KR20150044862A - Ionic cross-linked hyaluronic acid tissue restorative material - Google Patents

Abstract

The present invention improves the long-term persistence and maintain the shape of hyaluronic acid-based fillers. In the present invention, hyaluronic acids in fillers are cross-linked by using the ionic properties of hyaluronic acids so that at the time of injection, the physical resistance of the filler substances is reduced and, after injection, additional ionic cross-linking is produced in vivo. The increased number of the cross-linked hyaluronic acids delays the in vivo action of hyaluronidase and improves the retention of the in vivo shape.

Description

Ionic crosslinked hyaluronic acid tissue repair material {omitted}

The present invention relates to a gel formed by cross-linking between hyaluronic acids by an electrostatic force and a pulling force, and more particularly, to a gel formed by hyaluronic acid component approved by US FDA,

A hyaluronic acid solution having an ionic crosslinking number increased by adding a hyaluronic acid solution having predominant cationic properties under weakly acidic conditions to a solution having a predominant anionic property under vigorous conditions and then rapidly mixing to proceed a crosslinking reaction, It is possible to improve the disadvantage that the injected material in the body is rapidly decomposed by the body-degrading enzyme, and since the hyaluronic acid mixture is an initial stage of the cross-linking reaction when injecting the hyaluronic acid mixture into the body, And the shape retentive force is increased, thereby improving the weak shape retention of the conventional hyaluronic acid filler, and a method for producing the same.

In general, the soft tissue filling method is classified as dermal filling or subcutaneous filling. All of the common fillers used in the present invention include biosynthetic materials or heterogeneous proteins, not the active factors of the tissues themselves. Since the 1960s, a method of using an outer protein as a filling material has been developed. In particular, a filler using bovine collagen protein has a rapid effect, but has a problem that it is digested and absorbed in a tissue organ 3 to 6 months later.

The collagen protein was marketed as Zyderm I, II and III, but 90% of the healers were immunized and 1-3% of the healers were not satisfied with the allergic reaction. In order to solve this problem, the allergic reaction was solved by developing crosslinked collagen protein after filtration using glutaraldehyde, but the glutaraldehyde was not so excellent in the adhesiveness of the material obtained with glutaraldehyde. To remove the immune response of the bovine collagen protein, a method of taking collagen from the human placenta was marketed under the trade name Oturogen in the United States, but its effect lasted only a few months. Piglet, a collagen product extracted from pigs, is a mixture of gelatin and aminocaproic acid and a therapeutic plasma, which is effective for some patients but has a tendency to develop lumpy or ineffective treatment at the injection site.

Artecol has been used for several years as a complex of artificial synthetic particles and bovine collagen proteins. It has been shown to produce sustained effects by stimulating the local dermis to produce its own teaching material. However, the therapist can feel the particles through the touch, And the like.

Non-Animal Stabilized Hyaluronic Acid Gel, a biodegradable, non-anesthetic stabilized hyaluronic acid gel, is a wrinkle-removing injectable product that removes wrinkles and lips, and is approved by the US FDA on December 12, 2003 .

Medicis Pharmaceuticals' proprietary gel-type filler Resilane is also a hyaluronic acid formulation that focuses on improving wrinkles around the mouth and nose while Botox focuses on improving wrinkles on the forehead area.

Hyaluronic acid has also been released in more than 30 countries since its introduction in Europe in 1996, mainly based on hyaluronic acid extracted from the crests of birds such as chickens.

Juvederm from Elleran also removes wrinkles by injecting hyaluronic acid into the face.

In addition, Anica Therapeutics of Massachusetts has been authorized to release Eleven, a skin filler for wrinkles and wound healing, from the FDA.

Many of these materials have been used as skin fillers for wrinkle and wound healing, but many products have been approved by the US Food and Drug Administration (FDA) for the development of products that use proven hyaluronic acid.

Hyaluronic acid is a biomolecular substance of linear form of millions and tens of millions of Dalton lengths as discrete units of N-acetyl-D-glucosamine and D-glucuronic acid as discrete units, and is used in many applications such as ocular vitreous fluid, joint synovial fluid, exist.

Due to its excellent biocompatibility and viscoelasticity, hyaluronic acid is widely used for medical and medical purposes such as postoperative adhesion inhibitor, shaping aid, wrinkle improving agent, ophthalmic surgery aid, joint function improver, drug delivery material and eye drop, . However, by itself, it is easily decomposed in vivo or under acid and alkali conditions and its use is limited. Therefore, efforts to develop structurally stable hyaluronic acid derivatives have been made widely.

As one of hyaluronic acid derivatives, aluronic acid bridges in which hyaluronic acids are mutually covalently bonded using a crosslinking agent have excellent biocompatibility, physical stability and biodegradability, and a plurality of manufacturing methods are disclosed in this connection.

However, the hyaluronic acid bridges (hyaluronic acid derivatives) produced by the methods of these patents are made in vitro, so that they may encounter material limitations in order to avoid injection with the syringe into the body and application of large pressure to the top , The reactants involved in the crosslinking reaction may not be completely removed and thus toxicity may be caused. In addition, the components participating in the crosslinking reaction may be toxic due to leakage of the components themselves or in a modified form during the decomposition process.

Accordingly, an object of the present invention is to provide a method for producing a hyaluronic acid crosslinked product which can solve the problems of the prior art.

That is, an object of the present invention is to provide a gel prepared by using the pH-reactivity of hyaluronic acid, which can form a cationic state and an anionic state according to pH and form a crosslink by electrostatic interaction thereof, And it is possible to control the strength of the gel prepared by modifying the shape of the gel or by controlling the number of crosslinked water, or to dissolve the gel when necessary, and to remove the gel by the chemical, biochemical The present invention provides a method for manufacturing a crosslinked hyaluronic acid which is easy to process and which is safe, because the materials are not involved and thus the toxicity problem caused by them can be avoided.

It is an object of the present invention to provide a method and a device for injecting hyaluronic acid into a human body, which are problematic in terms of physical resistance at the time of injection, rapid degradation in the body, In order to solve this problem, we have completed the preparation of the tissue repair material with the problems by establishing the manufacturing conditions of the gel so as to increase the number of crosslinks even after the injection.

The present invention relates to a gel prepared by inducing crosslinking by the electrostatic attraction between hyaluronic acid molecules using a cationic form and an anionic form characteristic of hyaluronic acid according to pH and a method for producing the same.

Generally, hyaluronic acid molecules have repeating structures composed of D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units.

1) Deacetylation of the N-acetyl-D-glucosamine moiety in the disaccharide unit results in the formation of a D-glucosamine structure with free amine groups. When the hyaluronic acid molecule is placed under an acidic condition, the dissociation of the -COOH moiety of the D-glucuronic acid moiety is inhibited and the hydrogen ion binds to the amine of the deacetylated D-glucosamine. As a result, under acidic conditions, .

To have the form of ion anion ^condition-2) On the other hand, when alkali is about physiological conditions (normal position the hyaluronic acid molecule to pH 7.4), D- glucuronic acid portion of the -COOH is increased dissociation -COO It becomes hyaluronic acid.

Therefore, when the solution prepared in 1) is mixed with 2), a cationic hyaluronic acid molecule and an anionic hyaluronic acid molecule form an ion pair to form a gel.

In the present invention, the hyaluronic acid gel prepared through cross-linking by electrostatic attraction is easy to inject due to the less physical property due to less crosslinked water during injection, and the crosslinking water is continuously increased in the body after injection, thereby improving the point elasticity It is possible to provide a material capable of increasing the utilization in the human body application process such as cosmetic molding and wound restoration area by extending the formation and maintenance period of the intended shape by providing the gel for skin filling with excellent biocompatibility.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a chart of the rate of degradation of cross-linked hyaluronic acid gel (ion pair HA) and juvidum (control) when the gel formed by ionic cross-linking between hyaluronic acid was treated with hyaluronidase, an endolytic enzyme.
Figure 2 compares the viscosity of ionic crosslinked gel (ion pair HA) and juvidom (control) of hyaluronic acid.

The configuration of the present invention

(a) deacetylating hyaluronic acid;

(b) preparing a solution for maintaining deacetylated hyaluronic acid in a cationic state and a solution for keeping hyaluronic acid in an anionic state, respectively;

(c) stabilizing the cationic solution and the anion solution prepared in step (b) by maintaining the solution at 0 to 40 ° C for 1 to 72 hours; And

(d) reacting the stabilized cationic solution and the anionic solution in the step (c) to form a crosslink by electrostatic attraction.

Hereinafter, the present invention will be described in detail.

The gel according to the present invention is characterized in that a substance in a cationic state and a substance in an anionic state are produced by electrostatic attraction.

The process for preparing the gel of the present invention will be described step by step as follows.

The step (a) is a step of deacetylating hyaluronic acid in a steady state. The hyaluronic acid is dissolved in an aqueous solution, and then treated with hyaluronic acid deacetylase. The hyaluronic acid is deacetylated by enzymatic method in DN-acetylglucosamine of hyaluronic acid It is the process of removing acetyl group.

(B) is a step of preparing a solution for maintaining deacetylated hyaluronic acid in a cationic state and a solution for maintaining hyaluronic acid in an anion state, respectively, wherein deacetylated hyaluronic acid and normal hyaluronic acid are dissolved in a pH Are dissolved in a different solution to prepare a cationic solution and an anionic solution, respectively.

In step (b), each solution is stirred at 20 to 40 ° C, preferably at 25 ° C for 1 to 72 hours, preferably 12 hours.

The step (c) is a step of stabilizing the solution-state cationic material and the solution-state anion-state material prepared in step (b), and stabilizing the solution at 0 to 40 ° C, preferably at 4 ° C for 1 to 72 hours , More preferably for 48 hours to stabilize.

The step (d) is a step of binding the stabilized solution of the cationic material and the solution of the anionic material in step (c), and mixing the solutions in a volume ratio of 99: 1: 1 to 99 , Preferably in the same volume. The cationic material in the solution state and the anionic material in the solution state form a crosslink by the electrostatic attraction within a short time when the reaction proceeds.

At this time, when the solution in the cationic state in the acidic solution is mixed with the weakly basic anionic substance, the gel is formed while the ionic pair is formed in a short time while the solubility is decreased.

Since the ion-pair gel prepared by the above method is a highly safe compound composed entirely of hyaluronic acid molecules, it can be used as a cosmetic material such as a wound healing patch, wrinkle improvement, a cosmetic material, an arthritis remedy, a nerve, skin, bone and cartilage regeneration A support for tissue regeneration, and the like.

Since the gel is formed in a short time by simply mixing the two solutions, the solution can be applied to a treatment method requiring a solid function at the wound site by independently placing the solution in a different container and then meeting at the destination. Since the prepared gel has excellent biocompatibility, it can be applied as a filler for molding.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Production Example 1. Preparation of deacetylated hyaluronic acid

27.5 ml (0.1 g) of hyaluronic acid (0.363%) was added to 55 ml of 0.1 M Tris-HCl buffer (pH 8.5), and 68.75 ml of hyaluronic acid deacetylase isolated from Aspergillus nidulans was added, 96.25 ml of distilled water and 27.5 ml of 1 M KCl were added to adjust the total volume to 275 ml. Then, the reaction was carried out at 40 ° C. for 4 hours, and 99% ethanol (Merck, # 1.00983.1011) was added at a weight ratio of 1: 5 to the reaction product to precipitate deacetylated hyaluronic acid, followed by washing with 99% ethanol , Which was again dissolved in distilled water and dialyzed against distilled water. After 24 hours, it was lyophilized to obtain deacetylated hyaluronic acid.

The deacetylated hyaluronic acid deacetylated by hyaluronic acid according to the present invention has a higher degree of deacetylation than that of deacetylated hyaluronic acid by a chemical method so that the acetyl group of hyaluronic acid is more efficiently removed .

In addition, the deacetylated hyaluronic acid of the present invention is capable of inhibiting the initial degradation rate in vivo by hyaluronidase, minimizing the deterioration of molecular weight and viscosity, and reducing deacetylated hyaluronic acid having high molecular weight and high viscosity .

Production Example 2. Acidification of deacetylated hyaluronic acid

The deacetylated hyaluronic acid prepared in Preparation Example 1 was added to an acidic solution having a pH of 3.0 to 6.0 (the final concentration of the acid was 1 to 5 N). Then, the mixture was stirred at 30 to 80 DEG C for 1 to 10 hours so that deacetylated hyaluronic acid was sufficiently dissolved.

Preparation Example 3. Preparation of hyaluronic acid solution in physiological condition (pH 7.4)

The mixture was stirred at room temperature for 1 to 24 hours, and then the pH was adjusted to be physiological condition (pH 7.4). The pH was adjusted to 4 Lt; 0 > C for 24 hours.

Production Example 4. Gel Formation by Ionic Crosslinking of Hyaluronic Acid

The deacetylated hyaluronic acid solution (acidic solution of pH 3.0 to 6.0) prepared in Preparation Example 3 was prepared by stirring the hyaluronic acid solution in the physiological condition (pH 7.4) prepared in Preparation Example 4 at a proper rate, Go slowly.

This substance was allowed to react for 4 to 15 hours in a 37 ° C incubator, followed by dialyzing with PBS or neutralization with acetic acid.

Example 1. Measurement of resolution of ionic crosslinked hyaluronic acid gel

One of the gels prepared in Preparation Example 4 was selected and the resolution was measured at 0, 10, 20, 30, 40, 50 and 60 minutes by applying 40 Unit / ml of hyaluronidase. As a result, , It was confirmed that the ionic crosslinked hyaluronic acid gel (ion pair HA) was degraded more slowly than the commercially available hyaluronic acid gel, the juvidum product (control group).

Example 2: Viscosity change of ionic crosslinked gel of hyaluronic acid

The viscosity of the ionic crosslinked hyaluronic acid gel of the present invention prepared in Preparation Example 4 was measured according to a shear rate of 0.025 s ^-1 using a DV-II + P viscometer. As a result, as shown in FIG. 2, the gelabid product (control) crosslinked with hyaluronic acid alone showed a viscosity of 233,000 cp, whereas the ionic crosslinked hyaluronic acid gel of the present invention showed 198,000 cp, It was confirmed that the viscosity was decreased.

Claims (12)

Hyaluronic acid tissue repair material prepared by ionic crosslinking using hyaluronic acid as a main component. The method according to claim 1,
A part of the hyaluronic acid is deacetylated. 3. The method of claim 2,
Some of the hyaluronic acids include, but are not limited to, deacetylation reactions by chemical and enzymatic reactions. The method of claim 3,
The deacetyl hyaluronic acid comprises the steps of: (i) adding normal hyaluronic acid to a strong concentration of NaOH alkaline solution and stirring; And (ii) neutralizing the product after prolonged reaction at a temperature not lower than room temperature to obtain deacetylated hyaluronic acid having a degree of deacetylation of 1% to 95%. 5. The method of claim 4,
The alkaline aqueous solution is a sodium hydroxide solution having a concentration of 0.1 to 10 N NaOH. The method of claim 3,
The deacetyl hyaluronic acid process is a reaction in which hyaluronic acid deacetylase acts, and the deacetylase refers to an enzyme that removes the acetyl group of N-acetylglucosamine, but is not limited thereto. The method according to claim 1,
The condition for maintaining a cationic state is to maintain deacetylated hyaluronic acid in an acidic solution. 8. The method of claim 7,
The acidic solution containing deacetylated hyaluronic acid has a pH of 1.0 to 6.9. The method according to claim 1,
The condition for maintaining the negative ion state is to keep normal hyaluronic acid in an alkali solution. 10. The method of claim 9,
The pH of the alkaline solution containing normal hyaluronic acid is 7.1 to 14.0. The method according to claim 1,
The gel formed by cross-linking of hyaluronic acid is formed by crosslinking due to the charge characteristics of hyaluronic acid and derivatives thereof having cationic properties and hyaluronic acid and derivatives thereof having anionic properties. The method according to claim 1,
Wherein the hyaluronic acid ionic crosslinking material has a gel, sponge, bead, thread, or film shape.

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