KR102103180B1 - Anti-adhesion Composition Including Hyaluronic Acid Derivative, Pullulan and Carboxymethyl Cellulose and Manufacturing Method Thereof - Google Patents

Abstract

The present invention relates to a composition for preventing adhesion and a method for manufacturing the same, comprising a hyaluronic acid derivative, flurane and carboxymethyl cellulose.
The composition for preventing adhesion of the present invention has an improved adhesive force including flurane, thereby resisting gravity and forming a physical barrier on the surgical site, thereby preventing tissue adhesion due to surgery.
In addition, since the composition for preventing adhesion of the present invention has an improved retention effect in the body including carboxymethyl cellulose, the formed physical barrier protects the tissue for a sufficient time during which the surgical site can be healed, thereby preventing tissue adhesion.
In addition, the hyaluronic acid derivative of the present invention has a rapid in vivo decomposition and absorption property, thereby preventing adhesion after surgery and replacing the inferior hyaluronic acid in the wound at the surgical site, thereby improving the duration of hyaluronic acid in the body, thus healing the wound at the surgical site. It is effective to prevent tissue adhesion.
In addition, the composition for preventing adhesion of the present invention uses the content of flurane and carboxymethyl cellulose in 0.2 to 1% by weight and 1 to 5% by weight, respectively, and the mixing ratio of the flurane and carboxymethyl cellulose in a weight ratio of 1: 5. There is an effect that can be easily injected into the surgical site using a syringe while having sufficient adhesion and retention effect in the body.
The anti-adhesion composition of the present invention uses flurane, carboxymethyl cellulose and hyaluronic acid derivatives that are harmless to the human body and shows excellent anti-adhesion effect compared to the conventional anti-adhesion agent, so it is immediately applied to the clinic and can replace the conventional anti-adhesion agent. It is expected to be.

Description

Anti-adhesion Composition Including Hyaluronic Acid Derivative, Pullulan and Carboxymethyl Cellulose and Manufacturing Method Thereof}

The present invention relates to a composition for preventing adhesion and a method for manufacturing the same, comprising a hyaluronic acid derivative, flurane and carboxymethyl cellulose.

During the surgical procedure, tissue cells undergo treatment by themselves due to irritation, damage, abrasion, and inflammation caused by infection. Tissue adhesion is a process in which damaged tissue cells regenerate and adhere to other tissues in the course of natural treatment, which is called tissue adhesion, and this tissue adhesion creates a secondary problem for the patient in the recovery process after surgery.

During the healing process, vascular permeability increases, body fluid leaks, and inflammatory reactions progress. In the healing process, a viscous fibrous matrix is produced, which causes adhesion of tissues and organs. The fibrin matrix creates a cellular structure that contains blood vessels and nerves, which form stronger adhesions.

Adhesion in open surgery occurs in about 90% of patients, and complications from this are already at a serious level. When adhesion to the small intestine occurs, chronic abdominal pain is caused and obstruction of the small intestine occurs. In addition, if adhesion to the pelvis occurs, it can cause chronic pain and infertility. Therefore, if severe adhesions occur, reoperation is sometimes required to remove them, which causes economic and health problems for patients. Adhesion causing such problems can occur in all parts of the human body, such as the intestine, peritoneum, uterus, and pelvis, so a composition that prevents adhesion is essential in surgical procedures.

In addition to the basic method of physically covering the wound after surgery to prevent adhesion, there are pharmacological and physical barriers.

Pharmacological methods include anti-inflammatory drugs and aspirin, dexamethasone, heparin, L-arginine, pentoxifylline, methylene blue, or vitamin E to prevent adhesion between tissues. There are studies. However, the pharmacological method has a disadvantage in that its effectiveness is limited compared to a method of forming a physical barrier to prevent adhesion between tissues, mainly because a method of suppressing inflammation occurs in the recovery process.

As a method of forming a physical barrier, there is a method of using a film, a viscous solution, or a hydrogel. The method uses a biocompatible polymer that is harmless to the human body as a method to prevent physical contact between tissues by directly forming a physical barrier in the body. The biocompatible polymer prevents adhesion by forming a physical barrier between the surrounding tissue and the wound during the tissue healing period, and is absorbed and removed naturally in the body at the end of healing, so that no additional inflammation due to residual is formed. .

The method of using a film type anti-adhesion agent is a method of directly attaching a film made of a biocompatible polymer to an injured tissue site, and when the attachment site is changed, the process of detaching and pasting frequently occurs, so it is not easy to be widely used in various surgical methods. There are disadvantages.

When only the biocompatible polymer is used alone as an anti-adhesion agent, there is a problem in that the biocompatible polymer flows off the surface of the damaged tissue due to gravity, and thus the effect is halved. As a method for overcoming the above disadvantages, there is a method of increasing the injection amount of the anti-adhesion solution containing the biocompatible polymer, but the above method has a fear of stress, psychological burden, and abuse of the patient due to overdose. As a method for solving this, anti-adhesion agents of solutions or hydrogel formulations containing biocompatible polymers and having a high viscosity have been studied. The anti-adhesive agent has the advantage of being able to apply evenly to the damaged tissue due to a small amount of shedding due to gravity, and to effectively prevent tissue adhesion with a relatively small amount of use.

However, when using a biocompatible polymer and preparing an anti-adhesion agent in a solution phase with a viscosity, the adhesive strength is excellent, so that there is little dripping due to gravity, and it is necessary to maintain the viscosity that can be infused with a syringe. It should be possible to prevent tissue adhesion until tissue is recovered. In addition, if it contains pharmacological substances that can shorten the healing time of tissues, it is judged that it can effectively prevent tissue adhesion due to surgery.

The patent documents and references mentioned in this specification are incorporated herein by reference to the same extent that each document is individually and clearly specified by reference.

Korean Registered Patent # 1262321

1.Menzies D, Ellis H. Intestinal obstruction from adhesions: how big is the problem. Ann R Coll Surg Engl 1990; 72: 60-63. 2. Ellis H. The clinical significance of adhesions: focus on intestinal obstruction. The European journal of surgery 1997; 577: 59. 3. Diamond MP, Freeman ML. Clinical implications of postsurgical adhesions. Human Reproduction Update 2001; 7: 567-576. 4. Ling Li, Changyang Gong. Biodegradable and injectable in situ cross-linking chitosan-hyaluronic acid based hydrogels for postoperative adhesion prevention. Biomaterials 35 2014; 3903-3917. 5. Bart W. J Hellebrekers, Trudy C. M Trimbos-Kemper, J. Baptist M. Z Trimbos, Jef J Emeis, Teake Kooistra. Use of fibrinolytic agents in the prevention of postoperative adhesion formation. Fertility and Sterility 2000; 74: 203-212. 6.L. Muzii, R. Marana, L. Brunetti, F. Margutti, M. Vacca, S. Mancuso. Postoperative adhesion prevention with low-dose aspirin: effect through the selective inhibition of thromboxane production. Human Reproduction 1998; 13: 1486-1489. 7. Manse Kim, Youngmin Hwang, Giyoong Tae. The enhanced anti-tissue adhesive effect of injectable pluronic-HAhydrogel by poly (γ-glutamic acid). International Journal of Biological Macromolecules 2016; 1603-1611. 8.http: //www.food.gov.uk/safereating/chemsafe/additivesbranch/enumberlist accessed in January, 2009. 9. Hiroyuki Kono, Kenta Onishi, Taichi Nakamura. Characterization and bisphenol A adsorption capacity of β-cyclodextrincarboxymethylcellulose-based hydrogels. Carbohydrate Polymers 2013; 784-792. 10. D. R. Biswal, R. P. Singh. Characterization of carboxymethyl cellulose and polyacrylamide graft copolymer. Carbohydrate Polymers 2004; 379-387. 11. Douglasde Britto, Odilio B.G.Assis. Thermal degradation of carboxymethyl cellulose in different salty forms. Thermochimica Acta 2009; 115-122 12. Bernd H. A. Rehm. Microbial production of biopolymers and polymers precursors. Caister Academic Press. p. 230. 13.Vlahos A, Yu P, Lucas CE, Ledgerwood AM. Effect of a composite membrane of chitosan and poloxamer gel on postoperative adhesive interactions. Am Surg 2001 January; 67 (1): 15-21. 14. Daniela S.Couto, ZhongkuiHong, Joao F.Mano. Development of bioactive and biodegradable chitosan-based injectable systems containing bioactive glass nanoparticles. Acta Biomaterialia 2009 January; 115-123

In order to solve the problems of the composition for preventing adhesion in the present invention, the adhesive strength is improved by using flurane, carboxymethyl cellulose and hyaluronic acid derivatives to effectively form a physical barrier on the surgical site, and the residence time in the body is improved. A physical barrier can be sufficiently maintained for a period of time during which a wound is healed, and a composition for preventing adhesion and a method for manufacturing the same are provided to prevent tissue adhesion by promoting wound healing at a surgical site.

Accordingly, the object of the present invention is 0.2 to 1% by weight of acrylamide (acrylamide)-hyaluronic acid derivatives, 0.2 to 1% by weight of fluran (pullulan), 1 to 5% by weight of carboxymethyl cellulose (carboxymethyl cellulose) and 93 to It is to provide a composition for preventing adhesion and a method for manufacturing the same, comprising 98.6% by weight of distilled water.

Another object of the present invention is 0.4 to 1% by weight of 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether) -hyaluronic acid derivative, 0.2 to 1% by weight of fluran (pullulan), 1 to It is to provide a composition for preventing adhesion and a method for manufacturing the same, comprising 5% by weight of carboxymethyl cellulose and 93 to 98.4% by weight of distilled water.

Other objects and technical features of the present invention are presented in more detail by the following detailed description of the invention, claims and drawings.

The present invention relates to a composition for preventing adhesion, which is excellent in tissue adhesion, body retention time and inflammation prevention effect, and thus has excellent tissue adhesion prevention effect after surgery.

To this end, the present invention is 0.2 to 1% by weight of acrylamide (acrylamide)-hyaluronic acid derivative, 0.2 to 1% by weight of fluran (pullulan), 1 to 5% by weight of carboxymethyl cellulose (carboxymethyl cellulose) and 93 to 98.6 Provided is a composition for preventing adhesion, comprising distilled water by weight.

The anti-adhesion composition comprises: (a) preparing a hyaluronic acid solution containing 40-100 μM sodium hyaluronate and having a pH of 8-10; (b) adding an acryloyl chloride solution to the hyaluronic acid solution in a drop manner to prepare a hyaluronic acid-acryloyl chloride reaction solution having a concentration of acryloyl chloride of 80-200 μM and a pH of 8-10; (c) preparing an acrylamide-hyaluronic acid derivative by reacting the hyaluronic acid-acryloyl chloride reaction solution at 0 ° C; (d) filtering the reaction hyaluronic acid-acryloyl chloride reaction solution after completion of the reaction, dialysis in distilled water and lyophilizing to obtain an acrylamide-hyaluronic acid derivative powder; And (e) 0.2 to 1% by weight of the acrylamide-hyaluronic acid derivative powder, 0.2 to 1% by weight of pullulan, 1 to 5% by weight of carboxymethyl cellulose and 93 to 98.6% by weight. Mixing distilled water at room temperature to prepare a composition for preventing adhesion; prepared by a method for preparing a composition for preventing adhesion, in particular, the flurane and the carboxymethyl cellulose are contained in a weight ratio of 1: 5 and injected through a syringe. While this is possible, it has an optimum adhesion to the surgical site and produces an anti-adhesion composition having an excellent anti-adhesion effect.

In addition, the present invention is 0.4 to 1% by weight of 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether)-hyaluronic acid derivative, 0.2 to 1% by weight of fluran (pullulan), 1 to 5% by weight It provides a composition for preventing adhesion comprising a carboxymethyl cellulose (carboxymethyl cellulose) and 93 to 98.4% by weight of distilled water.

The anti-adhesion composition comprises (f) preparing a hyaluronic acid-sodium hydroxide solution comprising 150-250 μM sodium hyaluronate and 50 mM sodium hydroxide; (g) Hyaluronic acid-sodium hydroxide solution containing 99.5% by weight and 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether, BDDE) 0.5% by weight, pH 7 hyaluronic acid-sodium hydroxide- Preparing a BDDE reaction solution; (h) preparing a BDDE-hyaluronic acid derivative by reacting the hyaluronic acid-sodium hydroxide-BDDE reaction solution at 20 to 50 ° C; (i) filtering the reaction hyaluronic acid-sodium hydroxide-BDDE reaction solution after completion of the reaction, followed by dialysis in PBS buffer solution and distilled water, followed by lyophilization to obtain a BDDE-hyaluronic acid derivative powder; And (j) the BDDE-hyaluronic acid derivative powder 0.4 to 1% by weight, 0.2 to 1% by weight of pullulan, 1 to 5% by weight of carboxymethyl cellulose and 93 to 98.4% by weight of distilled water. Mixing at room temperature to prepare a composition for preventing adhesion; prepared by a method for preparing a composition for preventing adhesion, in particular, the flurane and the carboxymethyl cellulose are contained in a weight ratio of 1: 5 and injected through a syringe. It is possible to produce an adhesion-prevention composition having an excellent adhesion to the surgical site and having an excellent anti-adhesion effect.

The present invention relates to a composition for preventing adhesion and a method for manufacturing the same, comprising a hyaluronic acid derivative, flurane and carboxymethyl cellulose.

The composition for preventing adhesion of the present invention has an improved adhesive force including flurane, thereby resisting gravity and forming a physical barrier on the surgical site, thereby preventing tissue adhesion due to surgery.

In addition, since the composition for preventing adhesion of the present invention has an improved retention effect in the body including carboxymethyl cellulose, the formed physical barrier protects the tissue for a sufficient time during which the surgical site can be healed, thereby preventing tissue adhesion.

In addition, the hyaluronic acid derivative of the present invention has a rapid in vivo decomposition and absorption property, thereby preventing adhesion after surgery and replacing the inferior hyaluronic acid in the wound at the surgical site, thereby improving the duration of hyaluronic acid in the body, thus healing the wound at the surgical site. It is effective to prevent tissue adhesion.

In addition, the composition for preventing adhesion of the present invention uses the content of flurane and carboxymethyl cellulose in 0.2 to 1% by weight and 1 to 5% by weight, respectively, and the mixing ratio of the flurane and carboxymethyl cellulose in a weight ratio of 1: 5. There is an effect that can be easily injected into the surgical site using a syringe while having sufficient adhesion and retention effect in the body.

The anti-adhesion composition of the present invention uses flurane, carboxymethyl cellulose and hyaluronic acid derivatives that are harmless to the human body and shows excellent anti-adhesion effect compared to the conventional anti-adhesion agent, and is applied to the clinic immediately to replace the conventional anti-adhesion agent. It is expected to be.

1 shows the results of BHA hydrogen nuclear magnetic resonance analysis. BDDE and HA polymerized to show a new peak around 2.8ppm.
Panel A) of Figure 2 shows the results of the cytotoxicity test of acrylamide-hyaluronic acid derivative polymer using MDCK renal epithelial cell line, and panel B) is an anti-adhesion agent containing flurane and carboxymethyl cellulose using NIH-3T3 cell. The results of the cytotoxicity (MTT assay) experiment are shown.
3 is acrylamide-after hyaluronic adhesion prevention composition comprising an acid derivative, the Dix -SG ^® and Medina curtain ^® In the surgical field shows the result of comparing the degree of tissue adhesion after 21 days.
Figure 4 is a composition for preventing adhesion comprising acrylamide-hyaluronic acid derivative and fluran, 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether)-for preventing adhesion comprising hyaluronic acid derivative and fluran It shows the result of comparing the degree of tissue adhesion 21 days after the composition, Gadix-SG ^® and Medi curtain ^® were injected into the surgical site.
Figure 5 shows the results of comparing the degree was put into PBS and Medicare ^® curtains for the seven mice in the surgical tissue adhesion.
FIG. 6 shows the results of comparing the degree of tissue adhesion after 7 anti-adhesion compositions containing flurane and carboxymethyl cellulose were injected into the surgical site.
7 shows the degree of adhesion of mice using the composition for preventing adhesion of the present invention.

According to an aspect of the present invention, the present invention is 0.2 to 1% by weight of acrylamide (acrylamide)-hyaluronic acid derivative, 0.2 to 1% by weight of fluran (pullulan), 1 to 5% by weight of carboxymethyl cellulose (carboxymethyl cellulose) and 93 to 98.6% by weight of distilled water.

The acrylamide-hyaluronic acid derivative means a derivative of hyaluronic acid modified with acrylamide by the reaction of acryloyl chloride and hyaluronic acid. The hyaluronic acid is limited to use as a composition for preventing adhesion due to its rapid biodegradable property and high viscosity. The acrylamide (acrylamide)-hyaluronic acid derivative has the advantage of being applicable to a composition for preventing adhesion using a syringe or the like because it has a low viscosity and has a low biodegradation compared to hyaluronic acid.

According to an embodiment of the present invention, the acrylamide (acrylamide)-hyaluronic acid derivative of the present invention is included in 0.2 to 1% by weight. When the acrylamide-hyaluronic acid derivative is used in an amount of less than 0.2% by weight, the effect of preventing adhesion after surgery by hyaluronic acid and the wound healing effect of the surgical site is negligible, and compared to the composition for preventing adhesion used in the above range even when used in excess of 1% by weight Therefore, the degree of improvement in post-operative adhesion prevention and wound healing effect at the surgical site is negligible. Preferably, the acrylamide (acrylamide)-hyaluronic acid derivative is contained in 0.4 to 0.6% by weight, more preferably 0.5% by weight.

The pullulan (pullulan) is a polysaccharide produced in the metabolic process of Aureobasidium pullulans and discharged out of cells.It is a chain form in which three glucose is α-1,4 bound maltotriose is repeatedly bound to α-1,6 bond It is formed of glucan.

Microorganisms discharge waste products generated in the metabolic process of cells out of the cells, and at the same time produce polysaccharides and discharge them out of the cells together with the waste products. The polysaccharides are tacky and prevent drying of microorganisms and defend themselves.

The flurane of Aureobasidium pullulans It is a polysaccharide produced in the metabolic process in the cell and is discharged out of the cell and used for self-defense. The fluran is a natural polymer suitable for living organisms, which is soluble in water and has a property of increasing the adhesion of the solution when dissolved, and can be produced in large quantities through a fermentation process, and thus has a low production cost.

In addition, the fluran has a characteristic of forming a film, and thus is used not only as a main material for a film-type preparation, but also has no toxicity, and thus can be used as a food additive. Conventional Pullulan is formed by chain-like glucan in which three glucoses are α-1,4-linked maltotrioses are α-1,6-linked repeatedly. However, the fluran of the present invention is not formed of a repetitive structure only with maltotriose, but contains about 6% of maltotherose, and the terminal of the molecule is glucose.

Conventional film-type anti-adhesion agents are difficult to handle and have limitations in use depending on the affected area or surgical method. Since the composition for preventing adhesion of the present invention uses flurane, it is liquid and yet viscous, so it is easy to use, so there is almost no restriction on the surgical method or the affected area, and has the advantage of maintaining a sufficient physical barrier during the wound healing time of the surgical site. The fluran has the advantage of increasing the effectiveness of the physical barrier by the anti-adhesion composition in the body because it can overcome the disadvantage that the solution easily flows from the expected site of adhesion by gravity by improving the viscosity of the anti-adhesion composition. When the effectiveness of the physical barrier is increased, it is possible to prevent unnecessary contact between tissues during a time during which the damaged tissue is recovered, and thus has a great effect on preventing adhesion.

According to one embodiment of the invention, the composition for preventing adhesion of the present invention comprises 0.2 to 1% by weight of flurane. When the flurane is used in an amount of less than 0.2% by weight, the viscosity of the composition for preventing adhesion is weak, so the time to maintain the physical barrier at the surgical site is shortened, so the effect of preventing adhesion decreases.If the flurane is always used in excess of 1% by weight The viscosity of the anti-adhesion composition is too high, which may cause problems with injection using a syringe. Preferably, the anti-adhesion composition of the present invention comprises 0.4 to 0.6% by weight of flurane, more preferably 0.5% by weight of flurane.

The carboxymethyl cellulose (Carboxymethyl cellulose) is a cellulose derivative having a structure in which a carboxymethyl group is attached to the hydroxyl group of glucose, and has a water-soluble property unlike insoluble cellulose. The carboxymethyl cellulose has a characteristic that physical and chemical properties are changed according to a substitution rate of a carboxymethyl group per glucose, and thus the rate of absorption and decomposition in the body may be slowed down by controlling the substitution rate. In addition, the carboxymethyl cellulose is an eco-friendly biocompatible material that is approved as a food additive by the European Food Standards Agency, and has the advantage of being a safe material that is widely used in the food industry.

According to one embodiment of the invention, the composition for preventing adhesion of the present invention comprises 1 to 5% by weight of carboxymethyl cellulose. When the carboxymethyl cellulose is used in an amount of less than 1% by weight, the absorption and decomposition rate of the anti-adhesion composition is fast, so the time to maintain the physical barrier at the surgical site is shortened, so the effect of preventing adhesion decreases and the carboxymethyl cellulose is always 5% by weight. If it is used in excess of%, the absorption and decomposition rate of the body for preventing adhesions is excessively lowered, so that the patient stays in the surgical site for a long time in the recovery process, which may give the patient a feeling of rejection. Preferably, the composition for preventing adhesion of the present invention comprises 2 to 3% by weight of carboxymethyl cellulose, and more preferably 2.5% by weight of carboxymethyl cellulose.

According to another aspect of the present invention, the present invention is 0.4 to 1% by weight of 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether) -hyaluronic acid derivative, 0.2 to 1% by weight of furan ( It provides a composition for preventing adhesion, including pullulan), 1 to 5% by weight of carboxymethyl cellulose, and 93 to 98.4% by weight of distilled water.

The 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether) -hyaluronic acid derivative is 1,4-butanediol diglycidyl ether by reaction of 1,4-butanediol diglycidyl ether and hyaluronic acid, 1,4-butanediol diglycidyl ether It refers to a derivative of hyaluronic acid modified by diether. The 1,4-butanediol diglycidyl ether-hyaluronic acid derivative has an advantage that it can be used as an anti-adhesion agent by solving the disadvantages of hyaluronic acid like the acrylamide-hyaluronic acid derivative.

According to an embodiment of the present invention, 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether) -hyaluronic acid derivative of the present invention is contained in 0.2 to 1% by weight. If the 1,4-butanediol diglycidyl ether-hyaluronic acid derivative is used in an amount of less than 0.2% by weight, the effect of preventing adhesion after surgery with hyaluronic acid and wound healing at the surgical site is negligible. And even if it is used in excess of 1% by weight, the degree of improvement in post-operative adhesion prevention and wound healing effect at the surgical site is negligible compared to the anti-adhesion composition used in the above range. Preferably, the 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether) -hyaluronic acid derivative is contained in 0.4 to 0.6% by weight, more preferably 0.5% by weight.

The flurane and carboxymethyl cellulose have been described in one aspect, and thus will not be described in order to avoid duplication of the present specification.

According to one embodiment of the present invention, the composition for preventing adhesion described in one aspect and another aspect of the present invention is injected with a syringe or the like on the surgical wound site to prevent adhesion formation or to form only a thin film-like adhesion, resulting in adhesion between tissues It has the effect of preventing the occurrence of.

According to one embodiment of the present invention, the anti-adhesion composition of the present invention improves the viscosity and improves the retention time in the surgical site, and reduces the absorption rate and decomposition rate in the body, thereby improving the retention time in the surgical site. Cellulose is used in a weight ratio of 1: 5. If the weight ratio is exceeded, the viscosity of the anti-adhesion composition and the degree of absorption in the body may not be appropriate, so the anti-adhesion effect may be reduced or a feeling of rejection after surgery may occur.

In summary, the composition for preventing adhesion of the present invention replaces hyaluronic acid which is not easy to use as an adhesion preventing agent due to its rapid biodegradation properties and high viscosity, and the hyaluronic acid derivative (acrylamide) -hyaluronic acid derivative and Uses 1,4-butanediol diglycidyl ether (hyaluronic acid derivative), improves viscosity, increases absorption and decomposition time in the body, and has no toxicity, but is safe from flurane and carboxymethyl cellulose Since it uses a syringe, it has the advantage of being able to form a physical barrier for a sufficient amount of time in the surgical site, while having a liquid phase to the extent that it is possible to inject the surgical site using a syringe.

According to another aspect of the present invention, the present invention provides a preparation method comprising the following steps for an anti-adhesion composition comprising an acrylamide-hyaluronic acid derivative, pullulan, and carboxymethyl cellulose:

(a) preparing a hyaluronic acid solution containing 40-100 μM sodium hyaluronate and having a pH of 8-10;

(b) adding an acryloyl chloride solution to the hyaluronic acid solution in a drop manner to prepare a hyaluronic acid-acryloyl chloride reaction solution having a concentration of acryloyl chloride of 80-200 μM and a pH of 8-10;

(c) preparing an acrylamide-hyaluronic acid derivative by reacting the hyaluronic acid-acryloyl chloride reaction solution at 0 ° C;

(d) filtering the reaction hyaluronic acid-acryloyl chloride reaction solution after completion of the reaction, dialysis in distilled water and lyophilizing to obtain an acrylamide-hyaluronic acid derivative powder; And

(e) 0.2 to 1% by weight of the acrylamide-hyaluronic acid derivative powder, 0.2 to 1% by weight of pullulan, 1 to 5% by weight of carboxymethyl cellulose and 93 to 98.6% by weight of distilled water Mixing at room temperature to prepare a composition for preventing adhesion.

According to an embodiment of the present invention, the flurane and the carboxymethyl cellulose are included in a weight ratio of 1: 5.

According to another aspect of the invention the present invention is 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether)-hyaluronic acid derivatives, flulan (pullulan), and carboxymethyl cellulose (carboxymethyl cellulose) It provides a manufacturing method comprising the following steps for the composition for preventing adhesion:

(f) preparing a hyaluronic acid-sodium hydroxide solution comprising 150-250 μM sodium hyaluronate and 50 mM sodium hydroxide;

(g) Hyaluronic acid-sodium hydroxide solution containing 99.5% by weight and 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether, BDDE) 0.5% by weight, pH 7 hyaluronic acid-sodium hydroxide- Preparing a BDDE reaction solution;

(h) preparing a BDDE-hyaluronic acid derivative by reacting the hyaluronic acid-sodium hydroxide-BDDE reaction solution at 20 to 50 ° C;

(i) filtering the reaction hyaluronic acid-sodium hydroxide-BDDE reaction solution after completion of the reaction, followed by dialysis in PBS buffer solution and distilled water, followed by lyophilization to obtain a BDDE-hyaluronic acid derivative powder; And

(j) The BDDE-hyaluronic acid derivative powder 0.4 to 1% by weight, 0.2 to 1% by weight of pullulan, 1 to 5% by weight of carboxymethyl cellulose and 93 to 98.4% by weight of distilled water Mixing at room temperature to prepare a composition for preventing adhesion.

According to an embodiment of the present invention, the flurane and the carboxymethyl cellulose are included in a weight ratio of 1: 5.

The present invention will be described in detail through the following examples.

Example

Experiment method

1) Preparation of acrylamide modified haluronic acid derivative (AHA)

Acrylamide modified haluronic acid derivatives (AHA) were prepared as follows. First, 0.5 g of sodium hyaluronate (MW: 800 kDa) was dissolved in 100 ml of distilled water at 4 ° C. to prepare a hyaluronic acid solution. 2M sodium hydroxide (NaOH) was added to the hyaluronic acid solution to adjust the pH to 9 and mixed for 30 minutes at room temperature. To the mixed hyaluronic acid solution, 1 ml of 12 mM acryloyl chloride was added dropwise and adjusted to pH 9 using 2M sodium hydroxide. The reactions were carried out at 0 ° C. The reaction solution was sterilized by filtration, dialyzed using distilled water at room temperature, and freeze-dried at -70 ° C for 3 days to prepare AHA in powder form and stored at 4 ° C.

Using AVANCE 400MHz FT-NMR, it was confirmed whether acryloyl chloride and hyaluronate (HA) were well bonded. To this end, after dissolving AHA in D ₂ O at a concentration of 10 mg / ml, a hydrogen peak was confirmed (see FIG. 1).

2) 1,4- Butanediol diglycidyl ether  Modified hyaluronic acid derivatives (1,4- Butanediol Diglycidyl  Preparation of Ether modified hyaluronic acid derivative (BHA)

1,4-Butanediol diglycidyl ether modified hyaluronic acid derivative (1,4-Butanediol Diglycidyl Ether modified hyaluronic acid derivative, BHA) was prepared as follows.

First, 0.5 g of sodium hyaluronate (MW: 800 kDa) was dissolved in 30 ml of 0.05 M sodium hydroxide solution at room temperature to prepare a hyaluronic acid-sodium hydroxide solution. To the hyaluronic acid-sodium hydroxide solution, 0.5% by weight of 1,4-butanediol diglycidyl ether (1,4-Butanediol Diglycidyl Ether, BDDE) was added and mixed using a homogenizer at room temperature for 20 minutes to produce hyaluronic acid-hydroxylation. A sodium-BDDE solution was prepared. 1N hydrochloric acid was added dropwise to the hyaluronic acid-sodium hydroxide-BDDE solution to adjust the pH to 7. The hyaluronic acid-sodium hydroxide-BDDE solution adjusted to pH 7 was dialyzed in PBS buffer solution and distilled water for 3 days and 2 days, respectively, and freeze-dried at -70 ° C for 3 days to prepare a powdered BHA and stored at 4 ° C. .

Using AVANCE 400MHz FT-NMR, it was confirmed whether BDDE and hyaluronate (HA) were well bonded. To this end, after dissolving BHA in D ₂ O at a concentration of 10 mg / ml, a hydrogen peak was confirmed (see FIG. 1).

3) Preparation of anti-adhesion agents

First, in order to prepare an anti-adhesion agent comprising AHA or BHA, a pluronic-F127 (PF) polymer and a pullulan (Plu) were used. The adhesion inhibitor was classified based on the content of AHA or BHA, and the composition ratio (weight ratio) for production is shown in Table 1.

Sample name AHA and BHA content (% by weight) PF or Plu content (% by weight) Distilled water content (% by weight) AHA / PF0.25 AHA 0.25 wt% PF 20 wt% 79.75% by weight of distilled water AHA / PF0.5 AHA 0.5 wt% PF 20 wt% Distilled water 79.5 wt% BHA / PF0.25 BHA 0.25 wt% PF 20 wt% 79.75% by weight of distilled water BHA / PF0.5 0.5% by weight of BHA PF 20 wt% Distilled water 79.5 wt% AHA / Plu0.25 AHA 0.25 wt% Plu 20 wt% 79.75% by weight of distilled water AHA / Plu0.5 AHA 0.5 wt% Plu 20 wt% Distilled water 79.5 wt% BHA / Plu0.25 BHA 0.25 wt% Plu 20 wt% 79.75% by weight of distilled water BHA / Plu0.5 0.5% by weight of BHA Plu 20 wt% Distilled water 79.5 wt%

In order to prepare the Plu-CMC anti-adhesion agent, NaCl and sodium phosphate dibasic were added and stirred in double distillation water to prepare a biocompatible solvent. Plu was added to the prepared solvent to a concentration of 0.5% w / v, and the mixture was sufficiently stirred with a stirrer. When the Plu was completely dissolved, CMC was added to a concentration of 2.25 w / v% and 2.5 w / v%, respectively, and stirred sufficiently with a stirrer. If the two substances were sufficiently dissolved, impurities and microorganisms were removed through a filter and then stored in a refrigerator.

AHA powder or BHA powder was added to the prepared Plu-CMC anti-adhesive agent by adding 0.25% by weight or 0.5% by weight, followed by sufficient stirring.

4) Hazard evaluation of adhesion inhibitor

BHA is FDA-approved and is known to have little toxicity. Therefore, in vitro toxicity was evaluated for the AHA of the present invention and the prepared anti-adhesion agents. For the in vitro toxicity test, the MTT (3- (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide) assay was tested using NIH-3T3 cell (Korean Cell Line Bank, Korea). Through cytotoxicity was confirmed. MDCK cells were cultured under conditions of 37 ° C and 5% CO2 using DMEM medium containing 10% fetal bovine serum and 1% penicillin streptomycin. Medium was changed twice a week. Reagents required for cytotoxicity test (MTT assay) are DMEM (Corning, USA), bovine calf serum (Welgene, Korea), Penicillin-Streptomycin (Gibco, USA), Dulbecco's phosphate buffered saline (Gibco, USA), 3- ( 4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (Sigma-Aldrich, USA) and DMSO (Amresco, USA) were used. The concentrations of the prepared AHA or anti-adhesion solutions were 0.1, 0.25, 0.5, and 1 (unit: mg / mL), respectively, and used for cytotoxicity experiments. NIH-3T3 cells were seeded in 96 well plates at a concentration of 5 x 10 ⁴ . The prepared sample was added and cultured in an incubator for 24 hours. After incubation for 24 hours, 20 μL of MTT reagent prepared at 5 mg / ml was added, followed by incubation for 4 hours. After 4 hours, all the medium was slowly removed, and 100 μL of DMSO was added and reacted with a plate shaker for 30 minutes at room temperature with light blocked. After 30 minutes, the absorbance was measured at a wavelength of 590 nm using a Thermo Varioskan Flash Spectral Scanning Multimode Reader (ThermoFisher, USA).

5) Evaluation of adhesion prevention properties of anti-adhesion agents

Female Sprague-Dawley rats aged 4-7 weeks were randomly divided into groups (n = 6 or 7), and were regularly fed with water and food in a tree-free metal cage. The SD rats were intraperitoneally injected with 87 mg / kg ketamine and 13 mg / kg xylazine or anesthetized with ether. The hair of the abdomen of the anesthetized SD rat was removed and sterilized with alcohol. The abdominal wall of the SD rat was incised to a length of 3-4 cm and the peritoneum was incised about 4 cm to confirm the location of the appendix. A tissue adhesion model was made by performing a small midline incision of 0.5 cm x 0.5 cm in size near the ileocecal junction, or by wearing a 1.5 cm ² peritoneum in contact with the appendix with gauze to cause bleeding. The negative control SD rats were treated with 3 ml of PBS on the injured peritoneum and appendix and the peritoneum and skin were then sutured in sequence. Positive control SD rats have included as a main component an adhesion preventing agent, HA and hydroxyl ethyl starch (hydroxyethylstarch) Medina curtain ^® (Medicurtain ^ⓡ) or poloxamers (poloxamer) and sodium alginate (sodium alginate) comprising as a main component composition of the present invention Hadgardix-SG ^® (Guardix-SG ^ⓡ ) was injected. All the SD mice were sutured with abdominal incision and then supplied with appropriate food and water. After 2-3 weeks, the SD rats were sacrificed by cervical dislocation to check the degree of adhesion. The degree of adhesion was evaluated according to Blauer's 4 point scoring system and Vlahos A method (V. angie et al., The American Surgeon, 2001, 67, 15-21.). . The criteria for the scoring system are given in Table 2 below.

Score Adhesion degree Cohesion strength 0 No adhesion No adhesion One One thin film type adhesion Adhesion can be removed with very light force 2 Two or more film type adhesion Requires moderate force to remove adhesions 3 Thick, concentrated adhesions on the spot Significant force required to remove adhesions 4 Centralized thick adhesion on the plate Incision of the adhesion site is necessary to remove the adhesion 5 Very thick adhesions with blood vessels -

2. Experimental results

1) with AHA BHA  Manufacturing result

The synthesized BDDE modified HA polymer (BHA) and acrylamide modified HA polymer (AHA) are synthesized and confirmed by hydrogen nuclear magnetic resonance spectroscopy ( ¹ H-NMR). Did. ^{According to the 1} H-NMR result, BDDE and HA were polymerized, and a new peak was observed around 2.8 ppm from the methyl group (see FIG. 1). In addition, as the acrylamide and HA polymerized, a hydrogen peak of a vinyl group was observed around 5.8-6.2 ppm (see FIG. 1). Through this, it was confirmed that BDDE and HA or acrylamide and HA were successfully polymerized.

2) In vitro cytotoxicity evaluation result

BHA is FDA approved. Therefore, in the present invention, only cytotoxicity to the AHA polymer was evaluated. Panel A) of Figure 2 shows the results of the cytotoxicity test of the AHA polymer using MDCK renal epithelial cell line. As a result of increasing the concentration of AHA to 1 mg / ml in the MDCK renal epithelial cell line medium, cell growth was not inhibited by AHA. Therefore, it was confirmed through this result that the AHA of the present invention has no cytotoxicity. Cytotoxicity was confirmed for the anti-adhesion agent prepared above (see panel A in FIG. 2). Cytotoxicity experiments were confirmed through MTT assay analysis (see panel B in FIG. 2). As a result of the cytotoxicity test, it was confirmed that there was no significant difference between the control group not treated with the sample and the experimental group treated with the sample. It was confirmed that the cell viability was 85% or more using all concentration samples, and the cell viability was 90% or more in most of the samples. Through this, the anti-adhesion solution prepared was confirmed to have little cytotoxicity and was confirmed to be a biocompatible material applicable to the body.

3) Evaluation results of adhesion prevention properties of adhesion prevention agents

First, the effect of preventing adhesion by AHA and BHA was confirmed. Adhesion agent containing AHA or BHA To this end, the curtain Medi ^®, the Dix -SG ^® or PBS and then injected into the incision of the ileocecal valve of the SD rat was placed again all the tissues and organs in the peritoneum. The incision was closed and food and water were normally supplied. After 21 days, the degree of adhesion according to the use of AHA or anti-adhesion agent containing BHA was evaluated according to Blower's 4-point scoring system. Type approved by the FDA as a positive control were injected using a Medina curtain ^® and the Dix -SG ^® analyzed using PBS as a negative control.

First, in the negative control group using only PBS, solid adhesion was observed in the peritoneal tissue (see FIG. 3). Positive control with Medi curtain ^® is another positive control showed that the adhesion step 1 with a Dix -SG ^® showed a solid step 3 adhesion.

In contrast, the experimental group treated with AHA / PF 0.25 and AHA / PF 0.5, which are anti-adhesion agents containing AHA, showed negligible minimal tissue adhesion (see FIG. 3) and treated with AHA / Plu 0.25 and AHA / Plu 0.5. The experimental group also showed negligible levels of minimal tissue adhesion (see FIG. 4). In particular, as the content of AHA increased from 0.25% by weight to 0.5% by weight, the degree of adhesion tended to decrease, which is thought to improve the wound healing effect as the concentration of HA increased and decrease adhesion. Therefore, the anti-adhesion agent containing AHA of the present invention acts as a physical barrier between tissues after being injected into a sol (liquid) state at room temperature (25 ° C), and in particular, the degree of wound healing is improved by HA, thereby preventing adhesion of tissues and organs. It is judged to have an effect. The above results are also confirmed in the anti-adhesion effect by the anti-adhesion agent containing BHA (BDDE-HA). According to the results of FIGS. 3 and 4, BHA / PF 0.5 and BHA / Plu (BDDE-HA / Plu) 0.5 showed effective anti-adhesion properties, but BHA / PF 0.25 and BHA / Plu (BDDE-HA / Plu) 0.25 showed that Dix-SG ^® level showed a strong adhesion. It is interpreted that the anti-adhesion effect of the anti-adhesion agent was reduced because the amount of HA was reduced.

The effect of preventing adhesion by AHA and BHA described above is summarized in Table 3 below.

Sample name Hyaluronic acid or hyaluronic acid derivative content Score Positive control - Negative control (PBS) - 4 Medicurtain ^® HA 1.25 AHA / PF0.25 AHA 0.25 wt% 0.25 AHA / PF0.5 AHA 0.5 wt% 0 BHA / PF0.25 BHA 0.25 wt% 3.75 BHA / PF0.5 0.5% by weight of BHA 0.25 AHA / Plu0.25 AHA 0.25 wt% 0.25 AHA / Plu0.5 AHA 0.5 wt% 0 BHA / Plu0.25 BHA 0.25 wt% 3.25 BHA / Plu0.5 0.5% by weight of BHA 0.75

In order to confirm the anti-adhesion effect according to the ratio of Plu and CMC, the adhesion degree was evaluated after inserting the organic anti-oxidant into the incision using the above method using 7 SD mice for each sample. As a result of the evaluation, it was confirmed that in the SD mice treated with PBS with damage, the degree of adhesion was so severe that the organs adhered to each other (see panel A in FIG. 5). Organs of SD rats treated with PBS were difficult to remove adhesions. Commercially available Medicurton had less adhesion than PBS-treated mice, but adhesion was also confirmed to some extent (see panel B in FIG. 5).

On the other hand, in the case of SD mice (n = 7) using the adhesion inhibitor of the present invention containing Plu and CMC, adhesion was hardly found (see FIG. 6). For the anti-adhesion agent containing only CMC, the adhesion degree score was evaluated to be 0.5 to 0.75, and the adhesion strength score was evaluated to be 0.75 to 1 (see panels A) and B in FIG. 6). . In addition, in the case of an anti-adhesion agent containing both Plu and CMC, the adhesion degree score was evaluated to be 0.25 to 0.5, and the adhesion strength score was evaluated to be 0.26 to 0.55 (Panel C in FIG. 6) and D) See). In addition, the adhesion degree of the adhesion ratio of Plu: CMC of 1: 5 (0.5: 2.5) is 0.25 compared to the adhesion of Plu: CMC of 1: 4.5 (0.5: 2.25). The value was evaluated to be lower, and the adhesion strength score was evaluated to be lower than the value of 0.3. The results show that when Plu and CMC are used at the same time, the adhesion prevention effect is improved rather than using only CMC, and when the mixing ratio of Plu and CMC is 1: 5, it is confirmed that it shows the highest adhesion prevention effect.

3. Conclusion

The advantages of the adhesion preventing agent of the present invention are summarized as follows.

1) The anti-adhesion agent of the present invention contains a hyaluronic acid (HA) derivative, and thus has an effect of promoting wound healing in the surgical site, and has a rapid in vivo decomposition and absorption property, thereby preventing hyaluronic acid from having an anti-adhesion and wound healing effect. Acrylamide-hyaluronic acid derivative (AHA) or BDDE-hyaluronic acid derivative (BHA) is replaced to improve the duration in the body, so hyaluronic acid has an advantage of improved adhesion prevention and wound healing after surgery.

2) The flulan (plullan, Plu) and carboxymethyl cellulose (CMC) of the present invention can improve the viscosity of the anti-adhesion agent to effectively form a physical barrier (film) on the surgical site and the residence time of the anti-adhesion agent in the body It has the effect of preventing adhesion of the surgical site for a sufficient time until the wound on the surgical site is healed by improving.

3) When the Plu and CMC were mixed at a composition ratio of 1: 5, the adhesion strength by Plu and the retention time in the body by CMC were most effective, so the adhesion prevention effect of the surgical site was confirmed to be the best, and Plu and CMC were 0.5 each. It was confirmed that the use of weight% and 2.5% by weight shows an excellent anti-adhesion effect while having an appropriate viscosity that the anti-adhesion agent can be injected through a syringe.

4) Therefore, the composition of 0.5% by weight of acrylamide-hyaluronic acid derivative (AHA), 0.5% by weight of flulan, 2.5% by weight of carboxymethyl cellulose and 96.5% by weight of distilled water prevents adhesion. When the composition is prepared, the wound healing effect by hyaluronic acid is improved, the tissue adhesion force by flurane is improved, and the residence time in the body by carboxymethyl cellulose is improved, so that an anti-adhesion agent that can effectively prevent the swelling of the surgical site can be prepared. have.

5) In addition, 0.5% by weight of 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether) -hyaluronic acid derivative (BHA), 0.5% by weight of fluran (pullulan), 2.5% by weight of carboxy When a composition for preventing adhesion with a composition of methyl cellulose (carboxymethyl cellulose) and 96.5% by weight of distilled water is prepared, the wound healing effect by hyaluronic acid is improved, tissue adhesion by flurane is improved, and residence time in the body by carboxymethyl cellulose is improved. Becomes an anti-adhesion agent that can effectively prevent adhesion of the surgical site.

7) Since the composition for preventing adhesion of the present invention includes AHA or BHA, flurane and carboxymethyl cellulose, which are harmless to the human body, it is expected to be applied to the clinic immediately to solve the limitations of the conventional adhesion preventing agent.

The specific embodiments described herein are meant to represent preferred embodiments or examples of the present invention, and the scope of the present invention is not limited thereby. It is apparent to those skilled in the art that modifications and other uses of the present invention do not depart from the scope of the invention described in the claims of this specification.

Claims (8)

0.2 to 1% by weight of acrylamide (acrylamide)-hyaluronic acid derivative, 0.2 to 1% by weight of fluran (pullulan), 1 to 5% by weight of carboxymethyl cellulose (carboxymethyl cellulose) and 93 to 98.6% by weight of distilled water The composition for preventing adhesion, including the acrylamide (acrylamide) -hyaluronic acid derivative, flurane and the carboxymethyl cellulose in a weight ratio of 1: 1.
0.4 to 1% by weight of 1,4-butanediol diglycidyl ether-hyaluronic acid derivative, 0.2 to 1% by weight of flululan, 1 to 5% by weight of carboxymethyl Cellulose (carboxymethyl cellulose) and 93 to 98.4% by weight of distilled water, the 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether)-hyaluronic acid derivatives, flurane and the carboxymethyl cellulose 1 : 1: A composition for preventing adhesions contained in a weight ratio of 5.
The composition for preventing adhesion according to any one of claims 1 to 2, wherein the composition for preventing adhesion is injected into a surgical wound to prevent adhesion formation or form only a thin film-like adhesion.
delete (a) preparing a hyaluronic acid solution containing 40-100 μM sodium hyaluronate and having a pH of 8-10;
(b) adding an acryloyl chloride solution to the hyaluronic acid solution in a drop manner to prepare a hyaluronic acid-acryloyl chloride reaction solution having a concentration of acryloyl chloride of 80-200 μM and a pH of 8-10;
(c) preparing an acrylamide-hyaluronic acid derivative by reacting the hyaluronic acid-acryloyl chloride reaction solution at 0 ° C;
(d) filtering the reaction hyaluronic acid-acryloyl chloride reaction solution after completion of the reaction, dialysis in distilled water and lyophilizing to obtain an acrylamide-hyaluronic acid derivative powder; And
(e) 0.2 to 1% by weight of the acrylamide-hyaluronic acid derivative powder, 0.2 to 1% by weight of pullulan, 1 to 5% by weight of carboxymethyl cellulose and 93 to 98.6% by weight of distilled water Mixing at room temperature to prepare a composition for preventing adhesion, wherein the acrylamide-hyaluronic acid derivative, flurane and the carboxymethyl cellulose are included in a weight ratio of 1: 1 to 5;
Method for producing a composition for preventing adhesion comprising a.
delete (f) preparing a hyaluronic acid-sodium hydroxide solution comprising 150-250 μM sodium hyaluronate and 50 mM sodium hydroxide;
(g) Hyaluronic acid-sodium hydroxide solution containing 99.5% by weight and 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether, BDDE) 0.5% by weight, pH 7 hyaluronic acid-sodium hydroxide- Preparing a BDDE reaction solution;
(h) preparing a BDDE-hyaluronic acid derivative by reacting the hyaluronic acid-sodium hydroxide-BDDE reaction solution at 20 to 50 ° C;
(i) filtering the reaction hyaluronic acid-sodium hydroxide-BDDE reaction solution after completion of the reaction, followed by dialysis in PBS buffer solution and distilled water, followed by lyophilization to obtain a BDDE-hyaluronic acid derivative powder; And
(j) The BDDE-hyaluronic acid derivative powder 0.4 to 1% by weight, 0.2 to 1% by weight of pullulan, 1 to 5% by weight of carboxymethyl cellulose and 93 to 98.4% by weight of distilled water Mixing at room temperature to prepare a composition for preventing adhesion, the 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether) -hyaluronic acid derivative, flurane and the carboxymethyl cellulose are in a weight ratio of 1: 1 to 5 Included with;
Method for producing a composition for preventing adhesion comprising a.
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