KR101777910B1

KR101777910B1 - A composition for wound healing, method of producing the same and dressing using the same

Info

Publication number: KR101777910B1
Application number: KR1020150061656A
Authority: KR
Inventors: 김현정; 박일규; 조완진; 최환열; 강연주
Original assignee: 주식회사 제네웰
Priority date: 2015-04-30
Filing date: 2015-04-30
Publication date: 2017-09-13
Also published as: JP2018515478A; KR20160129459A; JP6501913B2; CN107708722A; WO2016175358A1

Abstract

The present invention relates to a composition for treating chronic wounds, a method for producing the same, and a dressing material for chronic wounds using the same, and more particularly, To a wound dressing material for chronic wounds.
According to the present invention, it has a continuous effect that it binds to multiple growth factors and is not easily decomposed for 7 days or more at the wound site, and has an effect of having properties suitable for cell culture in vitro and tissue regeneration after transplantation.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a composition for treating chronic wounds, a method for producing the same, and a dressing material for chronic wounds using the same.

The present invention relates to a composition for treating chronic wounds comprising a polymer complex capable of carrying multiple growth factors, a method for producing the same, and a dressing material for chronic wounds using the same, and more particularly, A method for manufacturing the same, and a dressing material for chronic wounds using the same.

The transition to an aging society, the development of medical technology, the increase in life expectancy, and changes in lifestyles are leading to a worldwide increase in the incidence of chronic diseases such as hypertension and diabetes. Wounds caused by chronic diseases are known to be slower and not healed than acute wounds. The use of growth factors to induce chronic wound healing has long been hypothesized because of the decreased expression of growth factors in chronic wounds such as pressure sores, chronic ulcers (lower leg ulcers, diabetic foot ulcers) Research has been conducted, some of which have been commercialized and used clinically. In addition, products containing epidermal growth factor (EGF) and fibroblast growth factor (FGF) are marketed in the market. However, these growth factor products are expensive, And there is a disadvantage in that the bioactivity is lowered when the frozen product is dissolved and used. In addition, the use of a wound dressing material after the use of a growth factor product on the wound has a problem that most of the growth factors are absorbed by the wound dressing and the effect is reduced.

In order to enhance the effect of the growth factor, Japanese Patent No. 3761816 discloses a method of stabilizing the epidermal growth factor (EGF) by using a polyvinyl alcohol hydrogel to improve the sustaining ability. However, since the growth factor is required to be stored in an aqueous solution, the activity of the growth factor is likely to be lowered, and thus the stability is not greatly improved.

In addition, U.S. Patent Publication No. 2013-054300 discloses a method of forming a covalent bond with a fibroblast growth factor (FGF) with a carboxy polysaccharide, and U.S. Published Patent Application No. 2014-0335045 discloses a method of forming a heparin- heparin mimicking polymer to form a covalent bond with a basic fibroblast growth factor (bFGF) to improve the stability of the growth factor.

However, since these patents are confined to the combination with single growth factors, it is difficult to treat chronic complications such as chronic wounds, which require various growth factors.

In order to solve the problems of the prior art as described above, it is an object of the present invention to provide a composition for treating chronic wounds and a method for producing the composition, which can stably bind multiple growth factors and act more effectively on chronic wounds.

It is another object of the present invention to provide a dressing material for chronic wounds using the composition.

The above and other objects of the present invention can be achieved by the present invention described below.

In order to achieve the above object, the present invention provides a composition for treating chronic wounds comprising an anionic polymer, a cationic polymer and a cross-linking agent, comprising a multiple growth factor as an active ingredient, and a method for producing the same.

The present invention also provides a dressing material for chronic wounds treatment using the composition.

According to the present invention, there is provided a composition for treating chronic wounds having a sustained effect that is not easily decomposed for 7 days or more at the wound site and has properties suitable for cell culture and tissue regeneration after transplantation in vitro, a method for producing the composition, and a method for treating chronic wounds There is an effect of providing a dressing material.

FIG. 1 is a graph showing the biodegradability evaluation results of the crosslinked matrix on which the multiple growth factors prepared in Examples 1 to 3 and Comparative Example 1 are mounted.
2 shows the results of measurement of the in vitro release behavior of the growth factors contained in the crosslinked matrices loaded with the multiple growth factors prepared in Examples 1 to 3 and Comparative Examples 1, 4, 5 and 6 Graph.
FIG. 3 is a graph showing the results of evaluating the performance of chronic wound healing of a crosslinked matrix loaded with multiple growth factors prepared in Example 3 and Comparative Examples 1 to 6. FIG.

Hereinafter, the present invention will be described in detail.

The composition for the treatment of chronic wounds of the present invention, a method for producing the same, and a dressing material for chronic wounds using the same are characterized by containing an anionic polymer, a cationic polymer and a cross-linking agent.

Growth factors vary in the isoelectric point (PI) at which positive and negative charges are equalized by the type and arrangement of 20 kinds of amino acids. Therefore, each growth factor has different charges depending on the isoelectric point in the neutral solution. For example, EGF has a PI value of about 4.5 to 6 and has a negative charge in a pH 7 solution, and the FGF has a PI value of about 9 to 11 to have a positive charge in a pH 7 solution. The negatively charged growth factor is ion-bound to a cationic polymer having NH ₃ ⁺ group (-NH ₃ ⁺ ) due to the formation of C00 ^- group (-C00 ^- ). The positively charged growth factor is NH ₃ ⁺ Group is formed and ion-bonded to the polymer forming the COO ^- group.

The anionic polymer may be, for example, hyaluronic acid, alginic acid or a mixture thereof.

The anionic polymer has a weight average molecular weight of 10,000 to 5,000,000 g / mol, 50,000 to 4,500,000 g / mol, or 100,000 to 4,000,000 g / mol, for example.

The anionic polymer may be contained in an amount of 39.8 to 98.9% by weight, 44.6 to 94.0% by weight, or 49.4 to 89.0% by weight, and the matrix surface can be uniformly formed within this range.

The cationic polymer is, for example, collagen, chitosan or a mixture thereof.

The cationic polymer has a weight average molecular weight of 1,000 to 500,000 g / mol, 5,000 to 450,000 g / mol, or 10,000 to 400,000 g / mol, for example, and has a crosslinking effect by the crosslinking agent within this range.

The cationic polymer may be contained in an amount of 0.9 to 59.9 wt%, 4.9 to 54.9 wt%, or 9.9 to 49.0 wt%, and has an effect of increasing cell growth within this range.

The anionic polymer and the cationic polymer have a weight ratio of 50 to 95: 50 to 5, 60 to 90: 40 to 10, or 65 to 85: 35 to 15, and anionic and cationic polymers Is suitable for use as a dressing material and is easy to handle.

The anionic polymer, cationic polymer and cross-linking agent are cross-linked scaffolds, that is, cross-linking matrices. In this case, they are excellent in stability and useful for the treatment of complex diseases.

The anionic and cationic polymers are crosslinked by the crosslinking agent to greatly improve the stability in the body. A cross-linking agent is mixed in the mixed solution of the anionic polymer and the cationic polymer, and the resultant is put in a mold of a predetermined size and lyophilized, followed by washing to prepare a crosslinked matrix, or a support composed of anionic and cationic polymers Crosslinking agent solution, crosslinked, and then washed to prepare a crosslinked matrix.

The crosslinking agent is at least one selected from the group consisting of butanediol diglycidyl ether (BDDE) and hexamethylene diisocyanate (HMDI).

The crosslinking agent may be contained in an amount of 0.099 to 40.93% by weight, 0.496 to 37.49% by weight, or 0.98 to 33.33% by weight, and the body fluid can be absorbed without dissolving in body fluids within this range, .

The multiple growth factor is a term referring to two or more kinds of growth factors.

The multi-growth factor is, for example, Epidermal Growth Factor (EGF) and Fibroblast Growth Factor (FGF). In this case, the multi-growth factor has a continuous effect on the wound region, There is an excellent effect on tissue regeneration.

The multi-growth factor may be contained in an amount of 0.00058 to 0.175% by weight, 0.003 to 0.156% by weight, or 0.006 to 0.133% by weight, and the therapeutic effect on chronic wounds is excellent within this range.

The multiple growth factor has a therapeutic effect on chronic wounds within a range of 1 to 9: 9 to 1, 2 to 8: 8 to 2, or 3 to 7: 7 to 3, great.

The multiple growth factors, particularly EGF and FGF-loaded crosslinking matrices, have an effect of maintaining the hydration state for the growth factors to act continuously on the wound region and being present in the wound region for more than 7 days.

The composition for treating chronic wounds may further include at least one member selected from the group consisting of stabilizers, excipients, isotonic agents, moisturizing agents, pH adjusting agents, antioxidants, antibacterial agents, and anti-inflammatory agents.

The composition for treating chronic wounds of the present invention can be used as a dressing material.

The method for preparing a therapeutic composition for chronic pancreatitis according to the present invention comprises the steps of adding an acid component to distilled water to adjust the pH to 3.0 to 5.0 and then adding a cationic polymer to prepare a mixed solution (I); Adding a base component to the mixed solution (I) to adjust the pH to 6.0 to 8.0, and then adding an anionic polymer to prepare a mixed solution (II); Mixing the cross-linking agent in the mixed solution (II) and crosslinking the cross-linking agent to prepare a cross-linked matrix; And introducing a multiple growth factor into the crosslinked matrix, freezing and lyophilizing the crosslinked matrix to prepare a crosslinked matrix on which the growth factor is mounted.

Examples of the multiple growth factors include epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF), vascular endothelial growth factor A growth factor (VEGF), a colony stimulating factor (CSF), and a platelet derived growth factor (PDGF), preferably EGF and FGF , Which has a long-lasting effect on the wound area and has excellent effects on cell culture in vitro and tissue regeneration after transplantation.

In the step of preparing the mixed solution (I), the cationic polymer is mixed with the anionic polymer in the step of preparing the mixed solution (II), followed by homogenization with a homogenizer.

The acid component may be at least one member selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid and nitric acid.

The basic component may be at least one selected from the group consisting of sodium hydroxide, calcium hydroxide, potassium hydroxide and ammonium hydroxide.

The anionic polymer is included at a concentration of, for example, 4 to 10 mg / g, 4.5 to 9.5 mg / g, or 5 to 9 mg / g, and has an effect of uniformly forming the matrix surface within this range.

The cationic polymer is included at a concentration of, for example, 0.1 to 6 mg / g, 0.5 to 5.5 mg / g, or 1 to 5 mg / g, and has an effect of increasing cell growth within this range.

Wherein the weight ratio of the anionic polymer and the cationic polymer is 50 to 95: 50 to 5, 60 to 90: 10 to 40, or 65 to 85: 35 to 15, and the anionic polymer and the cationic polymer So that it is suitable for use as a dressing material and is easy to handle.

The crosslinking matrix is prepared by mixing the crosslinking agent in the mixed solution (II) and crosslinking at 25 to 35 ° C for 3 to 24 hours.

The crosslinking agent is contained in a concentration of 0.01 to 7 mg / g, 0.5 to 6 mg / g, or 0.1 to 5 mg / g, for example, and is capable of absorbing body fluids without being dissolved in body fluids within this range. There is an exudate absorption effect.

A multi-growth factor is added to the crosslinked matrix to prepare a crosslinked matrix on which the growth factor is mounted.

The multi-growth factor is included in a concentration of 0.1 to 30 μg / g, 0.5 to 25 μg / g, or 1.0 to 20 μg / g, for example, and the therapeutic effect on chronic wounds is excellent within this range.

The weight ratio between the two growth factors of the multiple growth factor is, for example, 1 to 9: 9 to 1, 2 to 8: 8 to 2, or 3 to 7: 7 to 3, .

The crosslinked matrix on which the growth factor is loaded is injected into a mold of a certain type and then frozen at -85 캜 to -75 캜 for 4 to 12 hours and then dried in a freeze dryer at a temperature of -40 to -10 캜 for 12 to 35 hours .

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention. Such variations and modifications are intended to be within the scope of the appended claims.

[Example]

Example 1

Hydrochloric acid or sulfuric acid was added to 100 ml of distilled water to adjust the pH to 4.0, collagen (weight average molecular weight: 300,000 g / mol) was added in an amount such that the concentration became 1 mg / g based on the acid aqueous solution, . Sodium hydroxide was added to the mixed solution (I) to adjust the pH to 7.0 to 8.0. Then, hyaluronic acid (weight average molecular weight: 1,500,000 g / mol) was mixed in an amount such that the concentration became 9 mg / (II) was prepared. The crosslinking matrix was prepared by mixing 1,4-butanediol diglycidyl ether as a crosslinking agent at a concentration of 5 mg / g (relative to solid content) and crosslinking at 30 ° C for 12 hours. EGF and FGF were added to the crosslinked matrix in an amount of 5 쨉 g / g in a weight ratio of 5: 5, mixed, and then injected into a mold of a certain shape. The mold was frozen at -80 占 폚 for 6 hours and then dried at -20 占 폚 for 24 hours in a freeze dryer to prepare a crosslinked matrix loaded with multiple growth factors.

Example 2

The same procedure as in Example 1 was repeated except that collagen was used in an amount of 2 mg / g of the final concentration and hyaluronic acid of 8 mg / g of the final concentration in Example 1, A crosslinking matrix on which the growth factor was mounted was prepared.

Example 3

The procedure of Example 1 was repeated except that collagen was used in an amount of 5 mg / g of the final concentration of the collagen in Example 1 and 5 mg / g of hyaluronic acid in the final concentration. A crosslinking matrix on which the growth factor was mounted was prepared.

Example 4

The procedure of Example 1 was repeated except that EDC was used instead of 1,4-butanediol diglycidyl ether as a crosslinking agent in Example 1 to prepare a crosslinked matrix having multiple growth factors.

Example 5

The procedure of Example 1 was repeated except that HMDI was used instead of 1,4-butanediol diglycidyl ether as a crosslinking agent in Example 1 to prepare a crosslinked matrix having multiple growth factors.

Comparative Example 1

The procedure of Example 1 was repeated except that the crosslinking agent was not used in Example 1 to prepare a crosslinked matrix having multiple growth factors.

Comparative Example 2

The same procedure as in Example 1 was carried out except that EGF alone was used in an amount such that the concentration of EGF alone was 5 占 퐂 / g, thereby preparing a crosslinked matrix having the growth factor.

Comparative Example 3

The same procedure as in Example 1 was carried out except that FGF alone was used in an amount such that the concentration of FGF alone was 5 占 퐂 / g, thereby preparing a crosslinked matrix having the growth factor.

Comparative Example 4

A crosslinked matrix on which the growth factor was mounted was prepared in the same manner as in Example 1 except that dextrane was used in an amount of 1 mg / g instead of hyaluronic acid and collagen in Example 1.

Comparative Example 5

A crosslinking matrix on which a growth factor was mounted was prepared in the same manner as in Example 1, except that hyaluronic acid and collagen were replaced with hyaluronic acid in an amount of 10 mg / g.

Comparative Example 6

A crosslinking matrix on which the growth factor was mounted was prepared in the same manner as in Example 1, except that hyaluronic acid and collagen were used in an amount of 10 mg / g in place of hyaluronic acid and collagen.

[Experimental Example]

EXPERIMENTAL EXAMPLE 1. Biodegradability Evaluation of Crosslinked Matrix Equipped with Multiple Growth Factor

The crosslinked matrix on which the multi-growth particles prepared in Examples 1, 2 and 3 and Comparative Example 1 were mounted was cut into a size of 2 x 2 cm and put into a dish. 20 ml of phosphate buffer solution (PBS) containing 200 units of hyaluronidase and 200 units of collagenase were added to the above dish, and the mixture was stored at 37 ° C and 50 rpm. Then, 1, 3, 7, 14, and 21, the changed size of the crosslinked matrix on which the multiple growth factors were mounted was measured and shown in FIG.

As a result of the test, it was confirmed that the non-crosslinked matrix (Comparative Example 1) was rapidly decomposed and all decomposed before one day, but the crosslinked matrix (Examples 1 to 3) loaded with the crosslinked multi-growth factor was maintained for 7 days or more .

Experimental Example 2. Evaluation of crosslinking degree

The degree of crosslinking was evaluated by absorbance. The crosslinked matrix loaded with the multiple growth factors prepared in Examples 3 to 5 was cut to a predetermined size, and its initial weight was measured. The weight of the crosslinked matrix was measured after it was fully absorbed by a dish and put into distilled water. The absorption capacity was calculated by the following Equation 1, and the results are shown in Table 1.

[Equation 1]

Absorption capacity = (weight after absorption of distilled water (mg) - initial weight (mg)) / initial weight (mg)

Evaluation of absorption ability by crosslinking agent Initial weight Weight after absorption Absorption capacity Example 3 16.8 89.7 4.33 Example 4 17.5 105.6 5.03 Example 5 17.2 75.1 3.36 Comparative Example 1 13.9 Not measurable Not measurable

As a result of the experiment, it was confirmed that in Examples 3 to 5, water was not easily dissolved by moisture and water was absorbed. Therefore, the composition of the present invention can be maintained in a stable form on the top surface of the window, and can maintain exudate absorption and moisture environment, thereby providing an environment favorable for recovery of chronic wounds.

On the other hand, Comparative Example 1, in which a crosslinking agent was not used, was dissolved directly in water and measurement was impossible.

Experimental Example 3. Evaluation of stability of multiple growth factors

The in vitro release behavior of the growth factors contained in the cross-linked matrices loaded with the multiple growth factors prepared in Examples 1, 2 and 3 and Comparative Examples 1, 4, 5 and 6 was measured. The cross-linked matrix with multiple growth factors prepared in Example 1 and Comparative Examples 1, 4, 5 and 6 was cut into a 30 mm diameter circle and mounted on a Franz Cell. 14 ml of phosphoric acid buffer solution was added to Franzcell and stirred at 37 ° C at 50 rpm. On the 1st, 3rd, 7th, 14th, 21th, and 28th days, 1 ml was sampled and 1 ml of phosphate buffer solution was newly added.

Quantitative analysis of EGF and FGF was performed with an ELISA kit. The results are shown in FIG.

As a result of the experiment, it was confirmed that the crosslinking matrix on which the multiple growth factor prepared in Example 1 was mounted was continuously released for a long period of time as compared with the matrix prepared in Comparative Examples 1, 4, 5 and 6. Thus, the composition of the present invention improves the persistence of the growth factor, so that it can act more effectively on chronic wounds, thereby improving the recovery speed of the wounds.

Experimental Example 4. Evaluation of wound healing performance in a chronic wound animal model

The chronic wound healing performance of the cross-linked matrices loaded with the multiple growth factors prepared in Example 3 and Comparative Examples 1 to 6 was evaluated. To develop a model of type 1 diabetes using streptozotocin (STZ) and to induce a type 1 diabetes model, 6-week-old mice were stabilized for 1 week and then STZ 200 mg / kg. The blood glucose was measured every day for 1 week after injection to select the experimental animals with diabetes and to prevent deep contraction by inducing a deep wound at the center of the back region with a diameter of 1 cm ² The wound size was measured after suturing with a suture using a silicone ring. The healing performance was evaluated by the rate of area change of the wound on days 7 and 14, and the results are shown in FIG.

As a result, the fastest wound recovery rate was shown in Example 3 in which the multiple growth factor (EGF, FGF) was stably bound to the matrix. The neutron flux was constantly released even after the lapse of time, and the wound area of Example 3 was significantly reduced compared with Comparative Examples 1 to 6 on the 14th day.

Therefore, multiple growth factors can improve persistence by ionic bonding with cationic and anionic polymers, and multiple growth factors can increase the rate of wound recovery in chronic wounds compared to single growth factors.

Claims

Wherein the growth factor comprises two or more kinds of growth factors as an effective component and contains an anionic polymer, a cationic polymer and a crosslinking agent, wherein the two or more growth factors are 0.003 to 0.156 wt%, the anionic polymer is 44.6 to 94 wt%, the cationic polymer is 4.9 to 54.9 Wherein the anionic polymer and the cationic polymer have a weight ratio of 50 to 95: 5 to 50, and the two or more growth factors are selected from the group consisting of Epidermal Growth Factor EGF) and Fibroblast Growth Factor (FGF); The anionic polymer may be selected from the group consisting of hyaluronic acid; The cationic polymer includes collagen; Wherein the crosslinking agent is butanediol diglycidyl ether (BDDE), and the anionic polymer and the cationic polymer are crosslinked with the crosslinking agent to form a crosslinked support, wherein the anionic polymer has a weight average molecular weight of 100,000 to 4,000,000 g / mol, wherein the cationic polymer has a weight average molecular weight of 10,000 to 400,000 g / mol, and the weight ratio between the two growth factors is 1: 1.

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The method according to claim 1,
Wherein the composition further comprises at least one selected from the group consisting of stabilizers, excipients, isotonic agents, moisturizers, pH adjusting agents, antioxidants, antibacterial agents and anti-inflammatory agents.

A composition for treating chronic wounds, comprising a composition for treating chronic wounds according to any one of claims 1 to 12 as a dressing material.

Adding an acid component to distilled water to adjust the pH to 3.0 to 5.0, and adding a cationic polymer to prepare a mixed solution (I);
Adding a base component to the mixed solution (I) to adjust the pH to 6.0 to 8.0, and then adding an anionic polymer to prepare a mixed solution (II);
Mixing the cross-linking agent in the mixed solution (II) and cross-linking it to prepare a cross-linking matrix; And
Preparing a crosslinked matrix on which the growth factor is loaded by injecting two or more kinds of growth factors into the crosslinked matrix, freezing and lyophilization, wherein the two or more growth factors are selected from the group consisting of Epidermal Growth Factor EGF) and Fibroblast Growth Factor (FGF); The anionic polymer may be selected from the group consisting of hyaluronic acid; The cationic polymer includes collagen; Wherein the crosslinking agent is butanediol diglycidyl ether (BDDE), the anionic polymer has a concentration of 4 to 10 mg / g, the cationic polymer has a concentration of 0.1 to 6 mg / g, Wherein the weight ratio of the anionic polymer and the cationic polymer is 50 to 95: 5 to 50, and the weight ratio of the anionic polymer and the cationic polymer is in the range of 0.01 to 7 mg / g, and the concentration of the two or more growth factors is 0.1 to 30 g / The anionic polymer has a weight average molecular weight of 100,000 to 4,000,000 g / mol, the cationic polymer has a weight average molecular weight of 10,000 to 400,000 g / mol, and the cationic polymer has a weight- Wherein the weight ratio between the two growth factors is 1: 1.

15. The method of claim 14,
Wherein the acid component is at least one member selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid and nitric acid, and the base component is at least one selected from the group consisting of sodium hydroxide, calcium hydroxide, potassium hydroxide and ammonium hydroxide. &Lt; / RTI >

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