KR101003687B1 - Hyaluronic compound, its synthesis and use - Google Patents

Abstract

PURPOSE: A hyaluronic acid-based soft tissue filler with a hyaluronic compound is provided to ensure long biodegradation rate, to generate collagen although filler is biodegraded and to reduce needs to again inject filler to the applied soft tissue. CONSTITUTION: A hyaluronic compound with a structure of chemical formula 1 comprises the steps of: preparing pullulan-retinoic acid of following chemical formula 4 through an ester bond between pullulan with a structure of chemical formula 2 and retinoic acids with a structure of chemical formula 3; and cross-linking a methanol group and a methanol group of a hyaluronic acid through ether bond by reacting a pullulan-retinoic acid assembly of chemical formula 4, hyaluronic acid of chemical formula 5, and 1,4-butanediol diglycidyl ether as a cross-linking agent.

Description

Hyaluronic acid compound, its manufacturing method and use {Hyaluronic compound, its synthesis and use}

The present invention relates to novel hyaluronic acid-based compounds, and more particularly, to hyaluronic acid-based compounds useful as soft tissue fillers for the treatment of wrinkles and scars, methods for their preparation and use.

 In general, the method of filling human soft tissue is classified into dermal filling or subcutaneous filling, and all of the common fillers contain biosynthetic materials or heterologous proteins, not active factors of their own tissues. Paraffin was first used as a filling material in the 19th century, but many complications appeared and the effect was not good. In the 1960s, siloxane filling was used, but side effects are severe and are no longer used.

Since then, a method of using exogenous protein as a filling material has been developed. Particularly, fillers using bovine collagen protein showed a rapid effect, but had a problem of being digested and absorbed into tissue organs after 3 to 6 months. Construction and application of bovine collagen proteins are described in US Pat. Nos. 3,949,073, 4,424,208 and 4,488,911. Bovine collagen protein was marketed under the trade names Zyderm I, II and III at doses of 35 mg / ml and 65 mg / ml. The therapist was not satisfied with a clear allergic reaction. In order to solve this problem, allergic reactions were solved by developing a cross-linked collagen protein called Zyplast after filtration using glutaraldehyde (US Pat. No. 4,582,640 and JP 10-2007-0122316). , No. 4,642,117), the adhesiveness of the obtained material glutaraldehyde was too large, the treatment effect was not excellent. In order to eliminate the immune response of bovine collagen protein, the method of taking collagen from human placenta is disclosed in US Pat. Nos. 4,969,912 and 5,332,802 and marketed under the trade name AUTOLOGEN, but its effect is merely Only a few months were maintained. Fibrel, a collagen product derived from pigs, is a mixture of gelatin and aminocaproic acid and the plasma of therapist, which is effective in some patients but easily develops lumps at the injection site or does not maintain a therapeutic effect. It was.

Botox is a recently used short-term wrinkle removal injection that has been approved by the FDA in April 2004 for clinical use. The injections anesthetize facial motor muscles to achieve a therapeutic effect, with the advantage of simple manipulation and immediate effect. However, there is a problem that the facial expression is rigid after treatment, the duration of the therapeutic effect is short, and facial nerve paralysis is caused.

Artecoll is a complex of artificial synthetic particles and bovine collagen protein that has been in use for several years in Canada, Mexico, and Europe, and has a lasting effect by stimulating the local dermis to produce its own teachers. However, there is a problem that the therapist can feel the particles by the touch and swelling phenomenon appears in the treatment area.

Meanwhile, the biodegradable, non-animal stabilized hyaluronic acid gel, named Restyland (Q-Med Scandivavia, USA), mainly removes wrinkles and removes wrinkles that make the lips bulky. It was approved by the US FDA on 12 December 2003 as an injection. Hyaluronic acid, taken from the placenta as gluconate aldehyde, acts to provide nutrients to tissues and cells, provides a liquid environment for tissue metabolism, and makes the skin elastic and smooth.

According to the American Society of Aesthetic Plastic Surgery (ASAPS), cosmetic surgery using hyaluronic acid soft tissue fillers has been growing at an average annual rate of over 35%. The recently approved prebell silk is a colorless hyaluronic acid (HA) gel containing lytocaine, and clinical trials show that patients receiving freebell silk have less pain as compared to users of lidocaine-free hyaluronic acid products.

Medisys Pharmaceutical's injectable gel-type filler restillan is also a hyaluronic acid agent, while Botox focuses on improving the wrinkles on the forehead, while restylans focus on improving the wrinkles around the mouth and nose.

Hailafoam is a hyaluronic acid extracted from the crests of bird males such as chicken, and has been sold in more than 30 countries since its introduction in the European market in 1996.

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

In addition, Massachusetts-based Annika Terra-Futics has licensed Elevess, a wrinkle filler and wound healing skin filler from the FDA. Eleves is a soft tissue filler in the formulation of injectables that combines the local anesthetic lidocaine with chemically modified hyaluronic acid, which is patented by Anaca Therafutics.

The advantage of using hyaluronic acid for soft tissue filling is that it is a natural ingredient, safe and free of side effects, while its duration is as short as six months. In addition, when hyaluronic acid is used to remove wrinkles or perform a lip-rich procedure, there is an immediate effect, but there is a problem that the therapeutic effect is maintained for only about 6 months.

Hyaluronic acid is a linear natural mucopolysaccharide consisting of repeating duplexes of D-glucuronic acid and N-acetyl glucosamine, which are widely distributed in the skin. It is a material that has excellent biocompatibility and biodegradability, and has a lot of advantages over collagen, which is a popular filler, and serves to fill space, support structure, and protect cells in vivo.

When the skin having hyaluronic acid having such an action is stimulated by ultraviolet rays, it causes skin aging and elasticity is reduced, thereby losing the components of the skin, hyaluronic acid and collagen, causing wrinkles.

Hyaluronic acid was used to fill the wrinkles caused by ultraviolet rays. Since hyaluronic acid itself has a short half-life of 1 day, it is a crosslinking agent such as BDDE (1,4 butanediol diglycidyl ether) to hyaluronic acid. Attempts have been made to compensate for the drawback of crosslinking, which leads to the formation of gels by crosslinking hyaluronic acid. However, since the cross-linked hyaluronic acid is decomposed by the enzyme, radical, and hydration which is hyaluronidase, and gradually disappears, even if the cross-linked hyaluronic acid gel having a persistence of 6-12 months or less has to be reinjected after a certain period of time, There is a drawback of having a draw.

Accordingly, an object of the present invention is to provide a hyaluronic acid-based soft tissue filler having an increased durability compared to conventional hyaluronic acid-based soft tissue fillers.

The present inventors have studied the problem of the conventional hyaluronic acid-based soft tissue filler is low persistence and has to be re-injected to solve the problem, and in this study, when retinoic acid with collagen production is combined with pullulan, hyaluronic acid The discovery of the fact that it is capable of binding to and crosslinking, the soft tissue filler containing the crosslinked product has a longer biodegradation rate than the soft tissue filler using a crosslinked hyaluronic acid gel, and the retinoic acid component provides collagen production ability. Although the biodegradation of the resulting collagen maintains the skin, the present invention was completed based on the surprising finding that the need to re-inject the filler into the applied soft tissue is greatly reduced.

Therefore, according to the present invention, a hyaluronic acid compound having a structure of Formula 1 is provided.

Where n is an integer as the repeat unit.

In addition, according to the present invention as one of the preferred method for producing a hyaluronic acid compound having a structure of formula (1),

 Preparing a pullulan-retinoic acid conjugate of Formula 4 by ester bonding between pullulan having a structure of Formula 2 and retinoic acid having structure of Formula 3; And

1,4-butanediol diglycidyl ether as a pullulan-retinoic acid conjugate of formula (4), hyaluronic acid of formula (5) and a crosslinking agent to react 1,4-butanediol diglycidyl ether to react the methanol of the conjugate with the methanol of the hyaluronic acid There is provided a method for producing a hyaluronic acid compound having a structure of Formula 1 comprising the step of crosslinking the group by the ether bond by the crosslinking agent.

Where n is an integer as the repeat unit.

Where m is an integer as a repeating unit.

Preferably, the present invention provides a method wherein the pullulan and retinoic acid are dissolved and reacted in an organic solvent.

Preferably, according to the present invention, a method is characterized in that the solution of the pullulan-retinoic acid conjugate is mixed with a hyaluronic acid solution and reacted at 37 ° C. with the addition of a crosslinking agent to form a gel.

Preferably the present invention further provides a method comprising the step of neutralizing the gel.

Preferably, the present invention provides a method for producing a hyaluronic acid compound having a structure of Formula 1, wherein the pullulan-retinoic acid conjugate is ester-bonded with two retinoic acids per 609 glucose ring of the furan.

Further, according to the present invention, a human soft tissue filler containing a hyaluronic acid compound having a structure of Formula 1 is provided.

The soft tissue filler containing the hyaluronic acid compound of Formula 1 according to the present invention has a longer biodegradation rate than the soft tissue filler using a crosslinked hyaluronic acid gel, and the retinoic acid component provides collagen production ability even though the filler is biodegraded. Collagen maintains the skin, greatly reducing the need to re-inject the filler into the applied soft tissue, excellent therapeutic effect, natural and can be maintained for a long time, etc.

1 is a graph showing the results of measuring the viscosity of the compound gel of Formula 1 prepared in Example 3 according to the present invention before and after crosslinking,
Figure 2 is a graph showing the measurement results of the resolution of the conventional cross-linked hyaluronic acid gel and cross-linked hyaluronic acid / pullulan derivative-retinoic acid gel according to the present invention.

As used herein, the term 'hyaluronic acid' refers not only to hyaluronic acid, but also to any hyaluronic acid salt, including, for example, sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate and calcium hyaluronate, chemically modified or derivatized It is used to mean hyaluronic acid in the form.

 The term 'pullulan' refers to pullulan, as well as any pullulan salt, including, for example, pullulan sodium, pullulan potassium, pullulan magnesium, and pullulan calcium, in chemically modified or derivatized form It is used as a meaning containing.

The term 'retinoic acid' means not only retinoic acid, but also any retinoic acid salt, including, for example, sodium retinoic acid, potassium retinoic acid, magnesium retinoic acid and calcium retinoic acid, in the form of chemically modified or derivatized retinoic acid It is used as a meaning containing.

Hereinafter, the present invention will be described in detail.

Hyaluronic acid compound according to the invention is a compound having a structure of formula (1).

Preferably, the hyaluronic acid compound (1) according to the present invention is a compound in which hyaluronic acid, retinoic acid, pullulan and a crosslinking agent are combined.

Hyaluronic acid is a natural mucopolysaccharide that is widely distributed in the skin, and has excellent biocompatibility and biodegradability, and is a molded implant for maintaining a skin volume by forming a hydrogel having a three-dimensional network structure by crosslinking. As an FDA-approved material that is widely used as can be represented by the formula (5).

Pullulan is an soluble polysaccharide produced extracellularly by the Aureobasidium pullulans strain called black yeast. Pullulan is a linear glucan in which three maltotrioses, in which three glucoses are α-1,4, are repeatedly bonded in α-1,6 bonds. Pullulan is an odorless, amorphous white powder, which is toxic and mutagenic. There is no problem in the test results. Unlike general chemically synthesized materials, it is a non-toxic polymer that is easily biodegraded and converted to glucose by enterobacterial pullulase or isopululase.

Retinoic acid is a vitamin A derivative, a type of retinoids that reduces MMPs, an enzyme that degrades collagen, and promotes the growth of fibroblasts to synthesize new collagen to treat skin damage caused by UV rays. It may be used, and may be represented by the formula (3).

Since pullulan is a water-soluble natural polymer containing a large number of hydroxyl groups and having good reactivity, the pullulan may be activated by a catalyst to form a pullulan-retinoic acid conjugate of Formula 4 by ester bonding with a carboxyl group of retinoic acid.

According to the present invention, hyaluronic acid compound (1) in which 1,4-butanediol diglycidyl ether is bonded as a hyaluronic acid, retinoic acid, pullulan and a crosslinking agent is conjugated with pullulan and retinoic acid by an ester bond. The methanol group of a pullulan-retinoic acid conjugate and the methanol group of hyaluronic acid are bridge | crosslinked by the ether bond by the said crosslinking agent.

Retinoic acid may be toxic if large amounts are conjugated, so it is preferable to conjugate small amounts. The preferred degree of conjugation is to conjugate two retinoic acids per about 609 glucose rings.

The soft tissue filler containing the hyaluronic acid compound (1) according to the present invention has a long biodegradation rate compared to the soft tissue filler using a conventional crosslinked hyaluronic acid gel, and the retinoic acid component provides collagen production ability even though the filler is biodegraded. Collagen maintains the skin, greatly reducing the need to refill the applied soft tissue.

Hyaluronic acid-based compound of formula (1) according to the invention preferably comprises the steps of preparing a pullulan-retinoic acid conjugate of formula (4) by the ester bond between pullulan having a structure of formula (2) and retinoic acid having the structure of formula (3); And a 1,4-butanediol diglycidyl ether as a pullulan-retinoic acid conjugate of Formula 4, hyaluronic acid of Formula 5 and a crosslinking agent to react the methanol group of the conjugate and the methanol group of the hyaluronic acid with an ether bond by the crosslinking agent. It may be prepared through the step of crosslinking.

The pullulan and retinoic acid are preferably dissolved in an organic solvent and reacted. As the organic solvent, any organic solvent capable of dissolving both pullulan and retinoic acid may be used as long as it does not interfere with the formation of the pullulan-retinoic acid conjugate by ester bond formation of pullulan and retinoic acid. Although not particularly limited, a preferred example of such an organic solvent is dimethyl sulfoxide (DMSO).

For example, when the pullulan-retinoic acid conjugate, the hyaluronic acid, and the crosslinking agent are reacted, the solution of the pullulan-retinoic acid conjugate is mixed with the hyaluronic acid solution, and the crosslinking agent is added to react at about 37 ° C. for 4 to 15 hours. It is preferable to form.

In the crosslinking step, the pullulan-retinoic acid conjugate is dissolved in an alkaline aqueous solution, and hyaluronic acid is also dissolved in an alkaline aqueous solution so that the hydroxyl group of methanol is activated. At this time, the pH of the alkaline aqueous solution used is suitable 8 ~ 13, the crosslinking agent can be produced by the crosslinking of each hydroxy group by ether linkage, hyaluronic acid to a more robust gel. In addition, the activity of a crosslinking agent can be 25 degreeC-60 degreeC. The gel prepared under such conditions is alkaline, so it is preferable to perform a pH titration. The solvent used for pH titration is preferably an organic acid having a low pH, for example, acetic acid, and the titration is preferably about pH 7.2.

According to the present invention, the soft tissue filler containing the hyaluronic acid compound (1) contains the hyaluronic acid compound (1) in a gel state. The gel is preferably milled with a homogenizer to make the particles small and formulated into an injection. For this formulation the soft tissue fillers of the present invention may comprise additives such as inert, pharmaceutically acceptable carriers or diluents, thickeners, emulsifiers, preservatives and mixtures thereof. Suitable examples of such additives include formulations commonly used in pharmaceutical and skin care preparations. More preferably, examples of inert pharmaceutically acceptable carriers or diluents include saline and purified water. Suitable thickeners include acrylamide copolymers, carbomers, hydroxyethylcellulose, polyacrylic acid, polymethacrylic acid and polyvinyl alcohol. Suitable emulsifiers include capryl / capric triglyceride, stearate-7, cetyl alcohol, cetylphosphate, isostearate-11 and sodium isostearate. Preservatives confer resistance to the attack and toxicity of microorganisms. Suitable examples include alcohols, optional parabens, diazolidinyl urea, DMDM hydantoin, phenoxyethanol and iodoprovinyl butylcarbamate. Additives other than those listed above may also be used in the present invention as is commonly known to those skilled in the art.

Soft tissue fillers according to the present invention, when injected into the wrinkles caused by the ultraviolet light also helps collagen production, there is an effect of filling the wrinkles to give a sense of volume.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for the understanding of the present invention, and the scope of the present invention is not limited by them in any sense.

Example  One: Pullulan - Retinoic acid  Synthesis of Conjugates

1 g of pullulan (molecular weight 100,000 Da, Tokyo Chemical Industry) was added to 10 ml of dimethyl sulfoxide, stirred and dissolved, and 3 mmole of retinoic acid and N, N'-dicyclohexylcarbonylimide (DCC) and 4 -Dimethylaminopyridine (DMAP) was dissolved in 5 ml of dimethyl sulfoxide for 6 hours at a concentration of 6 mmoles, and then reacted with 5 ml of dimethyl sulfoxide solution in which retinoic acid was dissolved. The reaction product was added to the dissolved pullulan for 24 hours, and then dialyzed with distilled water for 3 days (Dialylsis membrane with MWCO 10,000 Da) to remove unreacted retinoic acid, followed by lyophilization to synthesize the pullulan-retinoic acid. Lan-retinoic acid conjugate).

Example  2: Pullulan - Retinoic acid  Synthesis Verification of Conjugates

In order to confirm that the pullulan-retinoic acid conjugate prepared in Example 1 was properly synthesized, it was confirmed by ultraviolet-visible spectrum-absorption spectroscopy (Uv-Vis spectroscopy). The results are shown in Table 1. Table 1 shows the synthesis ratio of the pullulan-retinoic acid conjugate.

 Code name  Retinoic acid amount bound to pullulan  Pullulan-retinoic acid2 (pr2) 2.13

Example  3: Pullulan - Retinoic acid Of Hyaluronic acid  Gel formation

In order to bind the pullulan-retinoic acid conjugate prepared in Example 1 to hyaluronic acid, BDDE (1,4-Butanediol Diglycidyl Ether) is used as a crosslinking agent. The ratio of hyaluronic acid and pullulan-retinoic acid conjugate was 100: 0, 98: 2, and 90:10 (w / v%). 10% hyaluronic acid (HA, 330kDa) was dissolved in 0.2N sodium hydroxide solution and 10% pullulan derivative-retinoic acid conjugate was dissolved 0.2N sodium hydroxide. The pullulan-retinoic acid conjugate is added dropwise to the tube in which the hyaluronic acid is dissolved, and mixed sufficiently by vortexing, and then the crosslinking agent BDDE is added at a molar ratio of hyaluronic acid and 1: 1 (HA: BDDE). Vortexing was performed. The material was reacted in a 37 ° C. incubator for 4-15 hours, neutralized with dialysis or acetic acid with PBS, and sufficiently inflated to break up the gel with a homogenizer to form small particles.

Integrating the reaction of Examples 1 and 3 represented by the reaction scheme is as follows.

Experimental Example  One: Bridged Pullulan - Retinoic acid Hyaluronic Acid Of gel (Formula 1)  Viscosity measurement

The gel prepared in Example 3 was measured with a shear rate of 0.025s¹ using a DV-II + P viscometer. As a result, the viscosity was 150,000 cps or less before adding the crosslinking agent as shown in FIG. 1, but the viscosity increased to 7,000,000 cps or less when the crosslinking agent was added. The cross-linked hyaluronic acid / pullulan-retinoic acid gel was higher in viscosity than that prepared with only hyaluronic acid, and the gel at 98: 2 ratio was higher than the gel at 90:10 ratio.

Experimental Example  2: Bridged Pullulan - Retinoic acid Hyaluronic Acid Of gel (Formula 1)  Resolution measurement

Among the gels prepared in Example 3, since 98: 2 is a gel having an amount of 1 μg to help collagen formation, one gel is selected to apply 40 unit / ml of hyaluronidase to 1,3,5, As a result of measuring the resolution of about 7 days, it was confirmed that the crosslinked hyaluronic acid / pullulan-retinoic acid gel is more slowly degraded than the crosslinked hyaluronic acid gel as shown in FIG.

Claims (9)

Hyaluronic acid compound having a structure of Formula 1:
Formula 1

Where n is an integer as a repeating unit.
delete In preparing a hyaluronic acid compound having a structure of Formula 1,
Preparing a pullulan-retinoic acid conjugate of Formula 4 by ester bonding between pullulan having a structure of Formula 2 and retinoic acid having structure of Formula 3; And
1,4-butanediol diglycidyl ether as a pullulan-retinoic acid conjugate of formula (4), hyaluronic acid of formula (5) and a crosslinking agent is reacted to crosslink the methanol group of the conjugate and the methanol group of the hyaluronic acid with an ether bond by the crosslinking agent Method for preparing a hyaluronic acid compound having a structure of Formula 1 comprising:
Formula 1

(Where n is an integer as a repeating unit)

(2)

(3)

Formula 4

Formula 5

In the above formulas 2, 4 and 5, n and m are integers as repeat units.
The method of claim 3, wherein the pullulan and the retinoic acid are dissolved and reacted in an organic solvent to react with the hyaluronic acid-based compound having the structure of Formula 1.
The method of claim 3, wherein the solution of the pullulan-retinoic acid conjugate is mixed with a hyaluronic acid solution and reacted at 37 ° C by adding a crosslinking agent to form a gel. Manufacturing method.
The method of claim 5, further comprising neutralizing the gel.
According to claim 3, wherein the pullulan-retinoic acid conjugate is a method for producing a hyaluronic acid compound having a structure of Formula 1, characterized in that two retinoic acid per ester of 609 glucose ring of the furan ester.
delete Human soft tissue filler containing the hyaluronic acid type compound of Claim 1.

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