KR20110107552A - Modified wnt10-derived peptides and uses thereof - Google Patents

Abstract

The present invention relates to a modified WNT10-derived peptide and a hair loss improving and skin improving composition comprising the same, and a composition for treating Wnt10 signaling-related diseases. The WNT10-derived peptide of the present invention has the same or similar function and stability as the native WNT10 protein, and is superior to the native WNT10 protein, and has excellent skin permeability. Thus, compositions comprising the peptides of the invention not only have very good efficacy in improving hair loss (eg, promoting hair growth or hair production), but can also exert excellent efficacy in treating WNT10 signaling-related diseases. In addition, the excellent activity and stability of the peptides of the present invention make it possible to be very advantageously applied to medicines, quasi-drugs and cosmetics.

Description

Modified WNT10-Derived Peptides and Uses Thereof}

The present invention relates to a WNT10-derived peptide, a hair loss improving and skin improving composition comprising the same, and a composition for treating WNT10 signaling-related disease.

Hair follicles are unique organs of mammalian skin, the organs of which the lower part of the primitive epidermis grows and extends into a deeper skin layer. At the base of the hair follicle is a plug of cells known as follicular or dermal papilla cells (Stenn and Paus, Physiol. Rev. , 81: 449 (2002)). The papilla is essential for normal circulation of hair follicles (Oliver, Embryol. Exp. Morph. , 15: 331 (1966); Oliver, Embryol. Exp. Morph. , 16: 231 (1966)) and hair shaft growth. The hair shaft is a tread-shaped structure made of tightly adherent epithelial cells filled with keratin filaments and filament aggregate proteins.

Human hair periodically undergoes a process of growing, degenerating, and resting, losing hair and regenerating it. The hair cycle is achieved through hormonal control and many growth factors. On the other hand, due to severe stress or malnutrition, the hair enters the resting stage early and causes severe hair loss (American Journal of Pathology, Vol, 162. No. 3, Stress and the Hair Follicle: Exploring the Connections (2003). )). In male baldness, the front and upper hair follicles of the scalp are much affected by androgens. Thus, in male baldness, the hair follicles become smaller than the hair follicles, but due to the excessive release of androgen, a male hormone, 5-alpha reductase is activated, which causes testosterone to be dihydrotestosterone. , Dihydrotestosterone, which causes hair loss by shortening the period of hair growth and miniaturizing the hair follicles, thereby reducing the number of thick, strong hairs. In addition, 20% of women with hair loss often show thinning hair on the top of the scalp, which is estimated to undergo some type of hair loss in their lifetime. As you age, hair loss spreads. In addition, different disease states such as, for example, scarring alopecia, burns, or scarring conditions associated with compression injuries can cause significant hair loss. Irrespective of the cause, women's entry into the modern society and men's attention to appearance can cause hair loss to eventually exert significant mental, social and sexual influences along with loss of self-esteem and self-esteem. To treat this hair loss phenomenon, various substances have been used as medicines until now, but the price is too expensive and various side effects are caused. In addition, these drugs require constant use and have the disadvantage of causing hair loss again when discontinued. In addition, individual differences in efficacy are severe, and side effects also vary in each individual disadvantages.

Other raw materials used in cosmetics have the advantage of low price, but because they are composed of plant extract-derived ingredients, the effect is not great. Thus, to make up for the shortcomings of bio-stable, effective and cost-effective parts, WNT10-derived peptides with similar functions as WNT10 are produced in large quantities at low cost compared to proteins using chemical synthetic methods. We developed a method that can do it, and developed a product that can see a great effect by using the nanosome technology to easily penetrate the skin, high penetration in the skin. The peptide induces the proliferation of stem cells in the hair follicles of skin tissues that can produce hair follicles, and induces differentiation to reach hair follicles, allowing new hair follicles to be produced. In addition, the peptide plays a role in promoting the growth stage, which is the period of hair formation and growth, and shows various effects of hair loss by inhibiting the cycle of hair to the stage of growth due to various environmental factors. It nourishes the hair and greatly affects healthy hair. The two available drugs known to date (minoxidil and finasteride) can delay further hair loss, but in reality nothing was available to induce regeneration of new hair follicles. In addition, many hair cosmetic products have been released to prevent hair loss using plant extracts, in particular, ginseng, red pepper, medicinal herbs, lettuce, green leaf, ginseng, licorice, peony, turmeric, fennel, cornus, garlic, etc. Addition of a composition containing xanthine and growth hormone improves the inhibition of cell metabolism caused by the excess of dihydrotestosterone, while growth hormone promotes hair growth and prevents hair loss and regenerates hair to promote hair growth Developed products containing minerals and vitamins, green tea, rosemary, mugwort and licorice extract to promote hair growth and hair growth, nourish scalp and hair, promote hair growth which is effective in preventing hair loss and promoting hair growth Products, such as vitamin B, vitamin C, vitamin D, vitamin E, nicotinic acid, pantothenic acid, biotin and folic acid By mixing the extracts to inhibit 5-alpha reductase in the human body to prevent the formation of dihydrotestosterone during the metabolism of male hormones and to help the hair metabolism, the development of male hair loss products, but also affects the production of new hair The product was hard to find. On the other hand, the company developed a product using corosolic acid, which was found to be effective in diabetes, by a clinical team of a research group at the University of Tokyo's Tokyo Society and the College of Health Medicine. Some products were developed with excellent features.

In the process of hair growth and degeneration, there are many factors linked to each other. Especially, among the various growth factors that encourage the hair cycle, human-derived WNT10 affects hair by transmitting signals from the cells. The pathway is activated by the interaction between secreted WNT10 and their receptor Frizzled protein, where LDL receptor-related proteins LRP5 and LRP6 act as co-receptors (Clin Cancer Res 2007; 13 (14) July 15, 2007, WNT Signaling Pathway and Stem Cell Signaling Network). Downstream effects of WNT10 signaling, including activation of DVL (Disheveled) proteins, are recruited to Axin-beta-catenin-GSK3beta-complexes after activating Akt (Fukumoto et al. , J. Biol. Chem. , 276) . : 17479-17483 (2001). GSK3beta is then phosphorylated and inactivated to inhibit phosphorylation and degradation of beta-catenin. Accumulated beta-catenin is translocated to the nucleus and interacts with transcription factors of the lymphoid enhancer-T cell factor (LEF-TCF) family to induce transcription of the target gene. These expressed proteins play an important role in hair growth and differentiation and allow the production of new hair cells. In addition, it has a function of preventing hair loss by lowering the function of DHT caused by testosterone.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The inventors have sought to develop materials that can perform the same or similar functions or actions as native WNT10, but have better activity, skin permeability and stability than WNT10 protein. As a result, WNT10 has excellent physiological activity (e.g., hair loss improvement, cell growth promoting ability, expression of fibronectin, etc.) among many peptide candidates, as well as stability (e.g., heat or pH stability) and skin permeability. By synthesizing the derived modified peptides, the present invention has been completed.

Accordingly, an object of the present invention is to provide a peptide of SEQ ID NO: 1 sequence.

Another object of the present invention to provide a composition for improving hair loss.

Still another object of the present invention is to provide a composition for improving skin conditions.

Another object of the present invention to provide a composition for improving or treating WNT10 signaling-related diseases.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, a peptide having the amino acid sequence of SEQ ID NO: 1 is provided.

According to another aspect of the present invention, there is provided a composition for improving hair loss comprising the peptide as an active ingredient.

The inventors have sought to develop materials that can perform the same or similar functions or actions as native WNT10, but have better activity, skin permeability and stability than WNT10 protein. As a result, WNT10 has excellent physiological activity (e.g., hair loss improvement, cell growth promoting ability, expression of fibronectin, etc.) among many peptide candidates, as well as stability (e.g., heat or pH stability) and skin permeability. Derived modified peptides were synthesized.

There are many factors linked to the process of hair growth and degeneration, and the present inventors have studied to promote the proliferation of keratinocytes of hair and induce differentiation into hair, which is the most important for hair root formation. A study was conducted using a series of growth factors that provided nutrients and promoted vascular endothelial growth factor activity. The inventors of the present invention show that WNT10 protein is very effective, but to obtain an active WNT10 protein, an additional step and time of refolding is required, and the purification process requires a complicated purification process to remove contaminants derived from E. coli. In order to compensate for the disadvantage of not easily jumping over the protective film of the hair due to the stability and high molecular weight and the low price due to the high price, we developed a similar peptide with WNT10 protein and applied it to the product. An analogue has been developed that can be economically competitive.

According to the results of our previous studies, cysteine-containing WNT10-derived peptides have very good physiological activity (eg, increased cell growth activity, increased beta-catenin and fibronectin expression, hair growth activity, etc.) compared to native WNT10. ), As well as excellent thermal stability and skin permeability (Korean Patent Application No. 10-2009-0081817). Based on this, the present inventors synthesized a peptide (SEQ ID NO: 1) in which a cysteine residue was substituted with a serine residue among the existing WNT10-derived peptide sequences containing cysteine.

Peptides of the invention have the same or similar physiological activity (eg, growth proliferative activity of HaCaT keratinocytes and NIH3T3 fibroblasts, increased beta-catenin and fibronectin expression, hair growth compared to conventional cysteine-containing WNT10-derived peptides). Activity, etc.), as well as thermal stability and skin permeability. Furthermore, the peptides of the present invention had better pH stability compared to conventional WNT10-derived peptides containing cysteine (see Figure 6b).

The peptide of the present invention comprises an amino acid sequence selected from the group consisting of WNT10-derived amino acid sequences. Preferably, the peptide in the present invention consists essentially of the amino acid sequence of SEQ ID NO: 1 sequence. As used herein, the term "peptide" refers to a linear molecule formed by binding amino acid residues to each other by peptide bonds.

Peptides of the invention can be prepared according to chemical synthesis methods known in the art, in particular solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85: 2149-54 (1963) Stewart, et al., Solid Phase Peptide Synthesis , 2nd.ed., Pierce Chem. Co .: Rockford, 111 (1984)).

Peptides of the present invention randomly synthesized several sites present in the WNT10 protein to search the binding site for the receptor protein first, and then select the peptide of the present invention by optimizing the amino acid sequence of the predicted site By screening the most active peptide among these candidate peptides, the peptide of the present invention is provided.

The first sequence peptide of SEQ ID NO: 1 is a peptide that performs a function similar to native WNT10 and binds to a receptor to perform a function such as a growth factor.

According to a preferred embodiment of the present invention, the peptide of the present invention has a growth promoting ability of keratinocytes and fibroblasts. According to a preferred embodiment of the present invention, the peptide of the present invention promotes the expression of fibronectin.

In addition, the peptide of the present invention has a function of activating WNT signaling. According to a preferred embodiment of the present invention, the peptide of the present invention delivers beta-catenin into the nucleus.

The peptide of the present invention is superior in stability to the native WNT10 protein by itself, but may be further improved by modification of amino acids.

According to a preferred embodiment of the invention, the N-terminus of the peptide is from the group consisting of acetyl group, fluorenyl methoxy carbonyl group, formyl group, palmitoyl group, myristyl group, stearyl group and polyethylene glycol (PEG) The protecting group selected is combined.

According to a preferred embodiment of the present invention, the peptide of the present invention is selected from the group consisting of acetyl group, fluorenyl methoxy carbonyl group, formyl group, palmitoyl group, myristyl group, stearyl group and polyethylene glycol (PEG) Protecting groups can be bound to provide peptides that are more stable than naturally occurring modified growth factors.

Modification of the above-mentioned amino acid serves to greatly improve the stability of the peptide of the present invention. The term "stability" herein means not only " in vivo" stability but also storage stability (eg, room temperature storage stability). The above protecting groups serve to protect the peptides of the present invention from the attack of protein cleavage enzymes in vivo.

According to another aspect of the present invention, the present invention provides a composition for treating or improving hair loss comprising the peptide of the present invention as an active ingredient.

According to another aspect of the present invention, the present invention provides a composition for improving skin conditions comprising the peptide of the present invention as an active ingredient.

Since the composition of the present invention includes the growth factor-related peptide of the present invention described above as an active ingredient, the common content between the two is omitted in order to avoid excessive complexity of the present specification.

According to a preferred embodiment of the present invention, the treatment or amelioration of hair loss by the peptides of the present invention is hair growth promotion or hair production.

As demonstrated in the following examples, the peptide of the present invention has the ability to promote fibroblast and keratinocyte growth, and promoted the beta-catenin signal which is a representative signaling of the WNT protein, confirming that the signal of the WNT works normally. The expression of fibronectin, a target gene of WNT, was also increased by the peptide treatment of the present invention. Animal experiments based on these results showed that the peptide of the present invention significantly promotes hair growth. Therefore, the composition of the present invention is very effective for improving hair growth and skin condition.

According to a preferred embodiment of the present invention, the improvement of the skin condition by the peptide of the present invention is wrinkle improvement, skin elasticity improvement, skin aging prevention, skin moisturization improvement, wound removal or skin regeneration.

According to another aspect of the present invention, the present invention provides a composition for improving or treating WNT10 signaling-related diseases comprising the above-described peptide of the present invention as an active ingredient.

Since the composition of the present invention includes the growth factor-related peptide of the present invention described above as an active ingredient, the common content between the two is omitted in order to avoid excessive complexity of the present specification.

According to a preferred embodiment of the present invention, the above-described WNT10 signaling-related disease is an eye disease, a lipid-modulated disorder, a bone disease or a tumor disease, more preferably a bone disease or a tumor disease.

According to a preferred embodiment of the present invention, the above-described bone diseases are osteogenic diseases, bone fractures, senile loss of bone, chondrotrophy, hypercalcemia, hyperosteoporosis, incomplete osteoplasia, osteomalacia, osteomyelitis, osteoporosis, Paget's disease, osteoarthritis And rickets.

According to a preferred embodiment of the invention, the tumor disease described above is colorectal carcinoma, breast carcinoma or melanoma.

According to a preferred embodiment of the present invention, the above-mentioned fat-regulating disease is heart condition, atherosclerosis, familial lipoprotein lipase deficiency, type 3 hyperlipoproteinemia, familial hypercholesterolemia, familial hyperneutral Lipolipidemia, multiple lipoprotein type hyperlipoproteinemia, fat increase by dialysis and / or diabetes, and familial apoprotein CII deficiency.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the growth factor-related peptide of the present invention described above; And (b) a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the efficacy or activity of the growth factor-associated peptide described above.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of extracts, powders, granules, tablets, capsules, or gels (eg, hydrogels), and may further include dispersants or stabilizers. .

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a cosmetically effective amount of the growth factor-related peptide of the present invention described above; And (b) a cosmetically acceptable carrier.

As used herein, the term “cosmetic effective amount” means an amount sufficient to achieve the skin improving efficacy of the composition of the present invention described above.

The cosmetic composition of the present invention may be prepared in any form conventionally produced in the art and may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant- , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The components included in the cosmetic composition of the present invention include components conventionally used in cosmetic compositions, in addition to peptides and carrier components as active ingredients, and include, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. Phosphorus adjuvant.

The features and advantages of the present invention are summarized as follows:

(Iii) Modified WNT10-derived peptides of the present invention may function the same or similar to native WNT10 proteins.

(Ii) The peptides of the present invention have very good stability compared to the native WNT10 protein and have very good skin permeability.

(Iii) Thus, compositions comprising the peptides of the present invention not only have very good efficacy in improving hair loss (e.g., promoting hair growth or hair production), but also exert excellent efficacy in treating WNT10 signaling-related diseases. have.

(Iii) The excellent activity and stability of the peptides of the present invention described above make it possible to be very advantageously applied to medicines, quasi-drugs and cosmetics.

1 is a graph showing the results of high performance liquid chromatography analysis of the peptide of SEQ ID NO: 1 prepared by the synthesis example of the present invention.
Figure 2a is a graph showing the cell growth promoting effect of keratinocytes treated with a peptide and cysteine-containing peptide prepared by the synthesis example of the present invention.
Figure 2b is a graph showing the effect of promoting cell growth of fibroblasts treated with a peptide and cysteine-containing peptide prepared by the synthesis example of the present invention.
Figure 3 is a microscopic picture of the cell growth promoting effect of keratinocytes and fibroblasts treated with the peptide and the cysteine-containing peptide of the present invention.
Figure 4a is a data measuring the expression of beta-catenin when the peptide of the present invention and the peptide containing cysteine.
Figure 4b is a photograph measuring the activity of beta-catenin when the peptide of the present invention and the peptide containing cysteine.
Figure 5 is a photograph measuring the activity of fibronectin when the peptide of the present invention and the peptide containing cysteine.
Figure 6a is a result of the temperature stability test of the peptide containing the peptide and cysteine of the present invention.
Figure 6b is a result of the pH stability test of the peptide and the cysteine-containing peptide of the present invention.
Figure 7 is a test result of the hair growth effect of the skin, such as a mouse treated with a peptide and cysteine-containing peptide of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1

Synthesis Example 1 Synthesis of Arg-Gln-Thr-Arg-Val-Gln-Arg-Ser-His-Ser (SEQ ID NO: 1)

700 mg of chloro trityl chloride resin (CTL resin, Nova biochem Cat No. 01-64-0021) was added to a reaction vessel, and 10 ml of methylene chloride (MC) was added thereto, followed by stirring for 3 minutes. The solution was removed, 10 ml of dimethylformamide (DMF) was added thereto, stirred for 3 minutes, and then the solvent was removed again. 10 ml of dichloromethane solution was added to the reactor, 200 mmole of Fmoc-Ser (trt) -OH (Bachem, Swiss) and 400 mmole of diisopropyl ethylamine (DIEA) were added to the mixture, followed by stirring to dissolve well. I was. After the reaction, the mixture was washed with methanol and DIEA (2: 1) in dichloromethane (DCM) for 10 minutes and washed with excess DCM / DMF (1: 1). The solution was removed, 10 ml of dimethylformamide (DMF) was added thereto, stirred for 3 minutes, and then the solvent was removed again. 10 ml of deprotection solution (20% of piperidine / DMF) was added to the reaction vessel and stirred at room temperature for 10 minutes to remove the solution. After adding the same amount of deprotection solution to maintain the reaction again for 10 minutes, the solution was removed and washed with DMF twice, once with MC, once with DMF for 3 minutes each to prepare Ser (trt) -CTL resin. 10 ml of DMF solution was added to a new reactor, 200 mmole of Fmoc-His (trt) -OH (Bachem, Swiss), 200 mmole of HoBt, and 200 mmole of Bop were stirred, and the mixture was dissolved. 400 mmole DIEA was added to the reactor twice in fractions and stirred for at least 5 minutes until all solids dissolved. The dissolved amino acid mixture solution was placed in a reaction vessel with deprotected resin and reacted with stirring at room temperature for 1 hour. The reaction solution was removed and stirred with a DMF solution three times for 5 minutes and then removed. A small amount of the reaction resin was taken and the degree of reaction was checked using a Nihydrin Test. His (trt) -Ser (trt) -CTL resin was prepared by deprotection twice in the same manner as above with the deprotection solution. After sufficient washing with DMF and MC, once again Kaiser test was performed, and the following amino acid attachment experiment was performed as above. Based on the selected amino acid sequence, Fmoc-Ser, Fmoc-Arg, Fmoc-Gln (trt), Fmoc-Val, Fmoc-Arg, Fmoc-Thr, Fmoc-Gln (trt) and Fmoc-Arg (pbf) The reaction was carried out. The Fmoc-protector was reacted twice with a deprotection solution twice for 10 minutes and then washed well. Acetylation of acetic anhydride, DIEA, and HoBt was carried out for one hour, and then the prepared peptidyl resin was washed three times with DMF, MC, and methanol, and dried slowly with nitrogen air, followed by vacuum under P ₂ O ₅ . After drying completely under reduced pressure, 30 ml of a fugitive solution (Trifluroacetic acid (95%, Trifluroacetic acid), 2.5%, distilled water 2.5%, Thianisole) 2.5%) was added thereto, and the reaction was kept at room temperature for 2 hours. The resin was filtered off and the resin was washed with a small amount of TFA solution and then combined with the mother liquor. Distillation was carried out using reduced pressure to half the total volume and 50 ml of cold ether was added to induce precipitation, followed by centrifugation to collect the precipitate and washing with two more cold ethers. The mother liquor was removed and dried sufficiently under nitrogen to synthesize 0.71 g of NH ₂ -Arg-Gln-Thr-Arg-Val-Gln-Arg-Ser-His-Ser-OH peptide 1 (yield: 91.5%) before purification. Molecular weight 1255.3 (theoretical value: 1255.1) was obtained when measured using a molecular weight meter.

number Amino acid sequence Analytical Values (Mass Spectrometer) Analytical value Theoretical value SEQ ID NO: 1 RQTRVQRSHS 1255.3 1255.1

Test Example 1 Identification of Cell Growth Effect Using Synthetic Peptides

In order to analyze the growth factor-like efficacy and growth factor-like efficacy and the inhibitory effect on the sequence peptide synthesized in Synthesis Example 1 compared to the peptide including the existing cysteine (Rizzino, et al. Cancer Res. 48 : 4266 (1988)) was measured using the SRB (Sulforhodamine B, Sigma) colorimetric method using HaCaT keratinocytes (Korea Cell Line Bank) and NIH3T3 fibroblasts (Korea Cell Line Bank).

HaCaT keratinocyte lines and NIH3T3 fibroblasts were cultured in Dulbecco's modied Eagle's medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (Sigma) using a 250 ml tissue culture flask. It was. The cultured cell lines were removed from the bottom of the culture vessel with 1% trypsin solution and centrifuged to collect only cell precipitates. The cultured cell lines were removed from the bottom of the culture vessel with 1% trypsin solution and centrifuged to collect only cell precipitates. After resuspending in DMEM culture medium containing no FBS, it was put in a 96-well tissue culture plate to be 3 X 10 ³ cells per well and incubated for 24 hours at 37 ℃, 5% CO ₂ conditions. After 24 hours, the medium was exchanged with the same culture except for the serum completely, and the blank sample and the synthetic peptide (1 ng / ml, 10 ng / ml, 100 ng / ml, 1 ㎍) sterilely dissolved in 10% DMSO for standardization. / ml and 10 μg / ml) and peptide complex (1 μg / ml) were incubated for 72 hours under the same conditions as above. After the incubation was completed, the culture supernatant was removed, the cells were immobilized with ethanol, and the cells were fixed and washed three times with PBS (phosphate buffer saline). After removing the washing solution and treated with colorimetric SRB solution and washed with 1% acetic acid sufficiently, the cells were observed under a microscope to observe the state of the viable cells, the absorbance was measured at 590 nm ultraviolet light to measure the viability of the cells.

Figure 2 shows the results of the growth of keratinocytes (Fig. 2a) and fibroblasts (Fig. 2b) after the peptide treatment, Figure 3 shows the morphological changes of the cells 72 hours after the peptide treatment cells in the keratinocytes And growth of fibroblasts.

As can be seen in Figure 2, the peptide of the present invention greatly enhanced the growth of keratinocytes and fibroblasts at the same concentration as the peptide containing the existing cysteine.

In Figure 3 it can be seen that the peptide of the present invention changes the growth and morphological appearance of keratinocytes and fibroblasts at the same concentration compared to the peptide containing the existing cysteine.

Test Example 2 Analysis of Increasing Amount of Beta-Catenin Expression of Synthetic Peptides

After 5 hours of treatment of the peptide synthesized in Synthesis Example 1 to HaCaT cells cultured for 48 hours, the expression level of beta-catenin, which is a representative signal of WNT protein and a signal necessary for promoting hair growth, was measured. The amount of expression was detected by Western blot using an antibody against beta-catenin (Santa Cruz, USA), and it was observed whether the beta-catenin was delivered into the nucleus by immunostaining hybridization using the same antibody. When the peptide of the present invention was treated, it was confirmed that the expression of beta-catenin was increased, which was observed to be equally effective when compared to the peptide containing the existing cysteine (FIG. 4A). In addition, the peptide of the present invention beta-catenin is translocated from the cytoplasm to the nucleus by the peptide treatment of the present invention when examined whether or not beta-catenin in the nucleus by immunostaining chemistry in HaCaT cells, In the cytoplasm still exist and could be confirmed to have activity (Fig. 4b).

In FIG. 4A, the beta-catenin expression is increased to the same level when treated with the peptide of the present invention compared to the peptide containing cysteine.

4b shows that the intranuclear delivery of beta-catenin when treated with the peptide of the present invention is delivered into the nucleus when compared with a peptide containing cysteine when observed by immunostaining chemistry.

In summary, the experimental results of Test Examples 1 and 2 show that the peptide of the present invention has the same function as that of the peptide containing cysteine. It can be seen that.

Test Example 3 Increase of Fibronectin Expression by Synthetic Peptides

In order to investigate whether the expression of fibronectin, a target protein of WNT of the peptide synthesized in Synthesis Example 1, was increased, fibroblasts were placed in a 6-well tissue culture plate to be 4 X 10 ³ cells per well. Incubated for 24 hours at 37 ℃, 5% CO ₂ conditions. After 24 hours, the medium was changed to the same culture except for the serum completely. The empty sample, the synthetic peptide, and the cysteine-containing peptide were sterilized in 10% DMSO to prepare the standard, and then treated with 1 ㎍ / ml of peptide. Then, incubated under the same conditions for 3 hours, 10 hours, 24 hours, 48 hours. After 72 hours of incubation, the culture solution was recovered, and the expression level of fibronectin was measured in the culture medium using a fibronectin ELISA kit (R & D systems, USA). As can be seen in Figure 5, the expression of fibronectin increased with the peptide treatment time of the present invention, which means that it has the same effect as the peptide containing cysteine.

FIG. 5 shows that the expression level of fibronectin increases to the same level over time when the peptide of the present invention is treated with fibroblasts over time to observe the expression level of fibronectin. Indicates.

This result indicates that the peptide of the present invention increases the expression level of the fibronectin, a hair growth promoting target protein, to the same level at the same concentration, compared to the peptide containing the cysteine, which means that the peptide of the present invention contains the existing cysteine. It means that it has the same activity as one peptide.

  Test Example 4: Stability of the prepared peptide

Peptides containing the existing cysteine was stable when the stability test was carried out for 70 days at 37 ℃. However, it was confirmed from the experimental results that the existence of an unstable form at neutral pH. This result confirms that the peptides containing two cysteine molecules are morphed by oxidizing cysteine at neutral pH. Therefore, the present invention was intended to prevent oxidation caused by cysteine at such a neutral pH, and invented a peptide that can maintain the activity as it is. This was to replace the cysteine serine in the amino acid sequence constituting the peptide to complement the pH stability. The peptide prepared in Synthesis Example 1 was dissolved in a phosphate buffer solution at a concentration of 0.1 mg / ml and then subjected to stability tests. The stability test was the first temperature stability test. The prepared solution was placed in a glass vial of 1 ml each and left at 37 ° C. The solution (pH 3-4), which was left at 37 ° C, was sampled at 0, 5, 10, 20, 30, 40, and 70 days, and centrifuged by date to remove the denatured peptide or protein. Quantification was carried out using (Fig. 6a). Secondly, a stability test for pH was conducted. In a 50 ml sterilized bottle, a peptide containing the peptide of the present invention and the existing cysteine at a concentration of 0.1 mg / ml was fixed at pH 7. After fixation, the peptide solution of pH 7 was transferred to 1 ml glass vials and allowed to stand at room temperature. Settled solutions were sampled on days 0, 5, 10, 20, 30, 40, and 70 days, centrifuged by date to remove denatured peptides, and supernatants were taken and quantified using HPLC (FIG. 6B).

As a result of the temperature stability test of the peptide of the present invention in FIG. 6a, it was observed that the peptide was stably maintained until the 70th day similarly to the peptide containing cysteine.

As a result of the stability test at pH 7 of the peptides of the present invention in FIG. 6b, it was confirmed that the cysteine-containing peptide was stably maintained at 70 days even at neutral pH at which oxidation of cysteine was suppressed.

Test Example 5: Hair growth effect of the mouse treated with the prepared peptide

Peptides prepared in Synthesis Example 1 and peptides containing cysteine were nanosomalized and applied twice a day to C57BL / 6 mice that had epidermal skin for 15 days. As a control, phosphate buffer solution was applied twice a day. On the 9th day of application, the back skin of the mouse appeared to be black in the group treated with the peptide of the present invention and the cysteine-containing peptide, and on day 15 after the application, the two peptides were treated compared to the control group. It was confirmed that a large amount of hair grows in the back skin of the mouse (FIG. 7).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> caregen <120> Modified WNT10-Derived Peptides and Uses Thereof <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> modified WNT10 peptide <400> 1 Arg Gln Thr Arg Val Gln Arg Ser His Ser   1 5 10

Claims (9)

Peptide having the amino acid sequence of SEQ ID NO: 1.
The peptide of claim 1, wherein the peptide is a human WNT10-derived modified peptide.
Hair loss treatment or improvement composition comprising the peptide of claim 1 as an active ingredient.
The composition of claim 3, wherein the treating or improving hair loss is promoting hair growth or hair production.
Composition for improving skin conditions comprising the peptide of claim 1 as an active ingredient.
The composition according to claim 5, wherein the improvement of the skin condition is wrinkle improvement, skin elasticity improvement, skin aging prevention, skin moisturization improvement, wound removal or skin regeneration.
WNT10 signaling-related disease improvement or treatment composition comprising the peptide of claim 1 as an active ingredient.
8. The composition of claim 7, wherein the WNT10 signaling-related disease is a bone disease or a tumor disease.
According to claim 8, wherein the bone disease, bone fracture, bone fracture, senile loss of bone, chondro dystrophy, hypercalcemia, hyperosteopathy, incomplete osteoplasia, osteomalacia, osteomyelitis, osteoporosis, Paget's disease, osteoarthritis and rickets Compositions selected from the group.

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