or emulsifying the peptide of the present invention together with a dispersant, a preservative, an isotonicity agent and the like in an aqueous solvent or an oily solvent. At this time, if necessary, additives such as a solubilizing agent, a suspending agent, a buffer, a stabilizer, a soothing agent, and a preservative may be used. Furthermore, in the case of producing a transdermal preparation, the peptide of the present invention is produced as a solid, semi-solid or liquid composition. For example, a solid composition can be produced by adding the active ingredient as it is, or by adding and mixing an excipient, a thickener and the like to give a powder. The liquid composition is manufactured in almost the same manner as in the case of injection. The semi-solid composition is produced, for example, by adding the peptide of the present invention to the above-mentioned base and mixing it, and by adding an emulsifier and the above-mentioned other components to the base if necessary. Good. Moreover, any of these compositions may contain a pH adjuster, a preservative, and the like. Furthermore, eye drops, for example, the desired components are dissolved or suspended in sterile water, an aqueous solvent such as physiological saline, or a non-aqueous solvent such as cottonseed oil, soybean oil, sesame oil, peanut oil, or the like, and a predetermined amount. It can be produced by adjusting the osmotic pressure of the above and subjecting it to sterilization treatment such as filtration sterilization. In addition, the formulation for direct application is prepared by, for example, dissolving or suspending the above-mentioned desired components in an aqueous solvent such as sterile purified water or physiological saline, adjusting to a predetermined osmotic pressure, and performing sterilization treatment such as filtration sterilization. It can be manufactured.

  The dose and frequency of administration of the pharmaceutical agent of the present invention vary depending on the administration form, age and weight of the patient, nature or severity of the condition to be treated, and for example, the peptide of the present invention as an active ingredient is directly applied to living tissue. When applied, the peptide of the present invention is usually administered once or several times a day so that the peptide of the present invention will be about 0.01 μg to 5 mg per adult, and more preferably, the peptide of the present invention will be about 0. It is administered once or twice a day so as to be 1 μg to 3 mg. In particular, when the retinal detachment is directly applied to the retina during vitreous surgery, about 0.5 μg to 50 μg of the peptide of the present invention is applied to the retina.

When the peptide of the present invention is used as an adjuvant, it is usually administered once or several times a day so that the amount of the peptide of the present invention is about 0.01 μg to 1000 mg and the amount of glycosaminoglycan is about 0.1 mg to 1000 mg per adult. More preferably, the peptide of the present invention is administered to an adult at about 0.01 μg to 1000 mg and glucosaminoglycan is administered at 0.1 mg to 30 mg once to four times a day. Furthermore, when the peptide of the present invention is transdermally administered as an adjuvant, the amount of the peptide of the present invention is usually about 0.01 μg to 1000 mg and the amount of glucosaminoglycan is about 0.1 mg to 1000 mg per adult. It is administered once to several times a day, more preferably once or twice a day so that the peptide of the present invention is about 0.01 μg to 1000 mg and glucosaminoglycan is 1 mg to 300 mg per adult.

  Furthermore, as described above, since glycosaminoglycan has an extremely high water retention ability, when applied to the skin, it can moisturize the skin and improve or maintain the luster and firmness of the skin. Therefore, the present invention also relates to a cosmetic containing (A) peptide, a derivative thereof, a pharmaceutically acceptable salt thereof or a solvate thereof, and (B) a glycosaminoglycan or a salt thereof. The cosmetic of the present invention may be composed of, for example, only the polypeptide of the present invention and glucosaminoglycan, as in the case of the above-mentioned pharmaceutical, and the peptide of the present invention, a derivative thereof, and a pharmaceutically acceptable thereof. It may be in the form of a composition comprising a salt or a solvate thereof, a glucosaminoglycan or a salt thereof, and a carrier used in cosmetics. Further, the cosmetic of the present invention may be in the form of a kit containing the peptide of the present invention and a glycosaminoglycan in separate formulations. In the above, examples of the “carrier used in cosmetics” include those similar to the pharmaceutical carriers used in the above-mentioned transdermal preparations, and their manufacturing methods and application methods are the same as those of the above-mentioned transdermal preparations. is there.

  Hereinafter, the present invention will be described more specifically by way of examples. These examples are provided for purposes of illustration and are not intended to limit the embodiments of the invention.

<Production Example 1> Production of various peptides The following Examples 1 to 9 were produced by the solid phase synthesis method (Fmoc method) in the same manner as the method described in Example 1 of Japanese Patent Publication No. 2005-538961. In the production of the peptides of each example, 1,3-diisopropylcarbodiimide / HOBt was used as the coupling reagent. The completion of the deprotection reaction and the peptide chain elongation reaction at each stage was confirmed by a ninhydrin test.
The peptides produced in each example were assayed by calculating the purity by high performance liquid chromatography (the conditions are described in detail in each example) and analyzing the molecular weight. As a result, the partial peptides produced in each Example had a purity of 95% or more, and the results of molecular weight analysis were in good agreement with the theoretical values. From these results, it was confirmed that each peptide was produced correctly.

[Example 1] Leu Gln Ser Asn Leu Thr Gly Phe Lys (SEQ ID NO: 3 or C1W)
· HPLC [Column: YMC-Pack ODS-AM ( 150 X 4.6 mm); solvent system _{A (0.02% TFA / H 2} O), B (0.02% TFA / CH 3 CN), 10% B 10 minutes; flow rate: 0.6 mL / min; UV at detection 214 nm]
Retention time: 6.9 minutes (Purity 98.9%)
MS (m / z) [ESI-MS]: 1007.8 (theoretical value: 1007.1)

[Example 2] Arg Gln Ser Asn Leu Thr Gly Phe Lys (SEQ ID NO: 4 or C1M (L / R))
· HPLC [Column: YMC-C18 (150 X 4.6 mm); solvent system _{A (0.02% TFA / H 2} O), B (0.02% TFA / CH 3 CN), 10% B 20 min Flow rate: 1 mL / min; UV at detection 214 nm]
Retention time: 7.2 minutes (purity 98.0%)
MS (m / z) [MALDI-TOF-MS]: 1050.8 (theoretical value: 1050.2)

[Example 3] Lys Gln Leu Glu Ile Leu Asn Phe Arg (SEQ ID NO: 5 or C2W)
· HPLC [Column: YMC-C18 (150 X 4.6 mm); solvent system _{A (0.02% TFA / H 2} O), B (0.02% TFA / CH 3 CN), 15% B 20 min Flow rate: 1 mL / min; UV at detection 214 nm]
Retention time: 11.6 minutes (Purity 99.2%)
MS (m / z) [MALDI-TOF-MS]: 1159.7 (theoretical value: 1160.4)
)

[Example 4] Lys Gln Leu Val Ile Leu Asn Phe Arg (SEQ ID NO: 6 or C2M (E / V))
· HPLC [Column: YMC-C18 (150 X 4.6 mm); solvent system _{A (0.02% TFA / H 2} O), B (0.02% TFA / CH 3 CN), 20% B 20 min Flow rate: 1 mL / min; UV at detection 214 nm]
Retention time: 6.9 minutes (purity 98.7%)
MS (m / z) [MALDI-TOF-MS]: 1130.6 (theoretical value: 1130.4)

[Example 5] Arg Arg Ser Asn Leu Thr Gly Phe Lys (SEQ ID NO: 7 or H1M (L / R))
· HPLC Column: YMC-C18 (150 X 4.6 mm); solvent system _{A (0.02% TFA / H 2} O), B (0.02% TFA / CH 3 CN), 10% B 20 min; Flow rate: 1 mL / min; UV at detection 214 nm]
Retention time: 6.5 minutes (purity 96.4%)
MS (m / z) [ESI-MS]: 1078.8 (theoretical value: 1078.2)

[Example 6] Leu Arg Ser Asn Leu Thr Gly Phe Lys (SEQ ID NO: 8 or H1W)
· HPLC [Column: YMC-Pack ODS-AM ( 150 X 4.6 mm); solvent system _{A (0.02% TFA / H 2} O), B (0.02% TFA / CH 3 CN), 10% B 20 minutes; flow rate: 1 mL / min; detection UV at 214 nm]
Retention time: 8.6 minutes (purity 98.1%)
MS (m / z) [ESI-MS]: 1035.2 (theoretical value: 1035.2)

[Example 7] Glu Gln Leu Glu Ile Leu Asn Phe Arg (SEQ ID NO: 9 or C2M (K / E))
· HPLC [Column: YMC-C18 (150 X 4.6 mm); solvent system _{A (0.02% TFA / H 2} O), B (0.02% TFA / CH 3 CN), 20% B 20 min Flow rate: 1 mL / min; UV at detection 214 nm]
Retention time: 9.2 minutes (purity 98.5%)
MS (m / z) [MALDI-TOF-MS]: 1162.0 (theoretical value: 1161.3)

[Example 8] Lys Gln Leu Ala Ile Leu Asn Phe Arg (SEQ ID NO: 10 or C2M (E / A))
· HPLC [Column: YMC-C18 (150 X 4.6 mm); solvent system _{A (0.02% TFA / H 2} O), B (0.02% TFA / CH 3 CN), 15% B 20 min Flow rate: 1 mL / min; UV at detection 214 nm]
Retention time: 11.0 minutes (Purity 99.6%)
MS (m / z) [MALDI-TOF-MS]: 1102.3 (theoretical value 1103.0)

[Example 9] Lys Gln Leu Val Ile Leu Asn Phe Arg Lys Gln Leu Val Ile Leu Asn Phe Arg (SEQ ID NO: 15 or C2M (E / V) 2)
· HPLC [Column: YMC-C18 (150 X 4.6 mm); solvent system _{A (0.02% TFA / H 2} O), B (0.02% TFA / CH 3 CN), 20% B 20 min Flow rate: 1 mL / min; UV at detection 214 nm]
Retention time: 10.8 minutes (Purity 98.7%)
MS (m / z) [MALDI-TOF-MS]: 2243.2 (theoretical value: 2242.8)

<Production Example 2> Production of various cyclic peptides The following Examples 10 to 11 were produced by the solid phase synthesis method (Fmoc method) in the same manner as the method described in Example 1 of JP 2005-538961A. In the production of the peptides of each example, 1,3-diisopropylcarbodiimide / HOBt was used as the coupling reagent. The completion of the deprotection reaction and the peptide chain elongation reaction at each stage was confirmed by a ninhydrin test. For Cys in the peptide, Fmoc-Cys (Acm) -OH (N-α- (9-fluorenylmethoxycarbonyl) -S-acetaminomethyl-L-cysteine) was used, and the peptide was prepared using iodine. By cutting out from the resin, the thiol group in the peptide was deprotected and the disulfide bond was generated.
The peptides produced in each example were assayed by calculating the purity by high performance liquid chromatography (the conditions are described in detail in each example) and analyzing the molecular weight. As a result, the partial peptides produced in each Example had high purity, and the results of molecular weight analysis were in good agreement with the theoretical values. From these results, it was confirmed that each peptide was produced correctly.

・ＨＰＬＣ［カラム：ＹＭＣ−Ｃ１８（１５０ Ｘ ４．６ ｍｍ）；溶媒系Ａ（０．０２％ ＴＦＡ／Ｈ₂Ｏ）、Ｂ（０．０２％ ＴＦＡ／ＣＨ₃ＣＮ）、２０％Ｂ ２０分；流速：１ｍＬ／分；検出２１４ｎｍのＵＶ］
保持時間：９．７分（純度８２．１％）
・ＭＳ（ｍ／ｚ）［ＭＡＬＤＩ−ＴＯＦ−ＭＳ］：１３３５．６（理論値１３３４．７） [Example 10]

· HPLC [Column: YMC-C18 (150 X 4.6 mm); solvent system _{A (0.02% TFA / H 2} O), B (0.02% TFA / CH 3 CN), 20% B 20 min Flow rate: 1 mL / min; UV at detection 214 nm]
Retention time: 9.7 minutes (purity 82.1%)
MS (m / z) [MALDI-TOF-MS]: 1335.6 (theoretical value 1334.7)

・ＨＰＬＣ［カラム：ＹＭＣ−Ｃ１８（１５０ Ｘ ４．６ ｍｍ）；溶媒系Ａ（０．０２％ ＴＦＡ／Ｈ₂Ｏ）、Ｂ（０．０２％ ＴＦＡ／ＣＨ₃ＣＮ）、２０％Ｂ ２０分；流速：１ｍＬ／分；検出２１４ｎｍのＵＶ］
保持時間：１０．３分（純度７５．２％）
・ＭＳ（ｍ／ｚ）［ＭＡＬＤＩ−ＴＯＦ−ＭＳ］：２４４８．７（理論値２４４７．０）試験例１：ビオチン化ヒアルロン酸と各ペプチドとの結合活性比較実験
各ペプチド5 nmolをニトロセルロース膜にImmunoBlot（商標、Hoefer社製）を用いてイムノブロットした。10％ nonfat milk in phosphate-buffered saline (PBS) containing0.1％ Tween 20 (PBS-Tween)溶液にてブロッキングを４２℃にて１時間実施した。ヒアルロン酸（ニワトリ鶏冠由来：生化学工業株式会社製）はビオチン化して、2 mg/mLの濃度で準備した。ビオチン化の行程は井上らの報告（Inoue Y, Yoneda M, Zhao J, Miyaishi O, Ohno-Jinno A, Kataoka T, Isogai Z, Kimata K, Iwaki M, Zako M. Molecular cloning and characterization of chick SPACRCAN. J Biol Chem. 2006;281:10381-8.）に従った。ヒアルロン酸をPBS-Tween溶液にて500倍希釈して室温にて３０分間インキュベーションし、膜上のペプチドと結合させた。PBS-Tween溶液にて室温にて３０分間washした。HRP-conjugated streptavidin (GE Healthcare Bio-Sciences Corp., Piscataway, NJ)に
て室温にて３０分間インキュベーションした。PBS-Tween溶液にて室温にて３０分間washした。WESTERN LIGHTNING (商標、PerkinElmer Life Sciences Inc., Boston, MA) にて発光させ、Ｘ線フィルムに感光させて検出した。
C1Wにおける発光強度を１として実施例２〜１１で合成したペプチド又は環状ペプチドにおける発光強度と比較した結果を図１に示す。C2M(E/V)2、Cycle C2M(E/V)、Cycle C2M(E/V)2がそれぞれ67.7、58.1、62.7を示し、C1Wと比べて非常に高いヒアルロン酸結合能が認められた。 [Example 11]

· HPLC [Column: YMC-C18 (150 X 4.6 mm); solvent system _{A (0.02% TFA / H 2} O), B (0.02% TFA / CH 3 CN), 20% B 20 min Flow rate: 1 mL / min; UV at detection 214 nm]
Retention time: 10.3 minutes (purity 75.2%)
MS (m / z) [MALDI-TOF-MS]: 2448.7 (theoretical value 2447.0) Test example 1: Comparative experiment of binding activity between biotinylated hyaluronic acid and each peptide 5 nmol of each peptide was placed on a nitrocellulose membrane Was immunoblotted with ImmunoBlot (trademark, manufactured by Hoefer). Blocking was carried out for 1 hour at 42 ° C. with a 10% nonfat milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-Tween) solution. Hyaluronic acid (derived from chicken crest: manufactured by Seikagaku Corporation) was biotinylated and prepared at a concentration of 2 mg / mL. The biotinylation process was reported by Inoue et al. (Inoue Y, Yoneda M, Zhao J, Miyaishi O, Ohno-Jinno A, Kataoka T, Isogai Z, Kimata K, Iwaki M, Zako M. Molecular cloning and characterization of chick SPACRCAN. J Biol Chem. 2006; 281: 10381-8.). Hyaluronic acid was diluted 500-fold with PBS-Tween solution and incubated at room temperature for 30 minutes to bind to the peptide on the membrane. It was washed with PBS-Tween solution for 30 minutes at room temperature. Incubation was carried out for 30 minutes at room temperature in HRP-conjugated streptavidin (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). It was washed with PBS-Tween solution for 30 minutes at room temperature. Western LIGHTNING (trademark, PerkinElmer Life Sciences Inc., Boston, MA) was used for light emission, which was then exposed to an X-ray film for detection.
The result of comparison with the luminescence intensity of the peptides or cyclic peptides synthesized in Examples 2 to 11 with the luminescence intensity of C1W being 1 is shown in FIG. C2M (E / V) 2, Cycle C2M (E / V), and Cycle C2M (E / V) 2 showed 67.7, 58.1, and 62.7, respectively, and a very high hyaluronic acid binding ability was recognized as compared with C1W.

Test Example 2: Comparative Experiment of Binding Activity between Biotinylated Heparan Sulfate and Each Peptide The same operation as in Test Example 1 was performed using heparan sulfate (derived from bovine kidney: manufactured by Seikagaku Corporation) instead of hyaluronic acid.
The results of comparison with the luminescence intensity of the peptides or cyclic peptides synthesized in Examples 2 to 11 with the luminescence intensity of C1W being 1 are shown in FIG. C2M (E / V) 2, Cycle C2M (E / V), and Cycle C2M (E / V) 2 showed 13.9, 4.9, and 25.8, respectively, and had a significantly higher heparan sulfate-binding ability than C1W.

Test Example 3: Binding activity comparison experiment between biotinylated chondroitin sulfate A and each peptide Chondroitin sulfate A (derived from whale cartilage: manufactured by Seikagaku Corporation) was used in place of hyaluronic acid, and the same operation as in Test Example 1 was performed. It was
FIG. 3 shows the results of comparison with the luminescence intensity of the peptides or cyclic peptides synthesized in Examples 2 to 11 with the luminescence intensity of C1W as 1. C2M (E / V) 2, Cycle C2M (E / V), and Cycle C2M (E / V) 2 showed 105.3, 57.6, and 41.3, respectively, showing a much higher chondroitin sulfate A-binding ability than C1W. ..

Test Example 4: Comparative experiment of binding activity between biotinylated chondroitin sulfate C and each peptide The same operation as in Test Example 1 was performed using chondroitin sulfate C (derived from shark cartilage: manufactured by Seikagaku Corporation) instead of hyaluronic acid. It was
FIG. 4 shows the results of comparison with the luminescence intensity of the peptides or cyclic peptides synthesized in Examples 2 to 11 with the luminescence intensity of C1W as 1. C2M (E / V) 2, Cycle C2M (E / V), and Cycle C2M (E / V) 2 showed 171.7, 142.9, and 154.2, respectively, showing a much higher chondroitin sulfate C-binding ability than C1W. ..

Test Example 5: Comparative Experiment of Hyaluronic Acid Outflow Inhibitory Ability Cycle C2M (E / V) 2 aqueous solution adjusted to 1 μg / mL was used to write “H” on the cornea of a pig eye (purchased from Marusho Foods) with a brush. 10 μg / mL Fluorescein-HA was applied to pig eyes. After the lapse of 3 minutes, the pig's eyes were washed by dropping physiological saline every 3 minutes. After washing 10 times, under the light irradiation condition by SFA-RB (manufactured by BEX), the fluorescence state of pig eye was photographed (camera: Nikon D5100 (manufactured by Nikon), lens: AF-S Micro NIKKOR 40mm 1: 2.8G (Manufactured by Nikon Corporation). In addition, physiological saline was used in place of the Cycle C2M (E / V) 2 aqueous solution, and "H" was written with a writing brush on the cornea of another pig eye, and the same operation was performed as a control.
A photograph of the control is shown in Fig. 5 (a), and a photograph of the test example is shown in Fig. 5 (b). Although the characters could not be recognized in the control, the character "H" could be recognized in the pig eyes of the test example, and the action of suppressing the outflow of hyaluronic acid from the cornea by Cycle C2M (E / V) 2 was recognized.

Test Example 6: Comparative experiment of chondroitin sulfate C outflow inhibitory ability Using a cycle C2M (E / V) 2 aqueous solution adjusted to 1 μg / mL, “C” was written on the cornea of a pig eye (purchased from Marusho Foods) with a brush. Writing, 10 μg / mL Fluorescein-CS was applied to a pig eye. After the lapse of 3 minutes, the pig's eyes were washed by dropping physiological saline every 3 minutes. After washing 10 times, under the light irradiation condition by SFA-RB (manufactured by BEX), the fluorescence state of pig eye was photographed (camera: Nikon D5100 (manufactured by Nikon), lens: AF-S Micro NIKKOR 40mm 1: 2.8G (Manufactured by Nikon Corporation). Also, Cycle C2M
A physiological saline solution was used instead of the (E / V) 2 aqueous solution, and "C" was written with a writing brush on the cornea of another pig eye, and the same operation was performed as a control.
A photograph of the control is shown in FIG. 6 (a), and a photograph of the test example is shown in FIG. 6 (b). Although the character could not be recognized in the control, the character "C" could be recognized from the pig eyes of the test example, and the action of suppressing efflux of chondroitin sulfate C from the cornea by Cycle C2M (E / V) 2 was recognized.

Test Example 7: Comparative experiment of peeling retinal adhesion ability (1) Preparation of pig eye A pig eye (purchased from Marusho Food Co., Ltd.) was incised along the ciliary body in the eyeball to remove the anterior segment and the vitreous body. Then, the posterior pole side of the eyeball was cut into an oval tissue of 1 cm x 1.5 cm size. Furthermore, the retina was peeled from the eye tissue, and the generated water was wiped off with Kimwipe.
(2) Comparison of peeling retinal adhesiveness A CycleC2M (E / V) 2 solution adjusted to 1 μg / mL was applied to the peeled retina in Test Example 7 to adhere the retina to the original eye tissue. After 1 minute, the above eye tissue was removed from the wood (20 cm x 2 cm x
It was fixed with a needle at a position 1 cm away from one end of the long side. Paperweights (132 mm x 12 mm x 12 mm) were placed perpendicularly to the wood at positions 1 cm and 17 cm away from the position where the eye tissue of the wood was fixed toward the center. At this time, the paperweight was made to intersect the wood at its center. Furthermore, the wood and the two paperweights were fixed with rubber bands. The wood to which the eye tissue and two paperweights were fixed was submerged in a 10L beaker containing 2L of PBS (-) solution, and the PBS (-) solution in the beaker was washed with a magnetic stirrer (set to 600 rpm) and a stirrer bar. The time until the retina detached from the eye tissue by 3/4 or more or flowed away with a water stream (time required for retina detachment) was measured. Further, physiological saline was used instead of the peptide aqueous solution to attach another eye cell to the detached retina, and the same operation was performed as a control.
FIG. 7 shows the time required for retinal detachment when the peptide aqueous solution and the control were used. The peptide aqueous solution was found to significantly extend the time required for retinal detachment.

  The present invention provides a peptide having a high binding ability to glycosaminoglycans. The peptide of the present invention is extremely useful as an active ingredient for preventing or treating various diseases, or as an auxiliary agent for improving the durability of glucosaminoglycan administered as an active ingredient, and is industrially applicable. have.

Claims (4)

Peptide represented by the following general formula (1), its those pharmaceutically acceptable salt thereof or a solvate thereof.
^{Y 1 -X 1 -X 2 -X 3} -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -Y 2 ( SEQ ID NO: 1)
(1 )
[In the formula,
X ¹ represents Lys ,
X ² represents Asn or Gln ,
X ³ and X ⁶ independently of each other represent Leu, Ile or Val ,
X ⁴ represents Leu, Ile or Val ,
X ⁵ represents Leu, Ile or Val ,
X ⁷ represents Asn or Gln ,
X ⁸ represents Phe ,
X ⁹ represents Arg ,
Y ¹ and Y ² simultaneously represent Cys , and the amino acids Y ¹ and Y ² form a disulfide bond] A peptide represented by the following general formula (2), a pharmaceutically acceptable salt thereof, or a solvate thereof.
Y ¹ -(X ¹ -X ² -X ³ -X ^Four -X ^Five -X ⁶ -X ⁷ -X ⁸ -X ⁹ ) ₂ -Y ² (SEQ ID NO: 2)
(2)
[In the formula,
X ¹ Indicates Lys,
X ² Represents Asn or Gln,
X ³ And X ⁶ Are, independently of each other, Leu, Ile or Val,
X ^Four Is Leu, Ile or Val,
X ^Five Is Leu, Ile or Val,
X ⁷ Represents Asn or Gln,
X ⁸ Indicates Phe,
X ⁹ Represents Arg,
Y ¹ And Y ² Are not present at the same time, or
Y ¹ And Y ² Are Cys at the same time, and Y ¹ And Y ² Amino acids form disulfides] Amino acid sequence represented by SEQ ID NO 27, peptide of claim 1, its those pharmaceutically acceptable salt or solvate thereof. The peptide according to claim 2, whose amino acid sequence is represented by SEQ ID NO: 15 or 28, a pharmaceutically acceptable salt thereof, or a solvate thereof.

Images