FORM 2
THE PATENTS ACT 1970
(39 of 1970)
AND
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rulel3)
1. TITLE OF THE INVENTION:
"A COMPOSITION FOR PREPARATION OF VISCOELASTIC
CROSSLINKED HYALURONIC ACID, AND CROSSLINKED HYALURONIC
ACID OBTAINED BY USING THE SAME"
2. APPLICANT:
(a) NAME: LG LIFE SCIENCES LTD.
(b) NATIONALITY: Republic of Korea
(c) ADDRESS: 58, Saemunan-ro, Jongno-gu, Seoul 110-783,
Republic of Korea.
3.PREAMBLE TO THE DESCRIPTION:
The following specification describes the nature of this invention and the manner
in which it is to be performed:

RELATED APPLICATION
The Present Application is taking priority of Korean Patent Application No. KR10-2013-0032956 filed on 27th March, 2013.
[DESCRIPTION]
[Technical Field]
The present invention relates to a composition for preparation of viscoelastic crosslinked hyaluronic acid, and crosslinked hyaluronic acid with both monophasic and biphasic characteristics obtained by using the same, and more specifically to a composition for preparation of viscoelastic crosslinked hyaluronic acid, which comprises hyaluronic acid alkaline aqueous solution of 15 to 25 weight % concentration and a crosslinking agent and has Tan 8 value of 0.5 to 1.5 at a frequency of 0.02 to 1 Hz, and crosslinked hyaluronic acid with both monophasic and biphasic characteristics obtained by using the same.
[Background Art]
Hyaluronic acid is biopolymer substance, and widely used as for medical, healthcare and cosmetic purposes due to its biocompatibility and viscoelasticity. However, hyaluronic acid alone is easily decomposed in vivo or under the conditions of acid or alkali, and the use thereof is limited. Therefore, efforts to develop a structurally stable hyaluronic acid derivative are widely being made (Laurent, T.C. "The chemistry, biology and medical applications of hyaluronan and its derivatives" Portland Press Ltd., London, 1998).
As the conventional preparation method of crosslinked hyaluronic acid, US Patent Publication No. 2003/0148995 discloses the method for forming gel by reacting hyaluronic acid at the concentration of 12.8% in the presence of alkali using relatively excess polyfunctional epoxide-based crosslinking agents.
In addition, Korean Patent Publication No. 10-2009-0085102 discloses the inset with the effect of inhibition against MMP expression and tissue restoration through in intradermal or subcutaneous injection by adding mannitol as an antioxidant in the mixture of crosslinked hyaluronic acid and free hyaluronic acid. In this case, while the usability can be improved by mixing additional free hyaluronic acid in crosslinked hyaluronic acid to

inhibit the expression of MMP, it is difficult to expect the role of free hyaluronic acid in restoration and maintenance of tissue because free hyaluronic acid has the disadvantage of easy decomposition by the enzyme reaction.
Putting the conventional techniques together, in the disclosed preparation methods for crosslinked hyaluronic acid using many types of crosslinking agents or through the various preparation processes, there was no mention of crosslinked product which exhibits both viscous and elastic characteristics with resistance to the enzyme. In addition, many of the existing preparation methods are not suitable for large-scale production.
Under such a background, the inventors of the present invention prepared a composition for preparation of viscoelastic crosslinked hyaluronic acid by adjusting a composition for preparation of viscoelastic crosslinked hyaluronic acid which is provided in the form of a mixture of hyaluronic acid alkaline aqueous solution of 15 to 25 weight % concentration and a crosslinking agent to have Tan 8 value of 0.5 to 1.5 at the frequency range of 0.02 to 1 Hz, before the crosslinking reaction takes place and identified that crosslinked hyaluronic acid with both viscous and elastic characteristics can obtained from the composition obtained by cross-linking a composition for preparation of crosslinked hyaluronic acid to complete the present invention.
[DISCLOSURE]
[Technical Problem]
One objective of the present invention is to provide a composition for preparation of
viscoelastic crosslinked hyaluronic acid.
Another objective of the present invention is to provide the crosslinked hyaluronic acid
with both monophasic and biphasic characteristics obtained by using the above
composition.
Another objective of the present invention is to provide the preparation method of the
cross linked hyaluronic acid with both monophasic and biphasic characteristics obtained
by using the above composition.
[Technical Solution]
In order to resolve the problems, the present invention provides a composition for

preparation of crosslinked hyaluronic acid, comprising hyaluronic acid alkaline aqueous solution of 15 to 25 weight % concentration and a crosslinking agent, and having Tan 8 value of 0.5 to 1.5 at the frequency range of 0.02 to 1 Hz.
The present invention is characterized in that a composition for preparation of crosslinked hyaluronic acid which is provided in the form of a mixture of hyaluronic acid alkaline aqueous solution of 15 to 25 weight % concentration and a crosslinking agent, has Tan 5 value of 0.5 to 1.5 at the frequency range of 0.02 to 1 Hz before crosslinking reaction.
Preferably, the composition has Tan 5 value of 0.5 to 1.5 at the frequency of 0.02 Hz.
The present invention can prepare viscoelastic crosslinked hyaluronic acid, which exhibits both viscous and elastic characteristics, by providing a composition with Tan 8 value of 0.5 to 1.5 at the frequency range of 0.02 to 1 Hz as a composition for preparation of crosslinked hyaluronic acid.
In the examples of the present invention, after preparing a composition with Tan 8 value of 0.7, 0.97, or J.39 at the frequency of 0.02 Hz, the rheological properties thereof were observed over the time, and the results identified that the composition has excellent viscoelasticity (Examples 1 to 3, Figure 1).
In addition, when using the composition for preparation of crosslinked hyaluronic acid of the present invention, viscoelastic crosslinked hyaluronic acid which has stability against enzyme, excellent tissue-restorability, and long duration can be prepared.
As used herein, the term "hyaluronic acid" refers to all of hyaluronic acid itself, salts thereof, or a combination of these. The hyaluronic acid salt includes inorganic salts such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate and organic salts such as tetrabutylammonium hyaluronate. In the present invention, hyaluronic acid can be used as hyaluronic acid itself alone, or as salt thereof alone; or hyaluronic acid or a salt thereof can be used in combination of two or more of hyaluronic acid itself or salt thereof.

In the present invention, the molecular weight of hyaluronic acid can be 100,000 to 5,000,000.
In the present invention, the alkaline aqueous solution can be NaOH solution, but is not limited thereto. Hereupon, NaOH solution can be used at the concentration of 0.25 N to 5
N.
In the present invention, the cross linking agent can be 1,4-butanediol diglycidyl ether, but is not limited thereto.
In addition, the present invention provides crosslinked hyaluronic acid obtained by crosslinking the composition for preparation of crosslinked hyaluronic acid which comprises hyaluronic acid alkaline aqueous solution of 15 to 25 weight % concentration and a crosslinking agent, and has Tan 5 value of 0.5 to 1.5 at the frequency range of 0.02 to 1 Hz.
In the present invention, as above mentioned, crosslinked hyaluronic acid with both monophasic and biphasic characteristics can be obtained after obtaining crosslinked hyaluronic acid by cross-linking the composition with specific range of Tan 8 values at the specific frequency range.
In other words, in the present invention, the final crosslinked hyaluronic acid comprises both monophasic and biphasic characteristics. Also, the final crosslinked hyaluronic acid comprises stability against enzyme, excellent tissue-restorability, and long duration.
In the example of the present invention, the result of investigation on resistance against enzyme of crosslinked hyaluronic acid obtained by cross-linking the mixture with Tan 5 value of 0.97 identified excellent resistance against hyaluronidase.
In addition, the present invention provides the preparation method for crosslinked hyaluronic acid comprising
1) obtaining crosslinked hyaluronic acid by cross-linking a composition for preparation of crosslinked hyaluronic acid, which comprises hyaluronic acid alkaline aqueous solution

of 15 to 25 weight % concentration and a crosslinking agent, and has Tan 5 value of 0.5 to 1.5 at the frequency of 0.02 to 1 Hz (Step 1).
Preferably, the preparation method for crosslinked hyaluronic acid of the present invention may comprise obtaining crosslinked hyaluronic acid slurry by crushing the crosslinked hyaluronic acid, cleansing with saline and swelling, and then grinding (Step 2) after the Step 1)
The step 1 for obtaining crosslinked hyaluronic acid by cross-linking a composition for preparation of crosslinked hyaluronic acid, which comprises hyaluronic acid alkaline aqueous solution of 15 to 25 weight % concentration and crosslinking agent, and has Tan 8 value of 0.5 to 1.5 at the frequency of 0.02 to 1 Hz is the step for obtaining initially viscoelastic crosslinked hyaluronic acid by cross-linking a composition for preparation of crosslinked hyaluronic acid with specific Tan 5 value at specific frequency range.
In the preparation method for crosslinked hyaluronic acid of the present invention, crosslinked hyaluronic acid with many characteristics can be prepared by inducing grafting rather than crosslinking by inducing decomposition of hyaluronic acid for some and by inducing crosslinking reaction for others during mixing process of raw material, which is one of the most important processes.
In order to induce the above reaction, the concentration of hyaluronic acid, and the range of Tan 5 value of the mixture may be important.
First, the concentration of hyaluronic acid alkaline aqueous solution used in crosslinking reaction needs to be adjusted to the range of 15 to 25 weight % concentration. If the concentration of hyaluronic acid alkaline aqueous solution is less than 10% by weight, degree of freedom of hyaluronic acid molecular chain increases and crosslinked product with viscosity is formed, and if higher than 25% by weight, viscoelasticity is high but biocompatibility is low, which may cause the side effects in the human body.
In addition, the above reaction can be completed by mixing and reacting under the heterogeneous condition during mixing raw material. Heterogeneous mixing can be

induced by adding a crosslinking agent after mixing an alkaline solution and hyaluronic acid first. The homogeneity of mixture can be checked by Tan 5 value of viscoelastic fluid and the degree of dissolution can be confirmed by controlling Tan 8 value since a Tan 5 value of close to 1 indicates a solution and that of close to 0 indicates an elastomer. In the present invention, crosslinked hyaluronic acid with both viscosity and elasticity can be prepared by using a composition for the preparation of crosslinked hyaluronic acid wherein Tan 8 value is adjusted to the range of 0.5 to 1.5 at the frequency of 0.02 to 1 Hz. Crosslinked hyaluronic acid of the present invention can be in the injectable form since it has both viscosity and elasticity as above-mentioned, and has an advantage of excellent stability and tissue-restorability without additional polymers.
In the present invention, in addition, the decomposition of hyaluronic acid exposed to alkaline solution can be minimized by controlling temperature increase caused by mixed frictional heat during mixing alkaline solution and hyaluronic acid. Hereupon, the temperature is preferably 10-50°C, more preferably 20-40°C, and most preferably 25-35°C.
The Step 2 for obtaining crosslinked hyaluronic acid slurry by grinding after cleansing with saline and swelling after crushing the crosslinked hyaluronic acid is the step for preparing the final crosslinked hyaluronic acid by post-processing the crosslinked hyaluronic acid.
In the present invention, the final crosslinked hyaluronic acid obtained as above comprises both monophasic and biphasic characteristics. In addition, the final crosslinked hyaluronic acid comprises stability against enzyme, excellent ability to restore tissue, and long duration.
[Effect of Invention]
The present invention can prepare viscoelastic crosslinked hyaluronic acid, which has both viscous and elastic characteristics, by preparing a composition for preparation of viscoelastic crosslinked hyaluronic acid with Tan 8 value of 0.5 to 1.5 at the frequency of 0.02 to 1 Hz. In addition, the present invention has an advantage of preparing viscoelastic crosslinked hyaluronic acid which comprises stability against enzyme, excellent ability to

restore tissue, and long duration by using the composition for preparation of crosslinked hyaluronic acid.
[Description of Figures]
Figure 1 represents the measurement results of the rheological properties according to the reaction time of a composition for preparation of crosslinked hyaluronic acid with rheometer. In figure 1, red dots indicate Example 1, blue dots indicate Example 2, yellow dots indicate Example 3, and green dots indicate the control group. Figure 2 represents the measurement results of viscoelasticity change during decomposing the crosslinked hyaluronic acid in Example 5 and Comparison Example 1 by hyaluronidase. In figure 2, blue dots indicate crosslinked hyaluronic acid of Example 5, and red dots indicate crosslinked hyaluronic acid of comparison Example 1.
[Specific mode for Invention]
The present invention is described in more detail through providing Examples as below. However, these Examples are merely meant to illustrate, but in no way to limit, the claimed invention.
Examples 1-3: Preparation of a composition for preparation of crosslinked hyaluronic acid and Investigation on the rheological properties thereof according to the reaction time
Hyaluronic acid 1 g (molecular weight: about 3 million daltons, manufacturer: LG Life Sciences) was dissolved in 0.25N NaOH solution at a concentration of 20% by weight, and premixed for the powder not to blow. The dye was applied to determine the degree of mixing, 1,4-butanediol diglycidyl ether was added as crosslinking agent to the mixture mixed homogeneously to be the same condition, and then mixture whose Tan 5 value in mixed state is 0.7 (Example 1), 0.97 (Example 2), or 1.39 (Example 3) at 0.02 Hz was prepared. The rheological properties of the mixture according to reaction time were observed using a rheometer (MCR301, Anton paar). For comparison, unreacted mixture prior to adding crosslinking agent was used as a control group. The results are shown in Figure 1.
Figure 1 shows that when Tan 5 value of mixture is 0.7, 0.97, or 1.39 at 0.02 Hz, viscosity change of reactant according to time is range of 40 million to 150 million cP, which

identifies that decomposition and crosslinking of some in the crosslinked mixture progress over the time.
Comparison Example 1: Preparation of crosslinked hyaluronic acid with monophasic characteristics
Hyaluronic acid (molecular weight: about 3 million daltons, manufacturer: LG Life Sciences) was dissolved in 0.25N NaOH solution at a concentration of 10% by weight, 1,4-butanediol diglycidyl ether was added as crosslinking agent, and then mixed. After the mixing is complete, the mixture was put in a constant temperature water bath, and gel lump was obtained by crosslinking reaction, followed by being cleansed with buffer solution and being swollen.
Examples 4-6: Preparation of crosslinked hyaluronic acid with both monophasic and biphasic characteristics
Crosslinked hyaluronic acid was obtained by cross-linking the mixture obtained in the Examples 1 to 3, crushed to the appropriate size, cleansed with saline, and swollen. Crosslinked hyaluronic acid was obtained after grinding using a grinding device.
Experimental Example 1: Investigation on the viscoelasticity of crosslinked hyaluronic acid with both monophasic and biphasic characteristics of the present invention
The value for viscoelasticity was obtained at frequency of 0.02 Hz after sterilizing the crosslinked hyaluronic acid of Comparison Example 1, and Examples 4 to 6, and then the analysis of Tan 5 was performed. The results are shown in Table 1. [Table 1]

Viscoelasticity after sterilization Tan 5 % Cohesiveness % Elasticity Phase
Example 4 0.34 108.8 74.7 Biphase
Example 5 0.20 116.9 83.1 Biphase
Example 6 0.27 113.6 78.8 Biphase
Comparison Example 1 0.40 101.78 71.1 Monophase

Table 1 identified that crosslinked hyaluronic acid of the present invention has superior cohesiveness, which corresponds to viscosity, and superior elasticity compared to Comparison Example 1. which confirmed that crosslinked hyaluronic acid of the present invention has excellent viscoelasticity. In addition, while crosslinked hyaluronic acid in Comparison Example 1 has monophasic characteristic, crosslinked hyaluronic acid of the present invention can be identified to have biphasic characteristic.
Experimental Example 2: Investigation on the resistibility against enzyme of crosslinked hyaluronic acid of the present invention
Crosslinked hyaluronic acid of Example 5 and Comparison Example 1 was finally
sterilized after being crushed, put into Eppendorf tube by 0.6 g, and hyaluronidase of 500
U/mL was added by 6 μl, mixed homogeneously, and then viscoelastic properties were
measured using a rheometer.
The result was represented in Figure 2.
Figure 2 identified crosslinked hyaluronic acid of the present invention has superior
viscoelasticity even under the treatment with hyaluronidase compared to Comparison
Example 1, which confirmed that crosslinked hyaluronic acid of the present invention has
an excellent resistance against the enzyme.

We Claim,
1. A composition for preparation of crosslinked hyaluronic acid, comprising hyaluronic acid alkaline aqueous solution of 15 to 25 weight % concentration and a crosslinking agent, and having Tan 5 value of 0.5 to 1.5 at the frequency of 0.02 to 1 Hz.
2. The composition for preparation of crosslinked hyaluronic acid according to claim 1, wherein Tan 8 value is 0.5 to 1.5 at the frequency of 0.02 Hz.
3. The composition for preparation of crosslinked hyaluronic acid according to claim 1, wherein the molecular weight of hyaluronic acid is 100,000 to 5,000,000.
4. The composition for preparation of crosslinked hyaluronic acid according to claim 1, wherein the alkaline aqueous solution is NaOH solution.
5. The composition for preparation of crosslinked hyaluronic acid according to claim 1, wherein the cross linking agent is 1,4-butanediol diglycidyl ether.
6. A preparation method for crosslinked hyaluronic acid comprising:
obtaining hyaluronic acid by cross-linking the composition for preparation of crosslinked hyaluronic acid of any one of claims 1 to 5 (Step 1); and obtaining crosslinked hyaluronic acid slurry by naturalizing through cleansing, and grinding the crosslinked hyaluronic acid (Step 2).
7. The method according to claim 6, which further comprises obtaining crosslinked
hyaluronic acid slurry by crushing the crosslinked hyaluronic acid, cleansing with
saline and swelling, and then grinding (Step 2) after the Step 1).