FILLER COMPRISING HYALURONIC ACID HYDROGEL HAVING EXCELLENT FILLING PROPERTIES

Abstract

[Summary]

The present invention has a special filling (WIE) property parameter value developed using microrheology technology, and thus exhibits improved high viscoelasticity flow properties, has low mobility when injected into skin whilst still maintaining the shape thereof, and thus has excellent soft tissue restoration properties, for example for the cheeks, breasts, nose, lips or bottom, and excellent volume expansion and wrinkle alleviation properties.

Claims

1. A filler comprising a hyaluronic acid hydrogel, wherein a winkle improvement efficiency (WIE) parameter in accordance with by Equation 1 as a value for filling properties is at least 0.6:
[Equation 1]
Wrinkle Improvement Efficiency (WIE) Parameter=([Average value of MSD slope]*[Absolute value of MSD at travel time of 0.1 s]*[Standard deviation of MSD slope value]*100).sup.−1 wherein in the Equation 1, MSD refers to a mean square displacement for a variable of tracer particles injected into the filler comprising a hyaluronic acid hydrogel.

2. The filler of claim 1, wherein the WIE parameter is 0.6 or more to 20 or less.

3. The filler of claim 1, wherein a value of an elasticity value times a cohesivity value is at least 600 Pa*N or more.

4. The filler of claim 1, wherein the filing properties comprises wrinkle improvement resulting from filling of biological tissues and wrinkles, remodeling of the face, or restoration or increase in the volume of soft tissues.

5. The filler of claim 1, wherein the hyaluronic acid hydrogel comprises a crosslinked hyaluronic acid.

6. The filler of claim 5, wherein the crosslinked hyaluronic acid is obtained by crosslinking a hyaluronic acid having an average molecular weight of at least 2,000,000 Da or a salt thereof.

7. The filler of claim 5, wherein the crosslinked hyaluronic acid is prepared using a hyaluronic acid or a salt thereof and a crosslinking agent, wherein the crosslinking agent includes at least one crosslinking agent selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), 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, 1,2-(bis(2,3-epoxypropoxy)ethylene, pentaerythritol polyglycidyl ether, and sorbitol polyglycidyl ether.

8. The filler of claim 1, further comprising an anesthetic agent.

9. The filler of claim 8, wherein the anesthetic agent is lidocaine or a salt thereof.

10. The filler of claim 8, wherein the filler is for skin injection.

11. A prefilled syringe filled with the filler comprising a hyaluronic acid hydrogel of claim 1.

12. A biomaterial for tissue restoration comprising the filler comprising a hyaluronic acid hydrogel of claim 1.

13. A method for tissue restoration, comprising injecting to a subject the filler comprising a hyaluronic acid hydrogel of claim 1.

14. A method for wrinkle improvement, comprising injecting to a subject the filler comprising a hyaluronic acid hydrogel of claim 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0056] FIG. 1 is a diagram briefly showing the properties of the fillers having phase properties of the present invention, i.e., monophasic filler and biphasic filler.

[0057] FIG. 2 is a diagram illustrating the result of analyzing the motion of tracer particles using image processing software (Matlab).

[0058] FIG. 3 is a graph showing the MSD measurement results of the monophasic filler (Comparative Example 4) and biphasic filler (Comparative Example 1).

[0059] FIG. 4 is a graph confirming the parameters such as high cohesivity/high elasticity/regularity based on Equation 1 according to the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0060] Hereinafter, the present invention will be described in more detail by way of Examples. However, these Examples are given for illustrative purposes only, and the scope of the invention is not limited to or by these Examples.

[0061] Example 1: Preparation of Filler Containing Hyaluronic Acid Hydrogel

[0062] In order to prepare a filler containing the hyaluronic acid hydrogel according to the present invention, the following process was carried out.

[0063] More specifically, sodium hyaluronate having an average molecular weight of 2.5 to 3.5 MDa, sodium hydroxide, and 1,4-butanediol diglycidyl ether (BDDE) as a crosslinking agent were weighed. During the reaction, the concentration of sodium hyaluronate was 16 wt %, and the mol % of BDDE was 4% relative to the unit of sodium hyaluronate added. Separately, a sodium hydroxide (NaOH) aqueous solution at a concentration of 0.25 N was prepared and filtered. The weighed sodium hyaluronate, 0.25N sodium hydroxide aqueous solution, and 1,4-butanediol diglycidyl ether (BDDE) were put in a mixer tank and mixed homogeneously, and the mixer tank was placed in a constant temperature water bath and reacted overnight at a temperature of 30° C. to complete a crosslinking reaction. Thereafter, the crosslinked hyaluronic acid hydrogel after the reaction was roughly cut.

[0064] Meanwhile, salts and anesthetic agents were dissolved at a concentration of 1.26 g/L of sodium hydrogen phosphate hydrate (dodecahydrate), 0.46 g/L of sodium dihydrogen phosphate monohydrate (monohydrate), 7 g/L of sodium chloride and 3 g/L of lidocaine hydrochloride in a buffer tank containing water for injection to prepare a buffer solution.

[0065] Some of the buffer solution was used as a primary buffer solution and transferred to a washing tank through a 0.22 μm filter. The cut hyaluronic acid hydrogel previously prepared was transferred to a washing tank containing a primary buffer solution and then stirred. The hyaluronic acid hydrogel was subjected to a primary washing and swollen, and then, when the swelling was completed, the washing solution was removed. Subsequently, the secondary buffer solution was transferred to a washing tank through a 0.22 μm filter and then stirred, and the hydrogel was subjected to a secondary washing and swollen, and then, when the washing and swelling were completed, the washing solution was removed. Thereafter, the tertiary buffer solution was transferred to a washing tank through a suitable 0.22 μm filter and then stirred, and the hyaluronic acid hydrogel was subjected to a third washing and swollen. Then, when the washing and swelling were completed, the washing solution was removed.

[0066] After completion of the third washing and swelling, it was confirmed whether the pH of the washing solution was in the neutral range, and after cutting the hyaluronic acid hydrogel gel in which washing and swelling was completed, it was transferred to an extruder tank to measure a weight, the buffer solution was added so that the weight of gel reaches a target weight, and a primary content correction was performed. When the primary content correction was completed, the hyaluronic acid hydrogel was extruded and ground in an extruder tank. Thereafter, the ground hyaluronic acid hydrogel was transferred to a sterilized tank and homogenized, after which the content was measured, and the buffer solution was added thereto to perform a secondary content correction. The hyaluronic acid hydrogel after the secondary content correction was heat-treated at a temperature of 121° C. or more for at least 1 minute, and degassing was performed by stirring the hyaluronic acid hydrogel under reduced pressure before loading into a syringe.

[0067] Thereafter, the hyaluronic acid hydrogel was vacuum-filled to each syringe by a predetermined amount and stoppered with a rubber stopper at the same time. The filled syringe was steam sterilized for at least 8 minutes at a temperature of 121° C. or higher in the final sterilizer.

[0068] Experimental Example 1: MSD measurement of Tracer Particles in Filler Containing Hyaluronic Acid Hydrogel using Microrheological Technique

[0069] For analyzing the properties of the prepared Example 1, the fillers containing commercial hyaluronic acid hydrogel of Comparative Examples 1 to 5 together with the hyaluronic acid hydrogel of Example 1 were used in the experiment, as shown in Table 1.

[0070] More specifically, the Brownian motion of the tracer particles dispersed in the sample was photographed using a video-microscope, and then the motion of the tracer particles was analyzed using an image processing software (Matlab) (FIG. 2).

[0071] Specifically, the test sample was prepared by dispersing 1 vol % of tracer particles (polystyrene particles having a diameter of 1 μm) in 1 mL of the filler of Example 1. Microrheological experiments were carried out by loading the filler sample in which the tracer particles were dispersed between transparent slide glasses, and recording the trajectory of the tracer particles moving into the filler through a camera connected to an optical microscope. The recording was performed 10 times for a minute each and averaged. The shooting speed was 38 frames per second. The recorded images were analyzed using an image analysis tool (Matlab), and how much the moving distance of the tracer particles changed with time, that is, the mean square displacement according to the travel time of the tracer particles, was plotted. In the same manner, the tracer particles were dispersed in the fillers containing the hyaluronic acid hydrogels of Comparative Examples 1 to 5 to prepare a test sample, and the behavior of the tracer particles was analyzed.

[0072] The characteristics of the mean square displacement (MSD) distribution of the tracer particles according to the phase properties of the hyaluronic acid hydrogel fillers were confirmed. Specifically, in the case of the biphasic filler of Comparative Example 1, the movements of the tracer particles located on the surface region and the tracer particles located in the crosslinked region were different, and the tracer particles located on the surface region had a relatively free movement, resulting in a large MSD slope value. In contrast, the particles which penetrated inside the particles and located in the crosslinked region had a limited movement, which exhibited a small MSD slope value. Meanwhile, the MSD distribution of the tracer particles for the monophasic filler of Comparative Example 4 was not divided into two regions, but showed similar slopes, thereby confirming that the particles formed a continuous shape without boundaries according to the region of the filler (FIG. 3). According to these results, it was confirmed that phase properties of the fillers could be inferred when the MSD value was measured by introducing the tracer particles into the fillers containing the hyaluronic acid hydrogel.

[0073] Furthermore, based on the relationship between the mean square displacement (MSD) slope value distribution of the tracer particles and the hydrogel phase, an attempt was conducted to derive parameters capable of reflecting the elasticity, cohesivity, and regularity of the filler structure, which are the most important physical properties of the filler for selecting excellent fillers.

[0074] In the case of the fillers having low cohesivity instead of high elasticity such as biphasic fillers, the structure of the fillers was not regular, and thus the movements of the tracer particles located on the surface region and the tracer particles located in the crosslinked region became different. Specifically, the tracer particles located on the surface region had a relatively free movement and thus exhibited a large value of MSD slope, and the tracer particles which penetrated into the filler and located in the crosslinked region had a limited movement and thus showed a small value of MSD slope. Accordingly, it was confirmed that these fillers had a large standard deviation of the MSD slope, and also shown that the average value of the MSD slope was large. In addition, it was confirmed that the absolute value of MSD at 0.1 seconds decreased as the elasticity increased. In contrast, in the case of fillers having low elasticity instead of high cohesivity such as monophasic fillers, the structure of the fillers was regular, and the standard deviation of the MSD slope value was small, and also, the tracer particles had a limited movement due to cohesivity, and thus showed a a small MSD slope value.

[0075] Based on the results of the tests, as a parameter capable of reflecting the elasticity, cohesivity, and regularity of the filler structure of the filler, the “wrinkle improvement efficiency (WIE) parameter” represented by Equation 1 was devised.

[Equation 1]

WIE Parameter=([Average of MSD slope]*[Absolute value of MSD at travel time of 0.1 s]*[Standard deviation of MSD slope value]*100).sup.−1

[0076] In Equation 1, MSD represents the mean squared displacement of tracer particles. The cohesivity of the filler represents the average value of MSD slope, the elasticity represents the absolute value of MSD at travel time of 0.1 seconds for particles, and the regularity of the filler structure represents the standard deviation of the MSD slope value, and these properties were all considered.

[0077] Accordingly, it was confirmed that the physical properties of the hyaluronic acid fillers could be measured by the WIE parameter using MSD, and the WIE parameter values of the fillers of Example 1 and Comparative Examples 1 to 5 were confirmed according to Equation 1 reflecting all of the cohesivity, elasticity and the regularity of the filler structure of the filler, and the results are shown in Table 1 and FIG. 4 below.

TABLE-US-00001 TABLE 1 Absolute Standard Average of value of deviation of MSD slope MSD at MSD slope WIE Symbol Sample value 0.1 s value value A Example 1 0.120 0.109 0.180 4.25 B Comparative Example 1 0.440 0.696 0.407 0.08 (Restylane Lyft with Lidocaine) C Comparative Example 2 0.279 0.358 0.260 0.38 (Juvederm Voluma with Lidocaine) D Comparative Example 3 0.255 0.416 0.214 0.44 (Teosyal Puresense Ultimate with Lidocaine) E Comparative Example 4 0.220 0.349 0.255 0.51 (Teosyal Puresense Ultra Deep with Lidocaine) F Comparative Example 5 0.420 0.507 0.236 0.2 (Teosyal Puresense Deep Line with Lidocaine

[0078] As shown in Table 1 and FIG. 4, the WIE parameter value of the filler according to Example 1 was 4.25, and the WIE parameter values of the fillers of Comparative Examples 1 to 5 were 0.51 or less. The fillers of Comparative Examples 1 to 5 had significantly lower WIE values compared to the filler of the Example according to the present invention, which did not satisfy the cohesivity, elasticity and regularity of the filler structure of the filler according to the present invention. In contrast, it was confirmed that the filler having a WIE parameter value of 0.6 or more could be selected as a filler having excellent filling properties (FIG. 4).

[0079] Specifically, in Comparative Example 1, the regularity of the filler structure was confirmed to be poor because a standard deviation of the MSD slope value was large as a typical biphasic filler, and it was also confirmed that the cohesivity was low because the average value of the MSD slope was large. In Comparative Example 2, the elasticity of the filler was confirmed to be low because the absolute value of the MSD (value at 0.1 s) was large as a monophasic filler.

[0080] In the case of Example 1, it was confirmed that the absolute value of the MSD (value at 0.1 s) was low, the average value of the MSD slope was low, and the standard deviation of the MSD slope value was low, and the cohesivity, elasticity, and regularity of the filler were superior. Therefore, the filler of Example 1 shows an excellent form-keeping ability at the injection site, a low possibility of movement to other sites during surgical operation, and a regular structure of the filler, and thus is expected to show excellent filling properties.

[0081] Experimental Example 2: Confirmation of Wrinkle-Improving Property of Filler Containing Hyaluronic Acid Hydrogel

[0082] In order to confirm whether the WIE parameter of Experimental Example 1 is substantially associated with the wrinkle-improving property, the wrinkle-improving property of the hyaluronic acid fillers according to Example 1 and Comparative Examples 1 to 5 shown in Table 1 was measured according to the following method.

[0083] In order to measure the lift capability, which is a parameter showing the wrinkle-improving property, the elasticity and cohesivity of the fillers were measured by a rheometer (ARES-G2, TA Instruments), and the two values were multiplied to calculate the lift capability. The results of confirming such elasticity, cohesivity, and lift capability are shown in Table 2 and FIG. 4 below.

[0084] Specifically, the elasticity was measured by determining how much stress was needed when the filler was loaded between the top and bottom of the circular geometry and a constant shear was applied thereto (shear strain=0.1%), the unit was expressed in Pa, and the rate at which shear was applied was 10 rad/s. The cohesivity was measured as the force applied when the filler was loaded between the top and bottom of the geometry and pulled vertically. As the cohesivity between filler particles was larger, the greater force was needed to pull. The stretching speed was 1 mm/s, and the unit was expressed in N.

TABLE-US-00002 TABLE 2 Storage Elastic Cohesivity Modulus (Pa) Lift WIE Example Type (N) @10 rad/s capability Parameter Example 1(A) Both monophasic 1.414 620.72 878 4.25 and biphasic Comparative Biphasic 0.590 598.264 353 0.08 Example 1(B) Comparative Monophasic 0.815 296.604 242 0.38 Example 2(C) Comparative Monophasic 1.346 391.664 527 0.44 Example 3(D) Comparative Monophasic 1.634 354.452 579 0.51 Example 4(E) Comparative Monophasic 1.186 235.074 279 0.20 Example 5(F)

[0085] From the results of the above Experimental Examples 1 and 2, it can be found that the filler containing the hyaluronic acid hydrogel according to the present invention exhibited a WIE parameter value of 4.25, which is greater than 0.6, and a high lift capability parameter, which is expressed as a product of elasticity and cohesivity, and represents the wrinkle-improving property, of 600 Pa*N or more, more preferably 800 Pa*N or more, and thus confirming that the filler of the present invention satisfied the elasticity and cohesivity at the same time. However, Comparative Examples 1 to 5, which are commercially available hyaluronic acid fillers, had a WIE parameter value of less than 0.6, did not show excellent elasticity and/or cohesivity, and had a lift capability of less than 600 Pa*N. Thus, it was confirmed that the wrinkle-improving property was not desirable as a filler compared to the filler of the present invention.

[0086] As a result, the filler (Example 1) having a WIE parameter value of 0.6 or more, as in the present invention, were excellent in cohesivity and elasticity, and exhibited a lift capability of 600 Pa*N or more, thereby being excellent in wrinkle-improving property.