KR20130055505A - Novel peptide derivative capable of synthesizing collagen and use thereof - Google Patents

Abstract

PURPOSE: A novel peptide derivative with collagen synthesis ability is provided to promote collagen synthesis and wound healing and to improve anti-aging and anti-wrinkling effects. CONSTITUTION: A peptide derivative contains a palmitoyl group at N-terminal of a peptide with 5-100 amino acids including YIGSR of sequence number 1. The peptide has 5-20 amino acids. The peptide has an amino acid sequence of sequence number 1. A composition for anti-aging, a cosmetic composition for anti-wrinkling, and a pharmaceutical composition for wound healing contain the peptide derivative or YIGSR peptide as an active ingredient. A skin filler contains the peptide derivative or YIGSR peptide as an active ingredient.

Description

Novel peptide derivative capable of synthesizing collagen and use about

The present invention relates to peptide derivatives and uses thereof, and more particularly to novel peptide derivatives with collagen synthesis ability and uses thereof.

As a person ages, skin aging occurs. The main symptom is wrinkles. Wrinkles are a phenomenon of age, and the most common cause of wrinkles is the degradation of collagen that forms the matrix in the dermis of the skin. Collagen in the skin decreases as aging progresses.

Conventional materials for promoting collagen synthesis include retinoids (RE36068), transforming growth factor (TGF-β), betulinic acid (JP8-208424), wild yam extract (Korean Patent Publication No. 2009-0055079), and the like.

Meanwhile, some of oligopeptides having a palmitoyl group attached to the N-terminus promote collagen synthesis and are used as antiwrinkle agents (Robinson et al ., Int. J. Cosmet. Sci . (2005). ) 27 (3): 155-160).

However, such a conventional collagen synthesis enhancer has a disadvantage of low efficiency, high cost as a recombinant protein, and a natural extract has a disadvantage of poor reproducibility.

The present invention is to solve a number of problems including the above problems, by effectively promoting collagen synthesis at a relatively low cost, new peptides that can be used as a cosmetic composition or wound treatment for reducing skin aging or improving wrinkles and Its purpose is to provide its use. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the invention, there is provided a peptide derivative having a palmitoyl group added to the N-terminus of a peptide consisting of 5 to 100 amino acids comprising the amino acid sequence (YIGSR) as set forth in SEQ ID NO: 1.

The peptide may be composed of 5 to 20 amino acids and may be a peptide composed of the amino acid sequence set forth in SEQ ID NO: 1.

According to another aspect of the invention, there is provided a composition for reducing skin aging comprising the peptide derivative as an active ingredient.

The composition for reducing skin aging may be provided in the form of a cosmetic composition.

According to another aspect of the invention, there is provided a cosmetic composition for improving wrinkles comprising the peptide derivative as an active ingredient.

According to another aspect of the invention, there is provided a wound healing agent containing the peptide derivative as an active ingredient.

According to another aspect of the present invention, a dermal filler containing the peptide derivative as an active ingredient is provided.

According to one embodiment of the present invention made as described above, by promoting the synthesis of collagen on the skin, it is possible to prevent skin aging and improve wrinkles, and further promote wound healing. Of course, the scope of the present invention is not limited by these effects.

1 is an immunoblotting analysis showing the concentration-dependent collagen synthesis activity of the peptide derivative of the present invention.
Figure 2 is an immunoblotting analysis comparing the collagen synthesis activity of the peptide derivative of the present invention and the comparative example.
3 is an immunoblotting analysis result comparing the Erk phosphorylation activity of the peptide derivative of the present invention and the peptide of the comparative example.
4 is a graph showing the concentration-dependent skin fibroblast proliferation activity of the peptide derivative of the present invention.
5 is a result of immunoblotting analysis comparing the collagen synthesis activity according to the exposure time of the peptide derivative of the present invention and the peptide according to the comparative example.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in more detail.

Peptide derivatives having a palmitoyl group added to the N-terminus of a peptide consisting of 5 to 100 amino acids comprising the amino acid sequence (YIGSR) set forth in SEQ ID NO: 1 are provided.

The peptide may be composed of 5 to 20 amino acids and may be a peptide composed of the amino acid sequence set forth in SEQ ID NO: 1.

The peptide derivative can be prepared by peptide synthesis. Such peptide synthesis methods are well known in the art (Merrifield, J. Am. Chem. Soc . (1963) 85 (14): 2149 ?? 2154)

According to another aspect of the invention, there is provided a composition for reducing skin aging comprising the peptide derivative or YIGSR peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

The composition for reducing skin aging may be provided in the form of a cosmetic composition.

According to another aspect of the present invention, there is provided a cosmetic composition for improving wrinkles comprising the peptide derivative or YIGSR peptide consisting of the amino acid sequence described in SEQ ID NO: 1 as an active ingredient.

According to another aspect of the present invention, there is provided a composition for treating wounds, comprising as an active ingredient the YIGSR peptide consisting of the peptide derivative or the amino acid sequence described in SEQ ID NO: 1.

Originally, YIGSR peptides are known as collagen binding motifs as sites well conserved in collagen binding proteins such as Laminin. The YIGSR peptide inhibits the growth of leukemia cells (Yoshida et al ., (1999) Br. J. Cancer, 80 (12): 1898-1904), and has been reported to be an anticancer agent (Graf et al . (1987) Cell, 48: 989-996). However, the collagen synthesis ability of the YIGSR peptide has not been reported, and the present inventors have diligently tried to find a peptide that enhances the collagen synthesis ability, and as a result, the YIGSR peptide and its palmitoylated derivative Pal-YIGSR peptide have improved collagen synthesis. By confirming that the present invention was completed, the present invention was completed.

The effective amount of Pal-YIGSR peptide or YiGSR peptide in the composition of the present invention may vary depending on the type of affected part, the site of application, the number of treatments, the treatment time, the dosage form, the condition of the patient, the type of adjuvant, and the like. The amount used is not particularly limited, but may be 0.01 μg / kg / day to 10 mg / kg / day. The daily dose may be administered once or in two or three times a day at appropriate intervals or may be administered intermittently at intervals of several days.

YIGSR peptide consisting of the peptide derivative or the amino acid sequence of SEQ ID NO: 1 in the composition of the present invention, may be contained in 0.1-100% by weight relative to the total weight of the composition.

Compositions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical or cosmetic compositions. In addition, for the preparation of pharmaceutical compositions or cosmetic compositions, additives for the preparation of solid or liquid may be used. The preparation additive may be either organic or inorganic.

Examples of the excipient include lactose, sucrose, saccharin, glucose, corn starch, starch, talc, sorbit, crystalline cellulose, dextrin, kaolin, calcium carbonate and silicon dioxide. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethylcellulose, methylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylcellulose, hydroxypropylmethylcellulose, calcium citrate, Dextrin, pectin, and the like. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any coloring agent may be used as long as it is usually allowed to be added to pharmaceuticals. These tablets and granules can be suitably coated with sugar (sugar coating), gelatin coating, and others as required. If necessary, preservatives, antioxidants and the like may be added.

The pharmaceutical compositions of the present invention may be prepared in any formulation conventionally prepared in the art (e.g., Remington's Pharmaceutical Science, latest edition; Mack Publishing Company, Easton PA), although the form of the formulation is not particularly limited. Preferably, it may be an external preparation. The external preparation of the present invention includes a sheet, a liquid coating, a spray, a lotion, a cream, a pape, a powder, a penetrating pad, a spray, a gel including a hydrogel, a pasta, a liniment, an ointment, an aerosol, a powder, a suspension. Formulations may include conventional external preparations such as transdermal absorbents. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour), a prescription generally known for all pharmaceutical chemistry.

In one example of the invention, the composition may be applied directly to the skin or wound site. That is, it may be scattered on the wound site. In the form of a sheet, it is applied to the wound site, where appropriate dressing is applied to the applied site to protect the wound while preventing the therapeutic effect of the active ingredient from decreasing. The dressing can be any commercially available or commonly known. Examples of commercially available dressings include Compeel, Duoderm, Tagaderm and Opsite.

When the peptide derivative of the present invention is used in the cosmetic composition, there is no particular limitation in the formulation, for example, a cosmetic having a formulation of softening longevity, nourishing longevity, massage cream, nourishing cream, pack, gel or skin adhesive cosmetic It may be a composition and may be a transdermal dosage form such as a lotion, ointment, gel, cream, patch or spray.

In the composition of the present invention, pharmaceutically or cosmetically orbitally acceptable carriers may be hydrocarbons such as petrolatum, liquid paraffin, gelled hydrocarbons (also called plastibases), depending on the formulation thereof; Animal and vegetable oils such as medium chain fatty acid triglyceride, lard, hard fat and cacao butter; Higher fatty acid alcohols and fatty acids such as cetanol, stearyl alcohol, stearic acid and isopropyl palmitate and esters thereof; Water-soluble bases such as macrogol (polyethylene glycol), 1,3-butylene glycol, glycerol, gelatin, white sugar and sugar alcohol; Emulsifiers such as glycerin fatty acid esters, polyoxyl stearic acid and polyoxyethylene-cured castor oil; Pressure-sensitive adhesives such as acrylate ester and sodium alginate; Propellants such as liquefied petroleum gas and carbon dioxide; Preservatives, such as paraoxybenzoic acid ester, etc. are mentioned, The external preparation of this invention can be manufactured according to a conventional method using these. In addition to these, stabilizers, flavors, colorants, pH adjusters, diluents, surfactants, preservatives, antioxidants and the like may be blended as necessary. The external preparation of the present invention can be applied to the topical wounds by conventional methods.

In addition, the pharmaceutical composition according to the present invention may be used adhered to a solid support such as a wound peeling cover of a conventional band-aid. In an aspect of the present invention, the solid support is first coated with an adhesive layer to enhance the attachment of the peptide derivative to the solid support. Examples of the pressure-sensitive adhesive may include polyacrylate and cyanoacrylate.

Many formulations of this type are commercially available, such as Smith & Nephew Ltd. with a non-adhesive wound release cover in the form of perforated plastic films, thin strips, patches, BAND-AIDs in the form of spots, plastic strips; Curity CURAD (Ouchless) Band-Aid from Colgate-Palmolive Co. (Kendall); And STIK-TITE elastic strips from American White Cross Laboratories, Inc .;

In one embodiment of the present invention, the composition according to the present invention may be formulated in the form of a liquid coating agent by mixing the peptide derivative and physiological saline in a constant volume ratio. As an example of the present invention, the pharmaceutical composition according to the present invention may be formulated in the form of an ointment by mixing the peptide derivative of the present invention with a water-soluble ointment and adding physiological saline thereto.

However, the amount of the pharmaceutical composition according to the present invention may be used in terms of the route of administration, the age, sex, weight of the patient, the severity of the patient, the type of wound, the site of application, the number of treatments, the treatment time, the dosage form, the condition of the patient, the type of supplement, and the like. The effective amount is not to be understood as limiting the scope of the present invention in any aspect because it is determined in view of various related factors.

In addition, the composition according to an embodiment of the present invention may be provided in the formulation of a dermal filler. The term 'dermal filler' or 'dermal filler' refers to a substance similar to skin tissue and is used in wrinkle improvement or contour correction by inserting in a specific area and expanding soft tissue. Collagen is commonly used as an injectable substance in soft tissue expansion. In addition, numerous other substances have been used as dermal fillers for injection, including proteins, fats, hyaluronic acid (HA), polyalcohols and other polymers such as carboxymethylcellulose, dextran. Examples of skin fillers based on collagen include EVOLENCE 30 (brand name of ColBar LifeScience skin filler), pig collagen as a main ingredient, Zyderm and Zyplast (above brand names of Inamed skin filler), and human collagen based on bovine collagen. CosmoDerm and CosmoPlast (trade name of skin fillers of Inamed Co., Ltd.) as main components are known. Main ingredients of hyaluronic acid include Rofilan (trade name of skin filler of Rofil / Philoderm), Perlane, Restylane (trade name of dermal filler of Medicis / Q-Med AB), Teosyal (trade name of dermal filler of Teoxane SA), Surgiderm (Trade name of dermal fillers of Corneal Laboratoire). Dermal filler according to an embodiment of the present invention is prepared by loading a suitable amount of the peptide according to an embodiment of the present invention in a known skin filler or by covalently or non-covalently binding the peptide to a polymer that is a skeleton of the skin filler. Can be.

Hereinafter, the present invention will be described in detail through Examples and Experimental Examples. It should be understood, however, that the invention is not limited to the disclosed embodiments and examples, but may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, It is provided to fully inform the owner of the scope of the invention.

Example

Example 1: Preparation of YIGSR

YIGSR peptide (SEQ ID NO: 1) was prepared by the peptide manufacturer (Anygen, Korea).

Example 2: Preparation of palmitoyl-YIGSR

Peptide derivative (Pal-YIGSR, molecular weight: 846) having a palmitoyl group attached to the N-terminus of the YIGSR peptide (SEQ ID NO: 1) of Example 1 in the same manner as in Example 1 using palmitoyl-tyrosine It was prepared (Anygen, Korea).

Comparative Example 1: Preparation of Pal-RGD

An RGD peptide having a palmitoyl group attached to the N-terminus was prepared in the same manner as in Example 1 using palmitoyl-arginine. RGD is a peptide having integrin binding activity and is used as a cell adhesive.

Comparative Example 2: Preparation of oleyl-YIGSR

A YIGSR peptide having an oleyl group added to the N-terminus instead of a palmitoyl group was prepared in the same manner as in Example 1 using oleyl-tyrosine.

Experimental Example

Experimental Example 1: Collagen Synthesis and Erk Phosphorylation

After treating the peptide derivatives prepared in Examples 1 and 2 and Comparative Example 2 to the cultured Hs27 dermal fibroblasts, the type 1 collagen expression in the cells was measured by immunoblotting assay. The Hs27 dermal fibroblasts were cultured under conditions of 95% humidity, 5% CO 2, 37 ° C. using DMEM medium (Lonza, USA) to which 10% FBS (Lonza, USA) was added. Peptides of each concentration (0, 2, 25, 50 and 100 μM) were treated with the Hs27 dermal fibroblasts for 24 hours or 100 μM peptides according to each time (0, 0.5, 6, 12 and 24 hours). Thereafter, the treated cells were pulverized ultrasonically in lysis buffer (150 mM NaCl, 1% Triton X-100, 10 mM Tris, 1 mM EDTA, pH 7.4), followed by centrifugation to separate the supernatant, and the Bradford assay (Bradford assay). Was quantified. The same amount of protein was mixed with 5x sample buffer and heated at 95 ° C. for 5 minutes, followed by electrophoresis on 6-15% gradient SDS-PAGE (SDS-Polyacrylamide gel electrophoresis) and adsorbed onto nitrocellulose membranes. Anti-type 1 collagen primary antibody (Rockland, USA), anti-phosphorylation-ERK primary antibody (Cell Signaling, USA) and anti-actin primary antibody (Santa Cruz, USA) comprising 5% scheme milk After diluting in TBST buffer (Tris buffered saline with 0.05% Tween 20, pH 7.6) for 16 hours, the mixture was washed 6 times with TBST for 10 minutes. Then, the mice were treated with anti-mouse rabbit peroxidase-binding secondary antibody (KPL, USA), reacted for 1 hour at room temperature, and washed six times with TBST again for 10 minutes, and enhanced chemiluminescent (ECL) solution (Amersham, USA). It was confirmed by color development).

As a result, the effect of Pal-YIGSR on collagen formation in dermal fibroblasts was increased collagen synthesis with increasing concentration, collagen synthesis was increased with increasing exposure time (Fig. 1). YIGSR was also weaker than Pal-YIGSR but increased collagen synthesis. However, in the case of oleyl-YIGSR, there was no significant difference in the degree of collagen synthesis from the control (Fig. 2).

In addition, induction of ERK phosphorylation by Pal-YIGSR in dermal fibroblasts was also observed (Fig. 3). However, YIGSR and oleyl-YIGSR had little effect on ERK phosphorylation.

Experimental Example 2: Analysis of the Effect of Palmitoyl Group

The inventors carried out experiments using RGD peptides known cell adhesion capacity to determine whether the collagen synthesis increased by Pal-YIGSR is purely palmitoyl.

Specifically, the experiment was performed in the same manner as in Experimental Example 1 using the Pal-RGD peptide prepared in Comparative Example 1 and the RGD peptide to which the palmitoyl group was not attached.

As a result, the RGD peptide and Pal-RGD peptide did not show a significant difference from the control group in the degree of coke synthesis (FIG. 5). This suggests that the increased collagen synthesis by the peptide derivative of the present invention is not simply due to the function of palmitoyl group itself.

Experimental Example 3: Measurement of Cell Proliferation

The Hs27 skin fibroblasts were sprinkled 40,000 in 24-well culture vessels and incubated at 5% CO ₂ at 37 ° C. for 24 hours to allow the cells to adhere to the culture vessel bottom. The next day after exchange with a medium containing FBS and incubated again for 24 hours, the peptide derivatives of Examples 1 and 2 and Comparative Examples 1 and 2 were treated by concentration and incubated again for 72 hours, followed by crystal violet assay. Proliferation of the cells was measured.

Specifically, the medium of each well is first removed, and then stained for 5 minutes by adding 500 ml of 0.1% crystal violet solution per well, and then the crystal violet solution is removed and washed four times until the wells are cleared with distilled water. It was. When the washed distilled water is cleared, the distilled water is removed, and 1 ml of 95% ethanol is added to dissolve the stained crystal violet on the cells while stirring for 20 minutes. 200 ml of this solution was dispensed in a 96-well container and absorbance was measured at 590 nm using an ELISA reader, and relative cell proliferation was calculated using the untreated group as a control.

As a result, Pal-YIGSR is 0.5 ?? Cell proliferation was observed in the 50 μM concentration range, and especially 13% and 23% cell proliferation in the 100 and 500 μM concentration range, respectively (Fig. 4).

As described above, the Pal-YIGSR peptide derivative of the present invention increased the production of type 1 collagen in cultured fibroblasts and promoted the proliferation of fibroblasts. It is to demonstrate that the peptide derivative of the invention can be used as an effective substance.

Manufacturing example

Preparation Example 1 Injection Solution

Injection solution containing 10 mg of Pal-YIGSR peptide derivative was prepared by the following method.

10 mg of Pal-YIGSR peptide derivative and 0.6 g of sodium chloride were dissolved in purified water to make 100 ml. This solution was sterilized by filtering with a 0.2 μm filter.

The components of the injection solution are as follows.

Component content (% by weight)

Purified Water To 100

Pal-YIGSR Peptide 0.01

Sodium Chloride 0.6

Preparation Example 2: Softener (Skin Lotion)

A flexible longevity containing Pal-YIGSR peptide was prepared according to a conventional method as follows.

Component Content (wt%)

Purified water To 100

Pal-YIGSR Peptide 0.01

Butylene Glycol 2.0

Propylene Glycol 2.0

Carboxyvinyl Polymer 0.1

Fiji-12 nonylphenyl ether 0.2

Polysorbate 80 0.4

Ethanol 10.0

Triethanolamine 0.1

Preservatives, colorings, flavors

Preparation Example 3: Nutrients (Milk Lotion)

Nutrients containing Pal-YIGSR peptides were prepared according to a conventional method as follows.

Component Content (wt%)

Purified water To 100

Pal-YIGSR Peptide 0.01

Beeswax 4.0

Polysorbate 60 1.5

Solbitan Sesquioleate 1.5

Liquid Paraffin 0.5

Caprylic / Capric Triglycerides 5.0

Glycerin 3.0

Butylene Glycol 3.0

Propylene Glycol 3.0

Carboxy Vinyl Polymer 0.1

Triethanolamine 0.2

Preservatives, colorings, flavors

Preparation Example 4 Nutrition Cream

A nutritive cream containing Pal-YIGSR peptide was prepared according to a conventional method as follows.

Component Content (wt%)

Purified water To 100

Pal-YIGSR Peptide 0.01

Beeswax 10.0

Polysorbate 60 1.5

Sebum 60 Cured Castor Oil 2.0

Solbitan Sesquioleate 0.5

Liquid Paraffin 10.0

Squalane 5.0

Caprylic / Capric Triglycerides 5.0

Glycerin 5.0

Butylene Glycol 3.0

Propylene Glycol 3.0

Triethanolamine 0.2

Preservatives, colorings, flavors

Preparation Example 5 Massage Cream

A massage cream containing Pal-YIGSR peptide was prepared according to a conventional method as follows.

Component Content (wt%)

Purified water To 100

Pal-YIGSR Peptide 0.01

Beeswax 10.0

Polysorbate 60 1.5

Sebum 60 Cured Castor Oil 2.0

Solbitan Sesquioleate 0.8

Liquid Paraffin 40.0

Squalane 5.0

Caprylic / Capric Triglycerides 4.0

Glycerin 5.0

Butylene Glycol 3.0

Propylene Glycol 3.0

Triethanolamine 0.2

Preservatives, colorings, flavors

Preparation Example 6: Pack

Packs containing Pal-YIGSR peptides were prepared according to conventional methods as follows.

Component Content (wt%)

Purified water To 100

Pal-YIGSR Peptide 0.01

Polyvinyl Alcohol 13.0

Sodium Carboxymethylcellulose 0.2

Glycerin 5.0

Allatoin 0.1

Ethanol 6.0

Fiji-12 Nonylphenyl Ether 0.3

Polysorbate 60 0.3

Preservatives, colorings, flavors

Manufacturing example  7: dermal filler ( dermal filler )

Dermal fillers containing Pal-YIGSR peptides were prepared according to conventional methods as shown in the table below.

Component content (% by weight)

Purified Water To 100

Pal-YIGSR Peptide 0.01

Human Collagen 3.5

Potassium Chloride (KCl) 0.02

Potassium phosphate mono (KH ₂ PO ₄ ) 0.024

Sodium Chloride (NaCl) 0.8

Sodium diphosphate (Na ₂ HPO ₄ ) 0.1145

Preservative

Although the present invention has been described with reference to the above Examples, Experimental Examples and Preparation Examples, these are merely exemplary, and it will be understood by those skilled in the art that various modifications and equivalent other embodiments are possible. will be. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

<110> NOVACELL TECHNOLOGY INC. <120> Novel peptide derivative capable of synthesizing collagen and use          the <130> PD12-0436 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> YIGSR petide <400> 1 Tyr Ile Gly Ser Arg   1 5

Claims (7)

Peptide derivative having a palmitoyl group added to the N-terminus of the peptide consisting of 5 to 100 amino acids comprising the amino acid sequence (YIGSR) described in SEQ ID NO: 1. The method of claim 1,
The peptide is a peptide derivative, consisting of 5 to 20 amino acids. The method of claim 1,
The peptide consists of an amino acid sequence set forth in SEQ ID NO: 1. The composition for reducing skin aging comprising the peptide derivative of any one of claims 1 to 3 or a YIGSR peptide composed of the amino acid sequence described in SEQ ID NO: 1 as an active ingredient. A cosmetic composition for improving wrinkles, comprising as an active ingredient a peptide derivative of any one of claims 1 to 3 or an amino acid sequence as set forth in SEQ ID NO: 1. A pharmaceutical composition for treating wounds, comprising as an active ingredient a peptide derivative of any one of claims 1 to 3 or an amino acid sequence as set forth in SEQ ID NO: 1. A dermal filler comprising the peptide derivative of any one of claims 1 to 3 or a YIGSR peptide composed of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

Images