WO2017213285A1

WO2017213285A1 - Filler composition for tissue augmentation comprising nucleic acid, chitosan, and hyaluronate, and preparation method therefor

Info

Publication number: WO2017213285A1
Application number: PCT/KR2016/007237
Authority: WO
Inventors: 김익수; 김한규; 홍철암; 이수연
Original assignee: 주식회사 파마리서치프로덕트
Priority date: 2016-06-07
Filing date: 2016-07-05
Publication date: 2017-12-14
Also published as: CN109195642A; CN109195642B; KR101710639B1

Abstract

The present invention relates to a filler composition for tissue augmentation comprising nucleic acid, chitosan, and hyaluronate, and a preparation method therefor. The injection of the composition of the present invention into a site in need of tissue augmentation increases the volume of the injection site and generates new tissues, leading to an excellent tissue augmentation effect.

Description

Specification

Title of the Invention: Filling composition for tissue strengthening comprising nucleic acid, chitosan and hyaluronic acid and method for preparing same

Field of technology

[1] The present invention relates to a layered composition for strengthening tissues containing nucleic acid, chitosan and hyaluronic acid and a method for producing the same.

Background

[2] anti-aging medically managing aging and maintaining youth

Various treatments for the future The anti-aging demand is rapidly increasing due to the aging population, the trend of appearance trends, the increase in income, and the social advancement of women, and the government policies and the bio and medical technology innovations that emphasize quality of life are As you add

The quality of anti-aging products and services is also developing (Kim Soo Bum, _2014;

Kim Cheol-young, 2015) .The anti-aging industry is emerging as a future growth industry, and the market for facial aesthetics, which includes botox, fillers, laser treatments, and dermabrasion, is drawing attention.

[3] Filler is a substance that can supplement skin tissue by means of 'filling, filling material'. It is used to restore the firmness of face, improve contour, and reduce facial wrinkles. It refers to a product that is intended to be filled and filled and to maintain its own volume without pharmacological action. It is a non-surgical treatment used without skin incisions or surgery.

These cosmetic fillers include injectable facial implants, dermal fillers, wrinkle fillers, and facial soft tissue fillers. or by various other names.

[4] Fillers are characterized by product characteristics (main ingredient, viscosity, etc.), procedure differences (injection amount, injection technology) and

Depending on the individual characteristics of the patient, there may be a difference in the duration of the effect. The characteristics of the filler can be divided into biphasic fillers and monophasic fillers. In the case of the everyday fillers, there is a lot of crosslinking agent, so it is sticky, and the mold is easy to make the surface smooth, but the durability is small.The ideal filler is elastic, and the deformation is small, but the surface may be bumpy (Hung Ki-woong, 2013) .

[5] Molding fillers can be divided into degradable and non-degradable, depending on their main components.

Degradable collagen, hyaluronic acid,

It contains natural polymers such as calcium hydroxyapatite as its main ingredient, and it has the advantage of being relatively easy to remove by using enzymes that decompose the ingredient. It has the advantage that the lasting effect is maintained for a long time by remaining, but it is difficult to remove after the procedure.

Poly (methylmethacrylate (PMMA) and crosslinked polyamide derivatives are used as main components. Some molding fillers contain traces of anesthetics to reduce the pain that occurs during the procedure.

(Medical Device Information Technology Support Center, 2015; Chao-Ha, et al., 2015).

Side effects of filler injection include edema, pain, tenderness, numbness, bruising, hematoma, redness, hemorrhoids, pigmentation, allergic reactions, itching, pruritus, asymmetry, irregularity, noticeable mass or nodule, infection, Granuloma, vascular compromise and Tyndall effect.

Therefore, the present inventors have studied the tissue-enhanced layer composition, and the gel formed by injecting a thermosensitive hydrogel composition containing nucleic acid, chitosan and hyaluronic acid into the dermal layer of skin, and the effect of continuous tissue enhancement The present invention could be accomplished by confirming that.

As a prior art, European Patent Publication No. 0696210 discloses hyaluronic acid and

A layer agent using a polydeoxyribonucleotide-based hydrogel has been disclosed, which is similar to the present invention, but does not describe the chitosan of the present invention. In addition, Korean Patent No. 1476381 discloses a DNA polymer isolated from fish sperm or eggs. Wrinkle-reducing face or skin fillers are included, but no chitosan or hydrogels are mentioned.

Detailed description of the invention

Technical task

It is an object of the present invention to provide a layered composition for strengthening tissues comprising nucleic acid, chitosan and hyaluronic acid and a method for producing the same.

Task solution

The present invention relates to a tissue composition filling composition comprising nucleic acids, chitosan and hyaluronic acid.

The composition may be a composition in which nucleic acids, chitosan and hyaluronic acid are mixed in a ratio of 10 to 1000: 1: 10 to 1000 by weight.

The composition may be a composition in which the nucleic acid, chitosan and hyaluronic acid are mixed in an increase ratio of 25 to 100: 1: 25 to 100.

The composition may be a composition in which the nucleic acid and the hyaluronic acid are mixed in a 1: 5 to 5: 1 weight ratio.

The content of the nucleic acid may be from 0 to 1% by weight to 3% by weight based on the total weight of the composition.

The nucleic acid may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a combination thereof. [16] The nucleic acid may have a molecular weight of lkDa to 100,000kDa.

[17] The nucleic acid may have a molecular weight of lOkDa to 10,000kDa.

The nucleic acid may have a molecular weight of 50 kDa to 3,500 kDa.

[19] The chitosan content of silver is 0.000% by weight, based on the total weight of the composition.

It may be 0.3% by weight.

The chitosan may have a molecular weight of 3 kDa to l, 000 kDa.

[21] The hyaluronic acid content is from 0 to 1% by weight based on the total weight of the composition

It can be 3% by weight.

The hyaluronic acid may have a molecular weight of 2 () kDa to 3000kDa.

In addition, the present invention comprises the steps of i) preparing a stock solution (nucleic acid); ii) preparing a chitosan stock solution; iii) mixing and stirring the nucleic acid storage solution of step i) and the chitosan storage solution of step ii); iv) adding the hyaluronic acid raw material to the nucleic acid and chitosan mixture of step iii) and mixing the mixture to stir the increase ratio of the nucleic acid, chitosan and hyaluronic acid to 10 to 1000: 1: 10 to 1000; and V) It is about the manufacturing method of the tissue composition filling composition which consists of a step of stirring the nucleic acid, chitosan, and hyaluronic acid mixed solution of step iv) and lowering to phase.

[24] The method of preparation comprises the following steps: i) adding nucleic acid to a buffer, at 70 ° C. to 80 ^° C.

Dissolving for 1 to 3 hours while stirring to prepare a nucleic acid storage solution; ii) dissolving chitosan in an acid buffer solution to produce a chitosan storage solution; iii) mixing the nucleic acid stock solution of step i) and the chitosan stock solution of step ii) for 20 minutes at 60 ^o C to 80 ^o C. Stirring for 1 hour; iv) adding the hyaluronic acid raw material to the nucleic acid and chitosan mixture of step iii) and mixing so that the weight ratio of the nucleic acid, chitosan and hyaluronic acid mixture is 10 to 1000: 1: 10 to 1000, and 55 ° C to 65 ° C Stirring for 1 hour to 2 hours; and V) lowering the temperature to room temperature while stirring the mixture of the nucleic acid, chitosan and hyaluronic acid of step iv) for 2 hours to 4 hours;

[25] The present invention will be described in detail below.

[26] The composition for enhancing tissue containing nucleic acid, chitosan and hyaluronic acid preferably has a weight ratio of nucleic acid, chitosan and hyaluronic acid in the range of 10 to 1000: 1: 10 to 1000. Nucleic acid, chitosan and hyaluronic acid It is more preferable that the weight ratio is ₂₅ to ₁₀₀ : i: 25 to 100.

[27] The composition contains a mixture of nucleic acid and hyaluronic acid in a ratio of 1: 5 to 5: 1 by weight.

It may be a composition.

The content of the nucleic acid may be 0 to 1% by weight to 3% by weight based on the total weight of the composition.

The nucleic acid may have a molecular weight of lkDa to 100,000 kDa. Preferably, it is lOkDa to 10,000kDa and most preferably 50kDa to 3,500kDa.

[30] The nucleic acid is deoxyribonucleic acid (DNA), ribonucleic acid (ribonucleic acid) acid, RNA) or a combination thereof, preferably deoxyribonucleic acid.

In addition, the deoxyribonucleic acid is an oligonucleotide (oligonuclotide),

Polynucleotides and

It may be a polydeoxyribonucleotide.

[32] The chitosan content is 0.000 wt% based on the total increase in the composition

It may be 0.3% by weight.

[33] The chitosan preferably has a molecular weight of 3 kDa to l, 000 kDa,

It is not limited.

[34] The hyaluronic acid content is 0.01% by weight based on the total weight of the composition

It may be 3% by weight.

The hyaluronic acid may have a molecular weight of 20 kDa to 3000 kDa.

[36] The method for preparing the tissue-enhanced layer composition includes i) dissolving the nucleic acid in a buffer and dissolving it for 1 hour to 3 hours while stirring at 70 ° C to 80 ° C.

Preparing a storage solution; ii) preparing a chitosan stock solution by dissolving chitosan in an acid buffer solution; iii) mixing the nucleic acid storage solution of step 0 and the chitosan storage solution of step ii) and stirring at 60 ° C. to 80 ° C. for 20 minutes to 1 hour; iv) Add the hyaluronic acid raw material to the nucleic acid and chitosan mixture of step iii) and mix so that the weight ratio of nucleic acid, chitosan and hyaluronic acid mixture is 10 to 1000: 1: 10 to 1000, and 55 ° 0 to 65 ° Stirring at C for 1 hour to 2 hours; and V) lowering the temperature to room temperature while stirring the nucleic acid, chitosan, and hyaluronic acid mixture solution of step) for 2 to 4 hours. have.

[37] The complete solution that can be used in the preparation of the nucleic acid stock solution is sodium phosphate dibasic dodecarate. 7}. Sodium phosphate dibasic dodecahydrate, sodium chloride, magnesium chloride,

Potassium chloride, phosphate buffered sline or HEPES (N- (2-hydroxyethyl) -piperazine-N'-2-ethanesulfonic acid) buffer solution, preferably sulphate dibasic basic decahydrate (sodium phosphate dibasic dodecahydrate) is available, but is not limited to this.

[38] An acid buffer solution that can be used in the preparation of the chitosan storage solution is used.

Acetic acid, hydrochloric acid, ascorbic acid, lactic acid, and nitric acid may be used. Preferably, acetic acid may be used, but the limitation is limited thereto. no.

[39] The filling composition for enhancing tissue containing nucleic acid, chitosan, and hyaluronic acid may be a temperature sensitive hydrogel. A temperature sensitive hydrogel is a sol gel or gel gel depending on the temperature. Phase transition

Refers to a hydrogel. [40] The filling composition for tissue augmentation not only shows the effect of tissue augmentation by injection, but also the formation of new blood vessels and collagen by proliferation of fibroblasts around the injection site. Filling the terrestrial and fascia may exhibit a sustained tissue-enhancing effect over existing tissue-enhancing filling compositions.

Effects of the Invention

[41] The present invention relates to a tissue-enhancing layer composition containing nucleic acid, chitosan and hyaluronic acid, and a method for manufacturing the same, wherein the layer-forming composition for enhancing tissue of the present invention is injected into the skin or subcutaneous region of the skin for tissue enhancement. The filling composition gelled to increase the volume at the injection site. Also, the histological changes of the injection site showed that new tissues were formed at the injection site.

[42] Through this, it is expected that the tissue enhancement layer composition including nucleic acid, chitosan and hyaluronic acid of the present invention can exhibit excellent tissue enhancement effect by treating the tissue enhancement layer in areas requiring tissue enhancement.

Brief description of the drawings

1 shows the elasticity (A), viscosity (B) and the composition of the filling composition for skin tissue and tissue augmentation

The result of checking the viscoelasticity (C) is shown.

FIG. 2 shows the structure of skin tissue and the administration site of the filling composition for tissue augmentation.

FIG. 3 shows the results of histological changes (A) and tissue stratification effects (B) following the injection of tissue-filling filling compositions.

4 shows the composition remaining in the flesh layer after the addition of the tissue-enhanced layer composition

Results are ascertained as to whether (A) and composition (B) remain in the epidermal and dermal layers.

Best Mode for Carrying Out the Invention

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the information presented here is to be thorough and complete, and to provide those skilled in the art to fully convey the ideas of the present invention.

Example 1 Preparation of Tissue Augmented Layer Composition Comprising Nucleic Acid, Chitosan and Hyaluronic Acid

[49] A stock solution was prepared for preparing a tissue-enhanced layered composition containing the nucleic acid, chitosan, and hyaluronic acid of the present invention.

[50] 130 mM sodium phosphate dibasic for nucleic acid stock solutions

Dodecahydrate (sodium phosphate dibasic dodecahydrate) was added to the buffer solution and prepared by dissolving for at least 2 hours using a heat stirrer at 75 ° C.

Chitosan stock solutions were prepared using 90 mM acetic acid. [52] The prepared nucleic acid and chitosan stock solutions were mixed and stirred for 30 minutes in a 70 ° C heat stirrer. The mixture of hyaluronic acid was further mixed with the mixed nucleic acid and chitosan solution for 1 hour in a thermal stirrer at 60 ° C for 3 hours. While stirring at room temperature, a tissue-enhancing filler composition containing nucleic acid, chitosan, and hyaluronic acid was prepared. The concentrations of the nucleic acid, chitosan, and hyaluronic acid were mixed so that the concentrations shown in Table 1 below.

[Table 1]

Comparative Example 1. Fabrication of tissue-strengthening layered composite for comparison

The composition for filling tissue for enhancing tissues was prepared so as to have a concentration corresponding to the comparative example of Table 2. The manufacturing method was the same as the method of Example 1 above.

Proceeded.

[Table 2]

Experimental Example 1. Measurement of Viscoelasticity of Tissue-Enhanced Layer Compositions

[58] The duration, volume and volume of the tissue-enhanced layered composition of the present invention

A rheometer was used to check the viscoelastic properties, which are related characteristics.The measurement conditions used were PU20, Gap lmm, 1Hz, 1%.

Ungryeok-strain (stress strain) while up to 1 minutes by 1 ° C from 25 ° C to 40 ^o C

G '(Pa) (elastic coefficient), G "(Pa) (viscosity coefficient) and G' (Pa) (viscoelastic coefficient) were measured.

[59] The viscoelasticity measurement was performed by visual inspection of the inventors in Example 1 and all the injection layers.

For comparison between Example 1-5 and the mixed composition, which performed the injection test with the best experience, Comparative Example 1-1, Comparative Example 1-2 and Comparative Example 1-3 were selected, and the measured results are shown in FIG. The results of the measurements at 36 ° C are shown in Table 3 below.

Indicated.

[60] [Table 3]

As shown in FIG. 1 and Table 3, Example 1-5 in which nucleic acid, chitosan and hyaluronic acid are mixed is Comparative Example 1-1 containing only nucleic acid, Comparative Example 1-2 in which nucleic acid and hyaluronic acid are mixed, and nucleic acid and chitosan. Compared with the composition of Comparative Examples 1-3, the elasticity (FIG. 1A) and the viscosity (FIG. 1B) and the viscoelasticity (FIG. 1C) were excellent, and the composition substructure of Example 1-5 was shown. It was found to be the best combination for use as a reinforcing layer composition. In addition, when chitosan was added to the composition consisting of nucleic acid and hyaluronic acid,

It was found that there was a change in the physical properties of the composition.

Experimental Example 2 Preparation of Experimental Animals

[63] Animal testing was conducted on 10-week-old Sprague-Dawley male rats (Rat).

Korea) and two rats were used for each experimental group.

Stainless steel wire cages (WxDxH, 260 匪 x305 隱 x210 麵) were quarantined, 2 dogs during the acclimation period and 1 dog during the observation period. The feeds were used for the laboratory animal solid feed (Harlan laboratories, Inc., US A). After feeding the solid feed into the feed and free intake. The drinking water was filtered with water filter oil sterilizer and irradiated with ultraviolet rays, and then freely ingested by the automatic water supply. The environment of the experimental animal breeding room was 23 ± 3 ° C and relative humidity. Luminance was maintained at 55 ± 15%, lighting time of 12 hours (8 am to 8 pm), and illuminance of 150 to 300 Lux.

[64] <Experimental Example 3 Injection of Tissue Filling Composition>

[0065] The rats reared in Experimental Example 2 were anesthetized using isoflurane. Cut off the hair of the anesthetized rat, disinfect the injection site with betadine cotton, and use 0.5cc disposable sterile syringe and 30 gage needle to inject 0.5cc of tissue layer-forming composition into the skin as shown in Fig. 2. intradermal, ID) or

Subcutaneous (SC) injections were performed in the feeding box for one week after the injection, and the effects, biocompatibility, and tissue enhancement were observed.

[66] <Experimental Example 4 Histopathological Observation>

[67] Tissue-enhanced layer composition of the present invention was injected subcutaneously, and after 7 days of sacrifice, rats were sacrificed with C0 ₂ gas, and the dorsal subcutaneous tissue was injected. The collected tissue was 10% neutral complete formalin (neutral). After 24 hours of fixation with buffered formalin, the tissues were dehydrated through normal tissue treatment and then infiltrated with paraffin. The tissues were infiltrated into 5 thick tissues. The prepared tissue sections were prepared in Mayer's hematoxyline (Sigma, USA) solution. After the initial reaction, the solution was washed for 10 minutes in running water. The reaction was then allowed to react with eosin (Sigma, USA) solution for 3 seconds. The stained tissue was dehydrated and encapsulated in Permount ^® (Fischer scientific, USA). Microscopy of H & E-stained tissue sections for histopathological changes

Observation and the results are shown in FIG.

[68] As shown in Fig. 3, (A) avoids the tissue composition filling composition of the present invention.

After 7 days after injection, the composition enhancement effect was shown by the injection of each composition.Comparative Example 1-1 showed the effect of tissue enhancement or stratification on the lower part of the flesh layer compared to the untreated group. On the other hand, when the compositions of Comparative Examples 1-2 and Comparative Examples 1-3 were injected, the tissue layer appeared thicker than that of the non-treated group, but between the two groups was large. No difference was seen. The treatment of the compositions of Examples 1-5 resulted in thicker portions of the upper layer and the formation of new tissue in place of the composition as compared to the treatments of the untreated and comparative examples. appear. (B) shows the effect of tissue stratification after injecting the tissue enrichment filling composition of Example 1-5. In other words, the layer was formed by filling the cells with the tissue enrichment filling composition and forming the layer. It is shown that the densification filling composition of the present invention does not merely gel and exhibit tissue enhancement effects, but shows tissue enhancement effects through tissue formation.

[69] Through this, the tissue-enhancing effect of the tissue enhancing filling composition of the present invention not only increases the volume of the tissue due to gelation of the composition, but also creates new tissues such as blood vessels and collagen at the composition injection site, thereby increasing the density of the tissue. It can be seen that the effect of tissue enhancement can be enhanced.

[70] <Experimental Example 5 Observation of Volume Change by Injection of Tissue Filling Composition>

[71] The composition of the present invention-operated augmentation filling composition was injected into the subcutaneous tissue, and the injection site was cut off on the 3rd or 7th day after the injection, and the remaining composition and remaining on the epidermis, dermis or terrestrial layer. Was confirmed, and the results are shown in FIG. 、

[72] As shown in Fig. 4, in (A), the injection layer of the tissue-enhanced layer composition is

Cut to separate the epidermis and dermis or the flesh layer,

After fixation with formalin, the cut was cut in half to observe the cross-section of the tissue. As a result of the increase in the volume of the denture, the cross section of the flesh layer was confirmed, and it was confirmed that the gel-like composition remained in the tissue-enhanced layer composition. In (B), it was observed whether the gel-formed composition remaining in the epidermal and dermal layers remained after 7 days of injecting the layer-forming composition for tissue enhancement. As a result, Comparative Example 1-1, Comparative Example 1-2, and Comparative Example 1_ _The composition remaining in the epidermal and dermal layers was not observed in the case of the injection of the tissue-enhancing filling composition of ₃ , whereas the transparent gel-like substance was injected into the epidermal and dermal layers when the tissue-enhancing filling composition of Example 1-5 was administered. It has been confirmed that it remains, and it can be seen that the effect of tissue enhancement by the tissue composition filling composition of the present invention can be maintained for a long time.

Claims

Claim

Claim 1 A tissue-filling filling composition comprising a nucleic acid, chitosan and hyaluronic acid.

2. The method of claim 1,

The composition is a layer strengthening composition for tissue strengthening, characterized in that the nucleic acid, chitosan and hyaluronic acid is 10 to 1000: 1 to 10 to 1000 increase ratio.

[Claim 3] The method according to claim 2,

The composition is a layer strengthening composition for tissue strengthening, characterized in that the nucleic acid, chitosan and hyaluronic acid is 25 to 100: 1: 25 to 100 weight ratio.

[Claim 4] The method according to claim 2,

The composition is a tissue-strengthening layer composition, characterized in that the ratio of 1: 5 to 5: 1 by weight of nucleic acid and hyaluronic acid.

5. The method of any of paragraphs 1 to 3,

The content of silver nucleic acid is from 0 to 1% by weight based on the total weight of the composition

A layer thickening composition for tissue augmentation characterized by being 3% by weight.

6. The method of any one of claims 1 to ₃ , wherein

The nucleic acid is a tissue enhancing filling composition characterized in that the deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a combination thereof.

[Claim 7] In any of paragraphs 1 to 3,

The nucleic acid has a molecular weight IkDa to 100,000kDa layer composition for strengthening tissues.

8. The method of claim 17, wherein

The nucleic acid has a molecular weight of 10kDa to 10,000kDa, layered composition for strengthening the tissue 조직

: Claim 9] The method according to claim 8,

The nucleic acid has a molecular weight of 50kDa to 3,500kDa characterized in that the filling composition for tissue reinforcement.

10. The method of any one of claims 1 to 3, wherein

The chitosan is a content enhancement layer precursor composition characterized in that the content is 0.00001% to 0.3% by weight based on the total weight of the composition. 11. The method of any one of claims 1 to 3,

The chitosan has a molecular weight of 3kDa to l, 000kDa characterized in that the layer precursor composition for tissue reinforcement.

12. The method of any one of claims 1 to 3, wherein

The hyaluronic acid is a content enhancement layer precursor composition characterized in that the content of 0. 2% to 3% by weight based on the total weight of the composition.

13. The method of any one of claims 1 to 3, wherein The hyaluronic acid has a molecular weight of 20kDa to 3000kDa layer thickening composition for tissue strengthening.

[Claim 14] A method for producing a tissue-enhanced layer composition as described in any one of claims 1 to 3.

i) preparing a nucleic acid stock solution;

ii) preparing a chitosan stock solution;

iii) mixing and stirring the nucleic acid storage solution of step i) and the chitosan storage solution of step ii);

iv) adding the hyaluronic acid raw material to the nucleic acid and chitosan mixture of step iii) and mixing the mixture, stirring and mixing so that the weight ratio of the nucleic acid, chitosan and hyaluronic acid is 10 to 1000: 1: 1 to 10 to 1000; and

V) lowering to room temperature while stirring the nucleic acid, chitosan and hyaluronic acid mixture of step iv);

Method for producing a layered composition for strengthening tissue, characterized in that it comprises a. 15. The method of claim 14,

The manufacturing method is

i) preparing a nucleic acid stock solution by dissolving the nucleic acid in a buffer and dissolving for 1 to 3 hours while stirring at 70 ° C. to 80 ^° C .;

ii) preparing chitosan stock solution by dissolving chitosan in an acidic buffer solution;

iii) mixing the nucleic acid storage solution of step i) and the chitosan storage solution of step ii) and stirring at 60 ° C to 80 ° C for 20 minutes to 1 hour; iv) Add the hyaluronic acid raw material to the nucleic acid and chitosan mixture of step iii) and mix so that the weight ratio of nucleic acid, chitosan and hyaluronic acid mixture is 10 to 1000: 1: 10 to 1000, and 55 ° C to 65 ⁰ Stirring for 1 hour to 2 hours at C; and

V) the mixture of the nucleic acid, chitosan and hyaluronic acid mixture of step iv) within 2 hours

Lowering the temperature to room temperature while stirring for 4 hours;

A method for producing a layered composition for strengthening tissues, comprising: a.