HYALURONIC ACID FILLER HAVING HIGH VISCOELASTICITY AND HIGH COHESIVENESS

Abstract

The hyaluronic acid filler according to the present invention exhibits improved high viscoelastic flow characteristics, and has advantages of monophasic hyaluronic acid fillers and biphasic hyaluronic acid fillers together, and therefore it maintains the shape while having low moving possibility when injecting into skin, and thus it is excellent when used for improving wrinkles, augmentation of soft tissues such as cheeks, breast, nose, lips and hips, etc., and correcting contours, and the duration time in the human body is increased and side effects are small, even if a small amount of crosslinking agent is used during the preparation process.

Claims

1. A hyaluronic acid hydrogel filler, comprising hyaluronic acid, or a salt thereof or a crosslinked hyaluronic acid, wherein the filler has a complex viscosity of at least 6×10.sup.4 cP at an angular velocity of 1 Hz as measured by a rheometer, a storage elastic modulus G′ of at least 400 Pa, and a cohesivity of at least 30 gf.

2. The hyaluronic acid hydrogel filler according to claim 1, wherein the filler has both properties of monophasic and biphasic hyaluronic acid hydrogel fillers.

3. The hyaluronic acid hydrogel filler according to claim 1, wherein the hyaluronic acid is a crosslinked hyaluronic acid.

4. The hyaluronic acid hydrogel filler according to claim 32, wherein the crosslinked hyaluronic acid is one in which a hyaluronic acid having a molecular weight of at least 2,500,000 Da is crosslinked.

5. The hyaluronic acid hydrogel filler according to claim 34, wherein the hyaluronic acid is crosslinked by a crosslinking agent selected from the group consisting of 1,4-butanediol diglycidyl ether: BDDE), ethylene glycol diglycidyl ether (EGDGE), hexanediol diglycidyl ether (1,6-hexanediol diglycidyl ether), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, tri-methylpropane polyglycidyl ether, bisepoxypropoxy ethylene (1,2-(bis(2,3-epoxypropoxy)ethylene), pentaerythritol polyglycidyl ether and sorbitol polyglycidyl ether.

6. The hyaluronic acid hydrogel filler according to claim 5, wherein the crosslinking agent is 1,4-butanediol diglycidyl ether.

7. The hyaluronic acid hydrogel filler according to claim 1, further comprising an anesthetic.

8. The hyaluronic acid hydrogel filler according to claim 7, wherein the anesthetic is lidocaine or a salt thereof.

9. (canceled)

10. (canceled)

11. (canceled)

12. A hyaluronic acid hydrogel filler, comprising a hyaluronic acid, a salt thereof, wherein the filler has a complex viscosity of at least 6×10.sup.4 cP at an angular velocity of 1 Hz as measured by a rheometer, a storage elastic modulus G′ of at least 400 Pa, and a cohesivity of at least 30 gf, and wherein the filler comprises a crosslinking agent at a concentration of 1 to 10 mol % based on 1 mol of N-acetyl-D-glucosamine and D-glucuronic acid in the hyaluronic acid or the its salt thereof.

13. The hyaluronic acid hydrogel filler according to claim 12, wherein the concentration of the crosslinking agent is 1 to 5 mol % based on 1 mol of N-acetyl-D-glucosamine and D-glucuronic acid in the hyaluronic acid or the salt thereof.

14. A preparation method of the hyaluronic acid filler according to claim 1, comprising the following steps: (a) preparing a crosslinked hyaluronic acid hydrogel by adding a hyaluronic acid or its salt and a crosslinking agent to an alkaline aqueous solution and stirring; (b) cutting the hyaluronic acid hydrogels prepared in the step (a); (c) providing a buffer solution; (d) washing and swelling the hyaluronic acid hydrogel cutted in the step (b) using the buffer solution of in the step (c); (e) grinding the washed and swollen hyaluronic acid hydrogels in the step (d); and (f) filling the hydrogels prepared in the step (e) into a syringe and then sterilizing.

15. The preparation method according to claim 14, wherein the alkaline aqueous solution is an aqueous solution of NaOH, KOH, NaHCO.sub.3, LiOH or a combination thereof.

16. The preparation method according to claim 15, wherein a concentration of the alkaline aqueous solution is 0.1N to 0.5N.

17. The preparation method according to claim 14, wherein the crosslinking agent is 1,4-butanediol diglycidyl ether.

18. The preparation method according to claim 14, wherein a concentration of the hyaluronic acid or its salt is 10 to 25% by weight as a weight ratio of the hyaluronic acid or its salt based on the total weight of a mixture of the hyaluronic acid or its salt and the alkaline aqueous solution.

19. The preparation method according to claim 14, wherein the step (a) is performed at a temperature range of 25 to 40° C. for 15 to 22 hours.

20. The preparation method according to claim 14, wherein the buffer solution of the step (c) comprises one or more selected from the group consisting of citric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, acetic acid, diethyl barbituric acid, sodium acetate, (tris(hydroxymethyl)methylamino)propanesulfonic acid)(TAPS), (2 bis(2-hydroxyethyl)amino)acetic acid)(Bicine), (tris(hydroxymethyl)ammonium methane)(Tris), (N-(2 hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine)(Tricine), (4 (2 hydroxyethyl)-1-piperazine ethanesulphonic acid)(HEPES), (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]methanesulfonic acid)(TES) and (piperazine-N,N′-bis(2-ethanesulfonic acid)(PIPES).

21. The preparation method according to claim 14, wherein the buffer solution further comprises an anesthetic and an isotonic agent.

22. A prefilled syringe comprising the hyaluronic acid hydrogel filler according to claim 1.

23. (canceled)

24. A method for augmenting soft-tissue comprising injecting to a subject the hyaluronic acid hydrogel filler according to claim 1.

25. A method for improving wrinkles comprising injecting to a subject the hyaluronic acid hydrogel filler according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG. 1 is a graph showing the result of measuring the storage modulus and resistance in compression of the hyaluronic acid fillers according to Examples 1 and 2, and Comparative examples 1 to 6.

[0056] Hereinafter, the present invention will be described in more detail by examples. However, these examples are intended to illustrate the present invention exemplarily, and the scope of the present invention is not limited by these examples.

EXAMPLES

[0057] For preparation of the hyaluronic acid filler according to the present invention, the following process was conducted.

[0058] Sodium hyaluronic acid, sodium hydroxide, and BDDE (1,4-Butanediol Diglycidyl Ether), having a molecular weight of 2.5 MDa to 3.5 MDa were weighed. The concentration of sodium hyaluronic acid during the reaction was 15 wt %, and the mol % of BDDE was 4 mol % based on the unit of the added sodium hyaluronic acid (namely, N-acetyl-D-glucosamine and D-glucuronic acid) of 1 mol. Separately, a sodium hydroxide aqueous solution at a concentration of 0.25N was prepared and filtered. The weighed sodium hyaluronic acid, 0.25N sodium hydroxide aqueous solution and BDDE (1,4-Butanediol Diglycidyl Ether) were added to a mixer container and were mixed homogenously, and this mixer container was put in a constant-temperature waterbath and the crosslinking reaction was completed at a temperature of 30° C. overnight. Then, the crosslinked hyaluronic acid hydrogels in which the reaction was completed were preliminarily ground. On the other hand, a buffer solution was prepared by dissolving salts and an anesthetic in a buffer tank filled with injection water at concentrations of sodium monohydrogen phosphate hydrates (12 hydrates) 1.26 g/L, sodium dihydrogen phosphate hydrates (monohydrates) 0.46 g/L, sodium chloride 7 g/L and lidocaine hydrochloride 3 g/L.

[0059] A part of the buffer solution was considered as the primary buffer solution and it was transferred to a washing tank through a 0.22 μm filter, and the preliminarily ground hyaluronic acid gel prepared earlier was transferred to the washing tank filled with the primary buffer solution and then was stirred to primarily wash and swell the hyaluronic acid gel, and then when swelling was completed, the washing solution was removed. Then, the secondary buffer solution was transferred into a washing tank through a 0.22 μm filter, and then it was stirred to secondarily wash and swell the hyaluronic acid gel. When the washing and swelling were completed, the washing solution was removed. Then, the tertiary buffer solution was transferred into a washing tank through a 0.22 μm filter, and then it was stirred to tertiarily wash and swell the hyaluronic acid gel. The washing solution was removed as soon as the washing and swelling was completed.

[0060] After completing the tertiary washing and swelling, whether the pH of the washing solution was in the neutral range was confirmed, and after cutting the hyaluronic acid gel in which washing and swelling was completed, it was transferred to an extruder tank and was weighed, and so as to reach a desired weight of the gel weight, the buffer solution was added to correct the primary content. When the primary content correction was completed, the hyaluronic acid gel was extruded and ground in the extruder tank. Then, the ground hyaluronic acid gel was transferred to a sterile tank and was homogenized, and then the content was measured and the buffer solution was added to conduct the secondary content correction. The hyaluronic acid gel in which the content correction was completed was heat-treated at a temperature of 121° C. or more, for 1 minute or more, and the hyaluronic acid gel before filling this was decompressed while stirring to conduct desaturation. Then, the hyaluronic acid gel in a fixed amount of filling was vacuumed/filled to each syringe and at the same time, it was stoppered with a rubber stopper. The filled syringes were steam sterilized in a final sterilizer at a temperature of 121° C. or more for 8 minutes or more.

Example 2: Preparation of the Hyaluronic Acid Filler According to the Present Invention

[0061] For preparation of the hyaluronic acid filler according to the present invention, the following process was conducted.

[0062] Sodium hyaluronic acid, sodium hydroxide, and BDDE (1,4-Butanediol Diglycidyl Ether), having a molecular weight of 2.5 MDa to 3.5 MDa were weighed. The concentration of sodium hyaluronic acid during the reaction was 16 wt %, and the mol % of BDDE was 4 mol % based on the unit of the added sodium hyaluronic acid (namely, N-acetyl-D-glucosamine and D-glucuronic acid) of 1 mol. Separately, a sodium hydroxide aqueous solution at a concentration of 0.25N was prepared and filtered. The weighed sodium hyaluronic acid, 0.25N sodium hydroxide aqueous solution and BDDE (1,4-Butanediol Diglycidyl Ether) were added to a mixer container and were mixed homogenously, and this mixer container was put in a constant-temperature waterbath and the crosslinking reaction was completed at a temperature of 30° C. overnight. Then, the crosslinked hyaluronic acid hydrogels in which the reaction was completed were preliminarily ground. On the other hand, a buffer solution was prepared by dissolving salts and an anesthetic in a buffer tank filled with injection water at concentrations of sodium monohydrogen phosphate hydrates (12 hydrates) 1.26 g/L, sodium dihydrogen phosphate hydrates (monohydrates) 0.46 g/L, sodium chloride 7 g/L and lidocaine hydrochloride 3 g/L.

[0063] A part of the buffer solution was considered as the primary buffer solution and it was transferred to a washing tank through a 0.22 μm filter, and the preliminarily cutted hyaluronic acid gel prepared earlier was transferred to the washing tank filled with the primary buffer solution and then was stirred to primarily wash and swell the hyaluronic acid gel, and then when swelling was completed, the washing solution was removed. Then, the secondary buffer solution was transferred into a washing tank through a 0.22 μm filter, and then it was stirred to secondarily wash and swell the hyaluronic acid gel. When the washing and swelling were completed, the washing solution was removed. Then, the tertiary buffer solution was transferred into a washing tank through a 0.22 μm filter, and then it was stirred to tertiarily wash and swell the hyaluronic acid gel. The washing solution was removed as soon as the washing and swelling was completed.

[0064] After completing the tertiary washing and swelling, whether the pH of the washing solution was in the neutral range was confirmed, and after grinding the hyaluronic acid gel in which washing and swelling was completed, it was transferred to an extruder tank and was weighed, and so as to reach a desired weight of the gel weight, the buffer solution was added to correct the primary content. When the primary content correction was completed, the hyaluronic acid gel was extruded and ground in the extruder tank. Then, the ground hyaluronic acid gel was transferred to a sterile tank and was homogenized, and then the content was measured and the buffer solution was added to conduct the secondary content correction. The hyaluronic acid gel in which the content correction was completed was heat-treated at a temperature of 121° C. or more, for 1 minute or more, and the hyaluronic acid gel before filling this was decompressed while stirring to conduct desaturation. Then, the hyaluronic acid gel in a fixed amount of filling was vacuumed/filled to each syringe and at the same time, it was stoppered with a rubber stopper. The filled syringes were steam sterilized in a final sterilizer at a temperature of 121° C. or more for 10 minutes or more.

Experimental Example 1: Investigation of Viscoelasticity Properties of the Hyaluronic Acid Filler Prepared by the Present Invention

[0065] For investigation of rheological properties of prepared Examples 1 and 2, analysis was conducted using a rheometer. For comparison with the filler of the present invention, viscoelasticity properties of commercially available filler preparations were also analyzed and compared. The commercially available filler preparations as comparative examples and analysis conditions were as follows.

COMPARATIVE EXAMPLES

[0066] Comparative example 1: Belotero Intense Lidocaine [0067] Comparative example 2: Belotero Volume Lidocaine [0068] Comparative example 3: Stylage L Lidocaine [0069] Comparative example 4: Stylage XL Lidocaine [0070] Comparative example 5: Juvederm Volift with Lidocaine [0071] Comparative example 6: Juvederm Voluma with Lidocaine.

[0072] <Analysis Conditions>

[0073] Analysis conditions of Oscillatory and Rotational Rheometer In case of storage elastic modulus (G′) and complex viscosity (rr) test [0074] (1) Test equipment: Rheometer (Anton Paar Ltd., MCR301) [0075] (2) Frequency: 1 Hz [0076] (3) Temperature: 25 r [0077] (4) Strain: 4% [0078] (5) Measuring geometry: 25 mm plate [0079] (9) Measuring gap: 1.0 mm

[0080] In case of resistance when compressed (Compression force) [0081] (1) Test equipment: Rheometer (Anton Paar Ltd., MCR301) [0082] (2) Gap: Initial position: 2.5 mm, Final position: 0.9 mm [0083] (3) Speed: 0.8 mm/min [0084] (4) Temperature: 25° C. [0085] (5) Measuring geometry: 25 mm plate [0086] (9) Normal Force Measuring gap position: 1.5 mm

[0087] Under the analysis conditions, the result values of the storage elastic modulus (G′), complex viscosity (η*) and resistance when compressed (Compression force) by frequency were shown in Table 1.

TABLE-US-00001 TABLE 1 Belotero Juvederm Comparative Comparative Stylage Comparative Comparative example 1 example 2 Comparative Comparative example 5 example 6 Example/ Belotero Belotero example 3 example 4 Juvederm Juvederm Comparative Present invention Intense Volume Stylage L Stylage XL Volift with Voluma with example Example 1 Example 2 Lidocaine Lidocaine Lidocaine Lidocaine Lidocaine Lidocaine Concentration 20 20 25.5 26 24 26 17.5 20 (mg/mL) Storage elastic 448 707 149 280 225 264 314 310 modulus (Pa, 1 Hz) Complex viscosity 7.18 11.3 2.54 4.57 3.65 4.28 5.08 4.97 (×10.sup.4 cP, 1 Hz) Compression 36 44 71 70 55 65 20 24 force (gf) Concentration- 448 707 117 215 188 203 359 310 corrected storage elastic modulus (Pa, 1 Hz) (storage elastic modulus* (20/sample concentration)) Concentration- 7.18 11.30 1.99 3.52 3.04 3.29 5.81 4.97 corrected complex viscosity (×10.sup.4 cP, 1 Hz) (complex viscosity* (20/sample concentration)) Concentration- 36 44 56 54 46 50 23 24 corrected Compression force (gf) (Compression force * (20/sample concentration))

[0088] As confirmed in the Table 1, it is determined that Examples 1 and 2 according to the present invention exhibit excellent viscoelasticity compared to Comparative examples 1 to 6. Moreover, regarding cohesivity despite of showing properties of monophasic fillers and biphasic fillers simultaneously, they show excellent compression force than the monophasic fillers, Comparative examples 5 and 6. Furthermore, considering both viscoelasticity and cohesivity, it can be seen that Examples 1 and 2 according to the present invention show excellent physical properties compared to Comparative examples 1 to 6.

Experimental Example 2: Analysis of the Particle Size of the Hyaluronic Acid Hydrogels According to the Present Invention

[0089] In order to confirm the particle size of the hyaluronic acid hydrogels of Examples 1 and 2 and Comparative examples 1 to 6 and distribution, the following test was conducted. The result of this test was shown in Table 2.

[0090] <Analysis Conditions>

[0091] Analysis Conditions of Laser Diffraction Particle Size Analyzer [0092] (1) Test equipment: Laser diffraction particle size analyzer (Malvern Ltd., Mastersizer 3000) [0093] (2) Dispersant: 0.9% NaCl solution [0094] (3) Stirrer rpm: 1,000 [0095] (4) Laser obscuration: 5-25%

TABLE-US-00002 TABLE 2 Belotero Juvederm Comparative Comparative Stylage Comparative Comparative example 1 example 2 Comparative Comparative example 5 example 6 Example/ Belotero Belotero example 3 example 4 Juvederm Juvederm Comparative Present invention Intense Volume Stylage L Stylage XL Volift with Voluma with example Example 1 Example 2 Lidocaine Lidocaine Lidocaine Lidocaine Lidocaine Lidocaine Particle 362 343 571 469 375 358 407 408 diameter, Dv(50) (μm)

Experimental Example 3: Analysis of Degree of Modification of the Hyaluronic Acid Hydrogels According to the Present Invention

[0096] In order to confirm the degree of modification of the hyaluronic acid hydrogels of Examples 1 and 2 and Comparative examples 1 to 6, a test was performed under the following conditions. The result of this test was shown in Table 3.

[0097] <Analysis Conditions>

[0098] Analysis Conditions of Nuclear Magnetic Resonance [0099] (1) Test equipment: FT-NMR System (Jeol Ltd., ECA500/ECZ400S), [0100] (2) Pulse: 30° [0101] (3) Scans: 512 [0102] (4) Relaxation time (delay): 5 s [0103] (5) Temperature: 25° C.

TABLE-US-00003 TABLE 3 Belotero Juvederm Comparative Comparative Stylage Comparative Comparative example 1 example 2 Comparative Comparative example 5 example 6 Example/ Belotero Belotero example 3 example 4 Juvederm Juvederm Comparative Present invention Intense Volume Stylage L Stylage XL Volift with Voluma with example Example 1 Example 2 Lidocaine Lidocaine Lidocaine Lidocaine Lidocaine Lidocaine Degree of 3.3 3.5 8.5 12.8 7.8 7.8 6.3 6 Modification (%)

[0104] As can be seen in the Table 3, it can be seen that the hyaluronic acid fillers of Examples 1 and 2 according to the present invention exhibit a low degree of modification despite of showing excellent physical properties as confirmed earlier, and this means that it is very biocompatible as a filler showing excellent physical properties can be provided even when using a small amount of crosslinking agent during preparation of a filler.