KR20180059308A - Anti-adhesion composition comprising hyaluronic aicd and poly-gamma-glutamate acid - Google Patents

Abstract

The present invention relates to an anti-adhesion composition comprising a hyaluronidase inhibitor and hyaluronic acid. The composition is characterized by being a liquid, and the hyaluronic acid and the hyaluronidase inhibitor are included in a weight ratio of 1: 0.005 to 2.550, or in a weight ratio of 1: 0.01 to 2.0. The present invention provides an anti-adhesion composition with a long in vivo duration time.

Description

HYALURONIC AICD AND POLY-GAMMA-GLUTAMATE ACID < / RTI > which comprises hyaluronic acid and polygamargumatic acid.

The present invention is directed to an anti-adhesion composition comprising hyaluronic acid and polygamma glutamic acid.

Adhesion refers to the formation of fibrous tissue during wound healing, such as inflammation, wound healing, friction, and wound healing, or the surrounding organs or tissues to be separated from each other It is a phenomenon that sticks.

Adhesion can occur after all types of surgery, which can lead to serious clinical sequelae.

One of the methods for preventing such adhesion is to use a barrier to form a physical barrier similar to a surfactant during the healing of tissue wounds and to prevent the formation of adhesion between adjacent tissues. Research is actively under way.

The anti-adhesion material used for the barrier may be divided into two types, one of which is a membrane type barrier including a film, a nonwoven fabric, and a sponge type, and the other is a solution type barrier containing a gel type 10-1624511).

Film-like adhesion preventive materials include oxidized regenerated cellulose, Expanded polyterafluoroethylene (hereinafter abbreviated as ePTFE), modified hyaluronic acid, sodium carboxymethylcellulose, and chemical crosslinking agents.

Examples of the solution type adhesion preventive material include a lactate ringer solution, a dextran-70 solution, a heparin solution, a carboxymethyl cellulose solution, a hyaluronic acid solution, a chondroitin sulfate solution, a polyethylene glycol, Solution, dextran-70 solution, heparin solution and the like. These were the main mechanisms by which the fiber-covered surfaces were floated to each other during the peritoneal healing process, which were used to separate the tissues from each other and to inhibit adhesion.

However, such a nucleus-type adhesion preventive material suffers from the fact that absorption in the abdominal cavity is excessively rapid, and it is not long-lasting in vivo, thereby making it difficult to effectively prevent adhesion.

It is an object of the present invention to provide an anti-adhesion composition having a long duration in vivo.

In order to achieve the above object, the present invention provides a hyaluronidase inhibitor and a composition for preventing adhesion comprising hyaluronic acid.

The anti-adhesion composition of the present invention has an effect of enhancing the persistence time in vivo and prolonging the anti-adhesion effect.

Fig. 1 shows the effect of preventing adhesion of Example 1 and Comparative Groups 1 and 2.
FIG. 2 is a photograph showing the naked eye observation of adhesion of tissues on the 14th day after application of the Example 1 and Comparative Groups 1 and 2 to the mouse ((a): Control group, (b): Example 1, Group 1).
Fig. 3 shows the in vivo residual amount of polygamma glutamic acid.
FIG. 4 shows the difference in the survival period of hyaluronic acid in vivo according to the ratio of hyaluronic acid and polygamma glutamic acid.

The present invention

A hyaluronidase inhibitor, and hyaluronic acid.

Also,

A hyaluronidase inhibitor, and a hyaluronic acid, which comprises the step of mixing a hyaluronidase inhibitor and hyaluronic acid.

Hereinafter, the present invention will be described in detail.

Hyaluronidase inhibitor

The hyaluronidase inhibitor of the present invention has an effect of inhibiting hyaluronic acid degradation by hyaluronidase. As a result, the hyaluronidase inhibitor of the present invention increases the duration of the hyaluronic acid in vivo by inhibiting the decomposition and release of the anti-adhesion composition applied to the wound, thereby improving the duration of the antiadherent composition.

The hyaluronidase inhibitor is preferably polygamma glutamic acid (γ-PGA). The polygamma glutamic acid provides high viscoelasticity and adhesive strength to effectively enhance the persistence of the anti-adhesion material in post-surgical tissue.

Hyaluronic acid

The hyaluronic acid of the present invention is not particularly limited as long as it is hyaluronic acid generally used in the composition for preventing adhesion.

Composition for preventing adhesion

The present invention relates to a hyaluronidase inhibitor and a composition for preventing adhesion comprising hyaluronic acid. The hyaluronic acid and the hyaluronidase inhibitor are contained in a weight ratio of 1: 0.005 to 2.550, and preferably the hyaluronic acid and the hyaluronidase inhibitor are contained in a weight ratio of 1: 0.01 to 2.0. By maintaining the above range, the duration of the hyaluronic acid used in the anti-adhesion composition is increased in vivo, and the adhesion efficiency is enhanced.

The composition for preventing adhesion of the present invention causes hyaluronic acid to remain in the body for 10 days or more when applied to a wound. Thereby enhancing the duration of the adhesion prevention of the present invention.

The composition for preventing adhesion of the present invention is a liquid. The liquid phase refers to a material having fluidity excluding a solid phase, and includes a gel phase.

The anti-adhesion composition of the present invention may further comprise an anti-inflammatory agent. Wherein the anti-inflammatory agent is at least one selected from the group consisting of dipyrinic acid, lorazolec, lanoxicam, loxoprofen, meclofenanic acid, mefenamic acid, meloxicam, monofluomate, benorylate, bromelain, salicylic acid imidazole, A prodrug, a prodrug, a prodrug, a prodrug, a prodrug, a prodrug, a prodrug, a prodrug, a prodrug, a prodrug, a prodrug, But are not limited to, ibuprofen, ibuproxam, indomethacin, zetofrofen, ketorolac, ketoprofen, nabumetone, naproxen, nimesulide, dexamethasone, dexibupropene, diclofenac, pyrazinobutarone, clonixin, , Tenoxycam, tolmetin sodium, tolpennamic acid, thiopropenoic acid, fenoprofen, fentiazac, felubiprofen, pranoprofen, piroxycam, progestogen, fluvifrophen, flufenamic acid , Glutamate, and proglumethasin It may be any one selected from the group.

The anti-adhesion composition of the present invention may further comprise an anti-blood coagulant. The anti-coagulant may be any one selected from the group consisting of enoxaparin, reefuridine, heparin, low molecular weight heparin, anchor rod, warfarin, and dinapharoid.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. It should be understood, however, that the invention is not limited to the disclosed embodiments, but is capable of many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, To fully disclose the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

≪ Materials and methods >

A mouse (Balb / C) was used as an experimental animal. These mice were supplied from Orient Bio Co., Ltd., and the temperature was 20 ± 3 ° C, the relative humidity was 40-70%, the ventilation frequency was 13-18 / hr, the illumination time was 12 hours (7:00 am to 7:00 pm) As a condition of 300 Lux, commercially available feed and drinking water were fed and kept isolated during the purification and test period.

Quantitative analysis of the composition of Example 1 in which the mouse was administered in vivo was carried out by attaching Gd to polygamma glutamic acid in the composition of Example 2 and measuring the concentration of Gd by ICP-MS (Inductivity Coupled Plasma Mass Spectrometer, X-series, Thermo Elemental, Research Institute of Environmental Sciences).

Polygamma glutamic acid was purchased from Bio Reader and Humidix, and hyaluronic acid was purchased from Shinpung Pharmaceutical and LG Life Science.

≪ Example 1 >

Bz-DTPA was dissolved in chloroform so as to have a concentration of 200 mg / ml, 450 쨉 l of aliquots were taken, and 1 ml of TFA was added thereto, followed by reaction at 4 째 C for 24 hours. After the reaction, the TFA was removed and 10 ml of 1M sodium bicarbonate was added. After freezing at -80 ° C for 6 hours, the particles were frozen at -80 to -85 ° C and dried for more than 48 hours at a pressure of 5 Pa. Respectively. The dried material was prepared at a concentration of 100 mg / ml, and 370 μl of a 100 mg / ml hyaluronic acid solution was added to 100 mM sodium acetate, mixed with the prepared solution, reacted for about 30 minutes, and then dried. 10 ml of a solution having a concentration of polygamma glutamic acid of 10 mg / ml was subjected to EDC / NHS reaction, and 10 ml of a hyaluronic acid solution was added to prepare a mixed solution of the present invention.

≪ Example 2 >

Bz-DTPA was dissolved in chloroform to a concentration of 200 mg / ml, 450 μl aliquotted, and 1 ml of TFA was added and reacted at 4 ° C for 24 hours. After the reaction, TFA was removed and 10 ml of 1 M sodium bicarbonate was added. After dialysis in PBS buffer solution, the particles were frozen at -80 ° C for 6 hours using a cryocooler, and maintained at -80 to -85 ° C and pressure of 5 Pa for 48 hours or more And dried. The dried material was prepared to have a concentration of 100 mg / ml, and 370 μl of a solution of 100 mg / ml of sodium gadolinium chloride dissolved in 100 mM sodium acetate was added to the prepared solution, followed by reaction for about 30 minutes. After the reaction, dialysis and drying were performed. Bz-DTPA-Gd was synthesized by adding 110 mg of dried Bz-DTPA-Gd to 10 ml of a solution having a concentration of polygamma glutamic acid of 10 mg / ml, followed by EDC / NHS reaction. The synthesized material was filtered with a 0.22 ㎛ membrane filter, and then dialyzed and dried.

<Comparative group 1>

Commercial adhesion inhibitors were purchased and used. It is an anti-adhesion agent containing 50 parts by weight of hydroxyethylstarch, 85 parts by weight of sodium chloride, 0.64 part by weight of sodium phosphate and dibasic, and 10000 parts by weight of purified water for injection, relative to 100 parts by weight of sodium hyaluronate.

&Lt; Comparative group 2 >

Commercial adhesion inhibitors were purchased and used. It is a product containing poloxamer, gelatin and chitosan.

Experimental Example 1 Evaluation of hyaluronidase in γ-PGA

The inhibition of hyaluronidase of γ-PGA was evaluated. First, the PGA solution was added to 400 units / ml of hyaluronidase so that the concentration of PGA was 0.002% to 0.008%, and the reaction was carried out at 37 ° C for 20 minutes with 0.4 mg / ml of hyaluronic acid. As a result, the IC50 was about 0.0025% . On the other hand, almost 100% inhibition activity was confirmed in the 0.008% γ-PGA solution.

<Experimental Example 2>

The gel retention time was evaluated for Example 1, Comparative Group 1 and Comparative Group 2. First, the above compositions were put into ampoules in an amount of 1 ml, and the residual volume of the gel was checked once a day regularly while storing in a closed room at room temperature, and the gel remaining time was measured. At this time, the time until the gel remaining in the ampule was not visible to the naked eye was defined as the gel remaining time.

As a result, it was confirmed that the composition of Example 1 remained longer than Comparative Groups 1 and 2. Therefore, it was judged that the degradation of the gel was inhibited and the sustaining power was increased by the inhibition of hyaluronidase of γ-PGA (Table 1).

Example Comparison group 1 Comparison group 2 Gel remaining time (days) 14 8 6

<Experimental Example 3> Anti-adhesion effect

The anti-adhesion effect of the anti-adhesion composition of the present invention was evaluated through animal tests.

First, the mouse was opened and the surface of the membrane of 1 x 1 cm was damaged to such an extent that hemorrhage occurred. The compositions of Example 1, Comparative Example 1, and Comparative Example 2 were applied uniformly to each damaged area by 1 ml each, and then the skin layer was continuously sutured. As a control group, a sample in which no sample was treated for adhesion prevention was used.

After 14 days of operation, the degree of adhesion of the tissue was visually evaluated. The degree of adhesion is represented by a number between 0 and 3.5. (0: no adhesion: 1: adhesion of about 30% of the damaged area, 2: adhesion of about 50% of the damaged area, and 3: adhesion of about 70% of the damaged area). As a result, the composition of Example 1 was superior to the comparative groups 1 and 2 in preventing adhesion (FIGS. 1 and 2, (a): control group, (b) ).

<Experimental Example 4> Bioabsorption and release test after prevention of adhesion formation

The composition of Example 2 was injected into the mice using the intramuscular administration method (total of 15 mice per 3 mice per time). After 6 hours, 1 day, 3 days, 7 days, and 14 days after the injection, the metabolic behavior in the body was analyzed by analyzing the distribution of the composition of Example 2 in vivo organs. At this time, organs (liver, spleen, lung, kidney) were extracted from the mouse after 6 hours, 1 day, 3 days, 7 days and 14 days and were dried and used in the composition using ICP-MS The distribution of polygamma glutamic acid was analyzed.

The concentration of Gd attached to poly-gamma glutamate before intramuscular injection was measured by ICP-MS and the adhesion rate was confirmed to be about 24%. Based on this, it was possible to quantify the amount of polygamma glutamic acid distributed in each organs over time in intramuscular injection.

As a result, general poisoning symptoms were not observed in the mice, and no abnormality was found in all the organs.

On the other hand, the polygamma glutamic acid introduced into the mouse through intramuscular injection accumulated in most of the liver in the early stage, and as a result of observation for 2 weeks, the accumulated amount of liver tended to decrease gradually with time. Similar trends were observed in the spleen and lung (FIG. 3).

Therefore, ICP-MS was able to confirm that the polygamma-glutamic acid introduced into the mouse through the intramuscular injection was continuously discharged from the kidney through the kidney, and the amount discharged was gradually decreased, It was confirmed that polygamma glutamic acid accumulated in liver, spleen, lung and the like was excreted through elongation by metabolism or decomposition in vivo. This means that the administered polygamma-glutamic acid is decomposed by metabolism in the body or excreted outside the body.

<Experimental Example 5> Evaluation of hyaluronic acid remaining ability according to the concentration of polygamma glutamic acid

The residual ability of hyaluronic acid was evaluated according to the ratio of polygamma glutamic acid to hyaluronic acid (weight ratio by weight%) in the adhesion inhibition composition. At this time, samples were applied to a living body (37 ° C) simulated environment, and the amount of the sample remaining after hemo-vacuum inhalation was evaluated. The residual amount of the sample was measured by absorbance measurement using a dye (Elisa absorbance analyzer).

As a result, it was confirmed that hyaluronic acid was kept particularly long in 0.50% by weight to 1.00% by weight of hyaluronic acid and 0.01 to 1% by weight of polygamma glutamic acid (Fig. 4, HA :, hyaluronic acid,? -PGA: polygamat glutamic acid) .

Claims (11)

A hyaluronidase inhibitor and hyaluronic acid.
The method according to claim 1,
Wherein the composition is in a liquid phase.
The method according to claim 1,
Wherein the hyaluronic acid and the hyaluronidase inhibitor are contained in a weight ratio of 1: 0.005 to 2.550.
The method according to claim 1,
Wherein the hyaluronic acid and the hyaluronidase inhibitor are contained in a weight ratio of 1: 0.01 to 2.0.
The method according to claim 1,
Wherein the hyaluronidase inhibitor increases the residence time of hyaluronic acid in vivo.
The method according to claim 1,
A composition for preventing adhesion, which further comprises an anti-inflammatory agent.
The method according to claim 1,
A composition for preventing adhesion, which further comprises an anti-blood coagulant.
The method according to claim 1,
Wherein the hyaluronidase inhibitor is polygamma glutamic acid.
The method according to claim 1,
Characterized in that hyaluronic acid remains for 10 days or more when applied to a wound.
The method according to claim 1,
And the duration of the adhesion prevention is improved.
A method for producing an anti-adhesion composition comprising mixing a hyaluronidase inhibitor and hyaluronic acid.

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