KR100753473B1 - Anti-wrinkle agents including oligonucleotides - Google Patents

Abstract

The present invention relates to an anti-wrinkle agent comprising an oligonucleotide. In particular, the present invention provides an antisense oligonucleotide for mRNA of the collagenase gene and an antisense oligonucleotide for mRNA of the c-Jun gene, which have an excellent anti-wrinkle effect by inhibiting collagen degradation.

Oligonucleotides, Anti-Wrinkle Agents

Description

Wrinkle improvement agent containing oligonucleotide {WRINKLE DECLINE AGENT CONTAINING OLIGONUCLEOTIDE}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to an anti-wrinkle agent comprising an oligonucleotide, and more particularly includes an antisense oligonucleotide for mRNA of a collagenase gene and an antisense oligonucleotide for mRNA of a c-Jun gene, and wrinkle improvement The effect is remarkable and relates to a wrinkle improver having no skin side effects.

[Private Technology]

Wrinkles are known to occur when the amount of collagen in the skin decreases and the structure is destroyed during natural aging or photoaging by ultraviolet rays. Reduction of collagen in the skin is caused by overexpression of collagenase, a collagen degrading enzyme, and especially expression of collagenase upon photoaging with ultraviolet rays rather than natural aging.

The gene that most influences the expression of collagenase so far studied is c-Jun, a proto-oncogene. c-Jun, together with another proto-oncogene, c-Fos, forms the transcription factor AP-1 transcription factor. The c-Fos gene is a gene that is constantly expressed in a cell at all times, and c-Jun is a gene whose expression is increased by stimulation such as ultraviolet rays. The expressed c-Jun and c-Fos bind to AP-1. By promoting the expression of various genes, including collagenase.

Since collagenase is a major factor in the wrinkle development process, researches to develop a natural extract that inhibits the activity of collagenase as an anti-wrinkle agent have been actively conducted. However, there is a problem that the use is limited due to skin irritation or product stability, or the wrinkle improvement effect is weak.

In order to solve the problems of the prior art, an object of the present invention is to provide an anti-wrinkle agent which is excellent in anti-wrinkle effect and can inhibit expression of a protein involved in collagen degradation.

Another object of the present invention is to provide an anti-wrinkle agent which is excellent in product stability and is not irritating to the skin.

In order to achieve the above object, the present invention provides an antisense oligonucleotide for mRNA of collagenase gene and antisense oligonucleotide for mRNA of c-Jun gene. Provide improvers.                     

Hereinafter, the present invention will be described in detail.

The present invention relates to oligonucleotides and their use as anti-wrinkle agents which can inhibit the expression of proteins involved in collagen degradation among wrinkle-causing factors, thereby improving wrinkle induction by collagenase expression.

The anti-wrinkle agent of the present invention includes an antisense oligonucleotide for mRNA of the collagenase gene and / or an antisense oligonucleotide for mRNA of the c-Jun gene as an active ingredient.

The collagenase gene and the c-Jun gene may be derived from animals including humans, and may be known genes derived from humans.

The antisense oligonucleotide is an oligonucleotide including a nucleotide sequence complementary to mRNA, the length of which may be 15 to 25 bp, but is not limited thereto. If the length of the antisense oligonucleotide is less than 15bp may not effectively inhibit the translation of the mRNA, if it exceeds 25bp may be difficult to penetrate the cell.

In one embodiment, the antisense oligonucleotide for mRNA of the collagenase gene is GeneBank accession no. It may be 15 to 25bp of nucleotides complementary to the mRNA for the sequence described in NM_002421, preferably at least one selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 1-9. Antisense oligonucleotides for mRNA of the c-Jun gene are described in GeneBank accession no. It may be a nucleotide of 15 to 25bp complementary to the mRNA for the sequence described in J04111, preferably may be selected from the group consisting of the nucleotide sequence set forth in SEQ ID NO: 10-18.

The antisense oligonucleotides of the present invention may be chemically synthesized, or may be synthesized in the form of phosphorothioate to further enhance stability in vivo.

The anti-wrinkle agent according to the present invention may include an antisense oligonucleotide selected from the group consisting of antisense oligonucleotides for mRNA of the collagenase gene, antisense oligonucleotides for mRNA of the c-Jun gene, and mixtures thereof as an active ingredient. Preferably, a mixture of antisense oligonucleotides for mRNA of the collagenase gene and antisense oligonucleotides for mRNA of the c-Jun gene may be included as an active ingredient.

The content of the active ingredient may be 0.00001 to 10.0% by weight of dry weight, but is not limited thereto. However, if the content of the active ingredient is less than 0.00001% by weight may have an insufficient wrinkle improvement effect, if it exceeds 10.0% by weight may be ineffective compared to the amount used. Preferably the content of the active ingredient may be 0.001 to 1.0% by weight.

When the antisense oligonucleotide to the mRNA of the collagenase gene and the antisense oligonucleotide to the mRNA of the c-Jun gene is used as the active ingredient, the mixing ratio thereof may be 1: 0.1 to 10 by weight.

The anti-wrinkle agent of the present invention inhibits the expression of collagen, that is, collagenase, and the antisense oligonucleotide to mRNA of collagenase gene inhibits the translation of collagenase mRNA to protein. Antisense oligonucleotides against mRNA of the c-Jun gene inhibit the transcription of the collagenase gene. Therefore, when the antisense oligonucleotide for the mRNA of the collagenase gene and the antisense oligonucleotide for the mRNA of the c-Jun gene is included as an active ingredient, the collagenase at the same time inhibits the translation of the mRNA from the collagenase to the protein By inhibiting the transcription of the gene, there is a significant wrinkle improvement effect. In addition, the anti-wrinkle agent of the present invention is a safe sample that does not cause skin irritation.

The present invention provides an antisense oligonucleotide for mRNA of the collagenase gene and / or an antisense oligonucleotide for mRNA of the c-Jun gene as an active ingredient. The anti-wrinkle agent may include only the active ingredient alone, but may further include a pharmacologically acceptable excipient according to the formulation and the method of use. The anti-wrinkle agent may include 0.00001 to 10.0% by weight of the active ingredient as dry weight, but is not limited thereto. However, if the content of the active ingredient is less than 0.00001% by weight may have an insufficient wrinkle improvement effect, if it exceeds 10.0% by weight may be ineffective compared to the amount used. Preferably the content of the active ingredient may be 0.001 to 1.0% by weight. As the active ingredient, antisense oligonucleotides for mRNA of the collagenase gene and antisense oligonucleotides for mRNA of the c-Jun gene may be used together, and a mixing ratio thereof may be 1: 0.1 to 10 by weight.

Excipients that can be mixed into the anti-wrinkle agent may include, but are not limited to, conventional pharmaceutical compositions, such as glycerin, starch, lactose, water, alcohol, propylene glycol, salicylic acid, dahyroacetic acid and saline.

Wrinkle improvement agents may be used orally or parenterally, preferably transdermal. The formulation of the anti-wrinkle agent can be properly adjusted according to the method of use, such as PLASTERS, GRANULES, LOTIONS, POWDERS, SYRUPS, and LIQUIDS. AND SOLUTIONS), AEROSOLS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS, SUSPENSIONS, INFUSIONS, TABLETS, INJECTIONS, CAPSELS CAPSULES) and PILLS, but are not limited thereto.

The dosage of the anti-wrinkle agent should be determined in consideration of the purpose of use, site, method, patient's age, sex, condition, absorption of the active ingredient in the body, inactivation rate, and the drug used in combination. It can be administered from 0.0001 mg / kg (body weight) to 500 mg / kg (body weight), based on.

In another aspect, the present invention provides a cosmetic composition for improving wrinkles comprising an antisense oligonucleotide for the mRNA of the collagenase gene and / or antisense oligonucleotide for the mRNA of the c-Jun gene as an active ingredient.

The cosmetic composition may be 0.00001 to 10.0% by weight of one or more active ingredients as a dry weight, but is not limited thereto. However, if the content of the active ingredient is less than 0.00001% by weight may have an insufficient wrinkle improvement effect, if it exceeds 10.0% by weight may be ineffective compared to the amount used. Preferably the content of the active ingredient may be 0.001 to 1.0% by weight. When the antisense oligonucleotide to the mRNA of the collagenase gene and the antisense oligonucleotide to the mRNA of the c-Jun gene is used as the active ingredient, the mixing ratio thereof may be 1: 0.1 to 10 by weight.

 The cosmetic composition of the present invention may include, in addition to the active ingredient, all kinds of ingredients usable for ordinary cosmetics, such as excipients, diluents, and the like.

The cosmetic composition of the present invention may be provided for the use of basic cosmetics, makeup cosmetics, body cosmetics, hair cosmetics, scalp cosmetics, shaving cosmetics or oral cosmetics. Examples of the basic cosmetics include creams, lotions, packs, massage creams, emulsions, etc. The makeup cosmetics include foundation, makeup base, lipstick, eyeshadow, eyeliner, mascara, eyebrow pencil, and the like. Examples include soaps, liquid cleansers, baths, sunscreen creams and sun oils. Hair care products include shampoos, rinses, hair treatments, hair mousses, hair liquids, pomades, hair colorants, hair bleaches, and colorants. There are rinses, hair tonics and scalp treatments as scalp cosmetics, aftershave and shaving creams as shaving cosmetics, and toothpaste and mouth washes as oral cosmetics.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention, and the present invention is not limited to the following examples.                     

Example 1-18: Oligonucleotide Synthesis Having an Anti-Wrinkle Effect

A total of 18 oligonucleotides of SEQ ID NOS: 1 to 18 were synthesized by BIONIA Co., Ltd., respectively, and synthesized with phosphorothioate DNA to increase stability in vivo. Each oligonucleotide prepared was as Examples 1-18. SEQ ID NOs: 1 to 9 are antisense oligonucleotides for mRNA of the collagenase gene, and SEQ ID NOs: 10 to 18 are antisense oligonucleotides for mRNA of the c-Jun gene.

SEQ ID NO: tgcttgaccctcagagacct

SEQ ID NO: 2 ctgcttgaccctcagagacc

SEQ ID NO: ttttcaacttgcctcccatc

SEQ ID NO: caccttcagggtttcagcat

SEQ ID NO: cagggtttcagcatctggtt

SEQ ID NO: 6 ctggttgaaaagcatgagca

SEQ ID NO: gagcatcccctccaatacct

SEQ ID NO: ccgcaacacgatgtaagttg

SEQ ID NO: ggtacatcaaagccccgata

SEQ ID NO: 10: cgtttccatctttgcagtca

SEQ ID NO: 11: cagggtcatgctctgtttca

SEQ ID NO: 12 gagggcatcgtcatagaagg

SEQ ID NO: 13: tgaggaggtccgagttcttg                     

SEQ ID NO: 14 gggcatcgtcatagaaggtc

SEQ ID NO: 15 cactgtctgaggctcctcct

SEQ ID NO: 16: gtgttctggctgtgcagttc

SEQ ID NO: 17 tgggttgaagttgctgaggt

SEQ ID NO: 18 cctgctcatctgtcacgttc

Experimental Example 1: Collagenase Expression Inhibitory Effect

 Each of the oligonucleotides of Examples 1 to 18 was added to the culture of human-derived fibroblasts at a concentration of 2 uM to test the inhibitory effect on increased collagenase expression induced by ultraviolet light. The measurement of collagenase was quantified using the BioTrak MMP-1 Assay Kit (Amersham Bioscience), and the inhibition rate (%) was calculated by substituting the following Formula 1. The results are shown in Table 1 below. The control group is a group irradiated with only ultraviolet light without treating oligonucleotides.

(Calculation 1)

Inhibition rate = (ultraviolet control-experimental group) / ultraviolet control X 100

Table 1

Example A ₄₅₀ + Std1 % Inhibition Negative control 0.551 + 0.077 - UV control 0.964 + 0.068 - Example 1 0.559 + 0.058 42 Example 2 0.569 + 0.056 41 Example 3 0.578 + 0.065 40 Example 4 0.560 + 0.064 42 Example 5 0.568 + 0.059 41 Example 6 0.579 + 0.067 40 Example 7 0.570 + 0.058 41 Example 8 0.561 + 0.071 42 Example 9 0.577 + 0.063 40 Example 10 0.694 + 0.069 28 Example 11 0.713 + 0.078 26 Example 12 0.723 + 0.057 25 Example 13 0.712 + 0.077 26 Example 14 0.704 + 0.066 27 Example 15 0.724 + 0.054 25 Example 16 0.714 + 0.061 26 Example 17 0.722 + 0.076 25 Example 18 0.706 + 0.062 27 * n = 3

As shown in Table 1, the oligonucleotides of Examples 1 to 9 had an inhibition of collagenase expression of at least 40% or more at 2 uM treatment concentration.

Experimental Example 2: Verification of the effect of mixing between oligonucleotides

The oligonucleotides of Examples 1 to 9 and the oligonucleotides of Examples 10 to 18 were mixed at the same concentration ratio, respectively, and treated with cells at a final concentration of 2 uM, and then the collagenase expression inhibition rate was measured in the same manner as in Example 1. It was. The results are shown in Table 2 below.

Table 2

Example A ₄₅₀ + Std1 % Inhibition Control 0.531 ± 0.077 - UV control 0.986 ± 0.068 - Example 1 + Example 10 0.217 ± 0.028 78 Example 3 + Example 12 0.237 ± 0.046 76 Example 7 + Example 15 0.227 ± 0.035 77 n = 3

In Table 2, when using the mixture of Examples 1 to 9 and Examples 10 to 18, it can be seen that the collagenase expression inhibition rate is significantly increased.

Example 19 Preparation of Skin Ointment

To the skin ointment was prepared in the composition of Table 3 (unit: wt%).

Table 3

Composition weight % Example 1 Example 10 Diethyl Sebacate Brazing Polyoxyethylene Oleyl Ether Phosphate Sodium Benzoate Vaseline 0.05 0.05 8 5 6 Suitable to 100

Example 20 Preparation of Cosmetics (Cream)

To prepare a cream in the composition of Table 4 (unit: wt%).

Table 4

Composition weight % Example 2 Example 13 Stearic Acid Cetanol Fiji-20 Sorbent Monostearate Sorbent Monostearate Mineral Oil Trioctanoate Triethanolamine Carbomer Glycerin Propylene Glycol Preservative Flavored Water 0.1 0.1 1.0 2.0 1.0 1.0 10.0 5.0 0.5 0.2 5.0 3.0 Proper quantity to 100

Example 21 Preparation of Cosmetic (Flexible Cosmetic)

To the flexible composition was prepared in the composition of Table 5 (unit: wt%).

Table 5

Composition weight% Example 5 Example 15 Ethanol Polyoxyethylene Cured Castor Oil Paraoxybenzoic Acid Methyl Glycerin 1,3-Butylene Glycol Flavored Dye 0.02 0.02 10.0 1.0 2 5.0 6.0 Proper quantity to 100

Example 22 Preparation of Cosmetic (Essence)

To the essence was prepared in the composition (unit: wt%) of Table 6.

Table 6

Composition weight% Example 1 Example 11 Propylene glycol glycerin sodium hyaluronate aqueous solution (1%) ethanol polyoxyethylene hardened castor oil paraoxybenzoic acid methyl carbomer triethanolamine flavored purified water 0.2 0.2 10.0 10.0 5.0 5.0 1.0 0.1 0.3 0.4 Suitable to 100

Example 23 Preparation of Cosmetics (Packs)

To prepare a pack in the composition of Table 7 (unit: wt%).

Table 7

Composition weight% Example 6 Example 16 Glycerine Propylene Glycol Polyvinyl Alcohol Ethanol Polyoxyethylene Cured Castor Oil Polyoxyethylene Oleethyl Ethyl Paraoxybenzoic Acid Methyl Flavored Dye 0.01 0.01 5.0 4.0 15.0 8.0 1.0 1.0 0.2 Proper quantity to 100

Example 24 Preparation of Cosmetics (Nutrition Makeover)

To nutrient cosmetics was prepared in the composition of Table 8 (unit: wt%).

Table 8

Composition weight% Example 7 Example 10 Polyoxyethylene Cured Castor Oil Paraoxybenzoic Acid Methyl Glycerin 1,3-Butylene Glycol Carbomer Triethanolamine Propylene Glycol Ethanol Carboxyvinyl Polymer Dye Flavored Purified Water 0.03 0.03 1.0 suitable 6.0 5.0 0.2 0.3 5.0 3.2 0.1 suitable quantity to 100

Comparative Examples 1 to 6

To the composition (unit: wt%) of the following Tables 9 to 14 skin ointments, creams, soft cosmetics, essences, packs, nutrient cosmetics, respectively.

Table 9

Comparative Example 1, Skin Ointment weight% Example 1 Example 10 Diethyl Sebacate Brazing Polyoxyethylene Oleyl Ether Phosphate Sodium Benzoate Vaseline --8 5 6 Suitable to 100

Table 10

Comparative Example 2, Cream weight% Example 2 Example 13 Stearic Acid Cetanol Fiji-20 Sorbent Monostearate Sorbent Monostearate Mineral Oil Trioctanoate Triethanolamine Carbomer Glycerin Propylene Glycol Preservative Flavored Water --1.0 2.0 1.0 1.0 10.0 5.0 0.5 0.2 5.0 3.0 Proper quantity to 100

Table 11

Comparative Example 3, Flexible Cosmetics weight% Example 5 Example 15 Ethanol Polyoxyethylene Cured Castor Oil Paraoxybenzoic Acid Methyl Glycerin 1,3-Butylene Glycol Flavored Dye --10.0 1.0 2 5.0 6.0 Proper amount to 100

Table 12

Comparative Example 4, Essence weight% Example 1 Example 11 Propylene glycol glycerin sodium hyaluronate aqueous solution (1%) ethanol polyoxyethylene hardened castor oil paraoxybenzoic acid methyl carbomer triethanolamine flavored purified water --10.0 10.0 15.0 5.0 1.0 0.1 0.3 0.4 Correction to 100

Table 13

Comparative Example 5, Pack weight% Example 6 Example 16 Glycerine Propylene Glycol Polyvinyl Alcohol Ethanol Polyoxyethylene Cured Castor Oil Polyoxyethylene Oleethyl Ethyl Paraoxybenzoic Acid Methyl Flavored Dye --5.0 4.0 15.0 8.0 1.0 1.0 0.2 Proper quantity to 100

Table 14

Comparative Example 6, nutrient lotion weight% Example 7 Example 10 Polyoxyethylene Cured Castor Oil Paraoxybenzoic Acid Methyl Glycerin 1,3-Butylene Glycol Carbomer Triethanolamine Propylene Glycol Ethanol Carboxyvinyl Polymer Dye Flavored Purified Water --1.0 appropriate 6.0 5.0 0.2 0.3 5.0 3.2 0.1 proper quantity to 100

Experimental Example 3: Verification of the wrinkle improvement effect

Wrinkle improvement effect for Example 19, Example 22, Comparative Example 1 and Comparative Example 4 was carried out as follows.

40 women from 35 to 50 years old were divided into two groups of 20 people, group 1 was Example 19 and Comparative Example 1, group 2 was Example 22 and Comparative Example 4, and exactly 1/2 of the left and right sides of the face. The left and right sides of the face were randomly assigned. Both the investigator and the subject did not know which product was an example or a comparative example. The test subject was to apply the test cosmetics after washing twice a day for 8 weeks.

After 8 weeks, the improvement of wrinkles was evaluated through visual evaluation and image analysis of wrinkles.

3-1. Visual evaluation

Visual evaluation was evaluated in three stages of no improvement, slight improvement, and significant improvement in comparison with before use regarding wrinkle improvement and elasticity improvement. The results are shown in Table 15 below.

Table 15

sample No improvement Minor improvements A significant improvement Comparative Example 1 13 5 2 Example 19 4 8 8 Comparative Example 4 11 6 3 Example 22 3 8 9

As shown in Table 15, it was judged that the samples of Examples 19 and 20 of the present invention had superior wrinkle improvement effects compared to Comparative Examples 1 and 4 during visual evaluation.

3-2. Wrinkle Image Analysis

Image analysis of wrinkles was performed by taking replicas under the eyes (Xantopren, Bayer) before the experiment began, and immediately after the experiment was finished, replicas were taken from the same area under the eyes. Dimensional analysis determined the density of wrinkles. As a result of measuring wrinkle density by image analysis, the reduction ratio for wrinkle density before use was obtained as an average, and the results are shown in Table 16 below.

Table 16

sample Wrinkle Density Reduction (%) Comparative Example 1 8% Example 19 43% Comparative Example 4 7% Example 22 45%

As shown in Table 16, as a result of image analysis, Example 19 and Example 22 was found to have a wrinkle reduction effect of 43% and 45%, respectively.

In addition, when the experiment on the human body did not show any side effects, it was found that the oligonucleotide of the present invention is very safe.

As described above, antisense oligonucleotides against the mRNA of the collagenase gene and the mRNA of the c-Jun gene of the present invention inhibit collagen degradation, thereby providing an excellent anti-wrinkle effect.

Attach sequence list electronic file  

Claims (8)

An antisense oligonucleotide for mRNA of the collagenase gene and an antisense oligonucleotide selected from the group consisting of antisense oligonucleotides for mRNA of the c-Jun gene. The anti-wrinkle agent of claim 1, wherein the antisense oligonucleotide consists of 15 to 25 bp. The anti-wrinkle agent according to claim 1, wherein the antisense oligonucleotide for the mRNA of the collagenase gene is at least one oligonucleotide selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 1-9. The antiwrinkle agent according to claim 1 or 3, wherein the antisense oligonucleotide for the mRNA of the c-Jun gene is at least one oligonucleotide selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 10 to 18. The anti-wrinkle agent according to claim 1, wherein the anti-wrinkle agent comprises antisense oligonucleotide for mRNA of the collagenase gene and antisense oligonucleotide for mRNA of the c-Jun gene in a ratio of 1: 0.1 to 10 by weight . The anti-wrinkle agent according to claim 1, wherein the anti-wrinkle agent is a cosmetic. The anti-wrinkle agent according to claim 1, wherein the anti-wrinkle agent comprises 0.00001 to 10.0 wt% of the active ingredient in dry weight. The anti-wrinkle agent according to claim 1, wherein the oligonucleotide is phosphorothioate oligonucleotide.