JP2021178803A - Adhesion preventive material and method for manufacturing adhesion preventive material - Google Patents

Abstract

To provide an adhesion preventive material that can be easily handled during a surgery, and a method for manufacturing an adhesion preventive material.SOLUTION: An adhesion preventive material contains modified hyaluronic acid and/or salt thereof which has hyaluronidase degradation resistance and is modified by a polyglutamyl group having the molecular weight of 750 or more and 20,000 or less. Further, a method for manufacturing an adhesion preventive material containing modified hyaluronic acid and/or salt thereof which is modified by a polyglutamyl group and has hyaluronidase degradation resistance, includes causing hyaluronic acid to react with polyglutamic acid having the molecular weight of 750 or more and 20,000 or less.SELECTED DRAWING: None

Description

The present invention relates to an adhesion-preventing material and a method for producing an adhesion-preventing material.

When performing open surgery, there is a high probability of "adhesions" in which organs and tissues that are not originally adhered to each other adhere to each other, which causes various complications such as chronic abdominal pain, intestinal obstruction, and infertility. .. Therefore, adhesion prevention materials are placed between organs and tissues to create a physical barrier, and adhesions are prevented by blocking direct friction and contact between tissues. In recent years, laparoscopic surgery for the purpose of minimally invasive treatment has become widespread, but it is also necessary to prevent adhesion formation in laparoscopic surgery. In clinical practice, anti-adhesion materials such as Genzyme's Sepra film (registered trademark, see Patent Documents 1 and 2) and Terumo's Adspray (registered trademark, see Patent Document 3) are used. Has been done.

Sepra film is a film-like adhesion-preventing material containing sodium hyaluronate and carboxymethyl cellulose as main raw materials. However, it has low strength and high hydrophilicity, and once it adheres to a non-target site, it is physically impossible to reattach it. In addition, since the sepra film at the time of drying has poor plasticity, it is difficult to deliver or indwell the sepra film into the abdominal cavity in laparoscopic surgery.

Adspray is a two-component mixed in-situ gel consisting of a solution of N-hydroxysuccinimide (NHS) -modified carboxymethyl (CM) dextrin / trehalose hydrate and a solution of sodium carbonate / sodium hydrogen carbonate. It is a type of anti-adhesion material. Therefore, Adspray can be used for laparoscopic surgery. However, the ad spray is a two-component mixed type and usually needs to be used within one hour after mixing. Therefore, it is not easy to handle because complicated solution preparation is required immediately before or during surgery.

Special Table No. 5-508161 Gazette Special Table 6-508169 Gazette Japanese Patent No. 6285413

An object of the present invention is to provide an adhesion-preventing material and a method for producing an adhesion-preventing material, which are easy to handle during surgery.

According to the aspect of the present invention, the modified hyaluronic acid and / or a salt thereof modified with a polyglutamyl group having a molecular weight of 750 or more and 20,000 or less is contained, and the modified hyaluronic acid and / or a salt thereof has hyaluronidase decomposition resistance. , Anti-adhesion material is provided.

In the above-mentioned adhesion-preventing material, the polyglutamyl group may be a γ-polyglutamyl group.

In the above-mentioned adhesion-preventing material, the number of polyglutamyl groups contained in one structural unit of hyaluronic acid may be 0.0015 or more and 0.5 or less.

In the above-mentioned anti-adhesion material, modified hyaluronic acid and / or a salt thereof may be dissolved in the solution.

In the above-mentioned anti-adhesion material, the modified hyaluronic acid and / or a salt thereof may be a powder.

Further, according to the aspect of the present invention, a modified hyaluronic acid having hyaluronidase decomposition resistance modified with a polyglutamyl group, which comprises reacting hyaluronic acid with polyglutamic acid having a molecular weight of 750 or more and 20,000 or less, and / Alternatively, a method for producing an adhesion-preventing material containing a salt thereof is provided.

In the above method for producing an adhesion-preventing material, hyaluronic acid and polyglutamic acid may be reacted in the presence of a carbodiimide catalyst.

In the above method for producing an adhesion-preventing material, the polyglutamyl group may be a γ-polyglutamyl group.

In the reaction of hyaluronic acid and polyglutamic acid in the above method for producing an adhesion-preventing material, 1 mol or more of polyglutamic acid may be grafted per 50 mol of one constituent unit of hyaluronic acid in terms of raw material ratio.

The method for producing an anti-adhesion material may further include preparing a solution containing modified hyaluronic acid and / or a salt thereof.

The method for producing an anti-adhesion material may further include powdering modified hyaluronic acid and / or a salt thereof.

According to the present invention, it is possible to provide an adhesion-preventing material and a method for producing an adhesion-preventing material, which are easy to handle during surgery.

It is a photograph showing the hyaluronidase decomposition resistance (state of time-dependent decomposition) of modified hyaluronic acid. It is a photograph which shows the comparative result of the hyaluronidase decomposition resistance of the modified hyaluronic acid by the difference of the graft amount. It is a photograph of a dissected rat. It is a graph which shows the average value of the scores of 3 adhesions in each evaluation target adhesion prevention material indwelling group. It is a photograph which stained the tissue piece on the peritoneal lumen side of a normal rat group with hematoxylin-eosin (HE), and the photograph which stained with Masson's trichrome (MT). Hematoxylin-eosin (HE) -stained photographs and Masson's trichrome (MT) -stained photographs of the tissue pieces on the peritoneal lumen side of the positive control group. Hematoxylin-eosin (HE) -stained photographs and Masson's trichrome (MT) -stained photographs of the tissue pieces on the peritoneal lumen side of the Sepura film indwelling group. Hematoxylin-eosin (HE) -stained photographs and Masson's trichrome (MT) -stained photographs of the tissue pieces on the peritoneal lumen side of the Adspray indwelling group. Hematoxylin-eosin (HE) -stained photographs and Masson's trichrome (MT) -stained photographs of the tissue pieces on the peritoneal lumen side of the modified hyaluronic acid indwelling group.

Hereinafter, embodiments of the present invention will be described in detail. However, the descriptions and drawings that form part of this disclosure should not be understood to limit the invention. Various alternative embodiments, examples and operational techniques should be apparent to those of skill in the art from this disclosure.

The anti-adhesion material according to the present embodiment contains modified hyaluronic acid and / or a salt thereof modified with a polyglutamyl group having a molecular weight of 750 or more and 20,000 or less. Modified hyaluronic acid and / or salts thereof are resistant to hyaluronidase degradation. The anti-adhesion material according to the present embodiment contains an effective amount of modified hyaluronic acid and / or a salt thereof for preventing adhesion. Hereinafter, the modified hyaluronic acid and / or a salt thereof may be simply referred to as "modified hyaluronic acid".

Hyaluronic acid is a linear polysaccharide having a repeating structure consisting of N-acetyl-D-glucosamine and D-glucuronic acid disaccharides. The origin of hyaluronic acid is not particularly limited, and examples thereof include lactic acid bacteria such as Streptococcus and Lactococcus, combs, and humans.

The characteristics, molecular weight, molecular weight distribution, etc. of hyaluronic acid are not particularly limited. Examples of the lower limit of the average molecular weight are 400 or more, 2,000 or more, 5,000 or more, 10,000 or more, 50,000 or more, 100,000 or more, 500,000 or more, 600,000 or more, 700,000. The above, or 800,000 or more can be mentioned. Examples of the upper limit of the average molecular weight are 1,000,000 or less, 8,000,000 or less, 6,000,000 or less, 4,000,000 or less, 3,000,000 or less, or 2,500,000. The following can be mentioned. Two or more kinds of commercially available hyaluronic acids having different average molecular weights may be mixed and used.

The average molecular weight of hyaluronic acid is, for example, a method of combining size exclusion chromatography and a multi-angle light scattering detector (SEC / MALS, for example, "National Institute of Pharmaceutical and Food Safety", 2003, Vol. 121, p.30-. It can be obtained by 33) or a combination of the Molecular-Elson method and the Carbazole sulfuric acid method (see JP-A-2009-155486).

The presence or absence of counter ions of hyaluronic acid is not particularly limited, and examples thereof include free type, sodium ion, potassium ion, calcium ion, magnesium ion, and ammonium ion.

In the modified hyaluronic acid, as shown by the following chemical formula (1), a polyglutamyl group such as a γ-polyglutamyl group (hereinafter, also simply referred to as “γ-PGA”) is bonded to the hyaluronic acid. The polyglutamyl group may be an L-polyglutamyl group. Modified hyaluronic acid can be obtained by reacting hyaluronic acid with polyglutamic acid using a water-soluble coupling agent and / or a coupling aid. Without being bound by theory, it is believed that the steric barrier of the polyglutamyl group suppresses the degradation of hyaluronic acid by degrading enzymes such as hyaluronidase.

Although polyglutamic acid is a safe substance that can be finally metabolized in the living body, for example, there may be a site in the body where a direct degrading enzyme using polyglutamic acid as a substrate does not exist, and the polypeptide becomes an antigen target. Modified hyaluron in view of the fact that it is easy, it is preferable not to give a large change in physicochemical properties such as the viscous property of hyaluronic acid even after modification, and it is preferable to maintain the physiological activity of hyaluronic acid even after modification. The polyglutamic acid used in the production of the acid is preferably of a low molecular weight type. Therefore, the molecular weight of polyglutamic acid is preferably 750 or more and 20,000 or less, preferably 750 or more and 10,000 or less, and more preferably 1,000 or more and 5,000 or less.

Water-soluble coupling agents and / or coupling aids for introducing polyglutamic acid into hyaluronic acid include, for example, water-soluble such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Carbodiimide can be used. Carbodiimide is capable of binding a carboxyl group to an amine. Further, N-hydroxysuccinimide (NHS) or a water-soluble analog thereof (sulfo-NHS) may be used to introduce an NHS ester into a carboxyl group and then bonded to an amine.

Alternatively, hyaluronic acid can be converted to a polyglutamyl group by deacetylating the acetylamino group of N-acetylglucosamine of hyaluronic acid to convert it into an amino group, and amide-bonding the amino group with the carboxyl group of polyglutamic acid. It is possible to modify.

Further, the method for producing modified hyaluronic acid can further include a step of obtaining a powder of modified hyaluronic acid by freeze-drying. Alternatively, the step of adding alcohol to the modified hyaluronic acid to obtain a precipitate can be further included. Here, examples of the alcohol include methanol and ethanol, and ethanol is preferable. By adding an alcohol to the modified hyaluronic acid to obtain a precipitate of the modified hyaluronic acid and / or a salt thereof, the modified hyaluronic acid separated from the remaining reagent can be obtained. Further, the purity of the modified hyaluronic acid can be further increased by dialysis in water or the like.

In the modified hyaluronic acid and / or a salt thereof, the number of polyglutamyl groups contained in one constituent unit of hyaluronic acid is preferably 0.0015 or more and 0.5 or less, preferably 0.002 or more and 0.5 or less. It is more preferable, and it is even more preferable that it is 0.01 or more and 0.2 or less. Here, "one constituent unit of hyaluronic acid" means one constituent unit composed of a disaccharide of glucuronic acid and N-acetylglucosamine.

In modified hyaluronic acid and / or a salt thereof, the number of polyglutamyl groups contained in one constituent unit of hyaluronic acid is 0.0015 or more and 0.5 or less, so that the resistance to decomposition of hyaluronidase and other degrading enzymes in the body is increased. It is obtained and tends to easily maintain the physiological activity and viscous properties inherent in hyaluronic acid.

Further, the larger the number of polyglutamyl groups contained in one constituent unit of hyaluronic acid, the longer the remaining time of the adhesion-preventing material arranged in the living body, and the longer the number of polyglutamyl groups contained in one constituent unit of hyaluronic acid. The smaller the amount, the shorter the remaining time of the adhesion-preventing material placed in the living body tends to be. Therefore, it is possible to adjust the remaining time of the adhesion-preventing material placed in the living body by adjusting the number of polyglutamyl groups contained in one constituent unit of hyaluronic acid according to the purpose.

In the modified hyaluronic acid and / or a salt thereof, the number of polyglutamyl groups contained in one structural unit of hyaluronic acid ^{can be identified by the Bicinchoninic acid (BCA) method or 1} H-NMR spectral analysis.

When producing a hyaluronic acid solution, the type of solvent that dissolves hyaluronic acid is not particularly limited as long as it is a liquid that uniformly dissolves hyaluronic acid, but water is preferably used.

The concentration of hyaluronic acid in the hyaluronic acid solution is not particularly limited as long as the hyaluronic acid can be dissolved. The higher the molecular weight of the hyaluronic acid used, the lower the upper limit of the concentration of hyaluronic acid that can be dissolved, for example, 0.001 to 10% (w / v).

The method for measuring the decomposition of the modified hyaluronic acid is not particularly limited, but for example, since the viscosity of the aqueous solution of the modified hyaluronic acid decreases as the molecular weight decreases, the method of measuring the viscosity of the modified hyaluronic acid aqueous solution is convenient. It can be used as a method. In addition, a method of combining size exclusion chromatography and a multi-angle light scattering detector and a method of combining the Morgan-Elson method and the Carbazole sulfuric acid method can also be used.

The kinematic viscosity of the aqueous solution of the modified hyaluronic acid and / or a salt thereof can be measured using an Ubbelohde viscometer (manufactured by Shibata Kagaku Kikai Kogyo Co., Ltd.). At this time, a Ubbelohde viscometer having a coefficient such that the number of seconds flowing down is 200 to 1,000 seconds is selected. ^{The kinematic viscosity (unit: mm 2} / s) can be obtained from the product of the number of seconds of flowing water of the aqueous solution measured by the Ubbelohde viscometer and the coefficient of the Ubbelohde viscometer.

The adhesion-preventing material according to the present embodiment may contain other natural materials, preservatives, functional materials and the like in addition to the modified hyaluronic acid.

The anti-adhesion material according to the present embodiment further contains a wetting agent, an emulsifier, a lubricant such as sodium lauryl sulfate and magnesium stearate, a coloring agent, a releasing agent, a coating agent, a preservative, and an antioxidant. May be good.

Examples of pharmaceutically acceptable antioxidants are (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogensulfate, sodium disulfate, and sodium sulfite, and (2) ascorbyl palmitate. , Oil-soluble antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol, and (3) citric acid, ethylenediamine tetraacetic acid (EDTA). , Sorbitol, tartrate acid, and metal chelating agents such as phosphoric acid.

The adhesion-preventing material according to the present embodiment may be provided as a powder or as a solution. When the adhesion-preventing material according to the present embodiment is provided as a powder, a solution obtained by dissolving the powder in a solvent such as physiological saline is used as an adhesion-preventing agent. When the adhesion-preventing material according to the present embodiment is provided as a solution, it can be used as it is. When the adhesion-preventing material according to the present embodiment is a solution, a one-component type may be used. Therefore, unlike the two-component type, it is not necessary to mix the two solutions immediately before surgery.

The adhesion-preventing material according to the present embodiment can be used for preventing adhesions after laparotomy and also for preventing adhesions after laparoscopic surgery. The method of applying the adhesion-preventing material according to the present embodiment is not particularly limited, but the adhesion-preventing material according to the present embodiment may be dropped, applied, or sprayed to a site where adhesion is desired to be prevented. It may be injected or it may be injected.

Hereinafter, the present embodiment will be described in more detail with reference to Examples and Comparative Examples. However, the technical scope of the present invention is not limited to the following examples.

(Example 1: Synthesis of modified hyaluronic acid and evaluation of hyaluronidase resistance)
In the following procedure, modified hyaluronic acid having a different content of γ-polyglutamyl group was synthesized by changing the theoretical compounding molar ratio of γ-polyglutamic acid and one constituent unit of hyaluronic acid. When the theoretical compounding molar ratio of γ-polyglutamic acid and one constituent unit of hyaluronic acid is 1: 5, the final concentration of hyaluronic acid (HA200, molecular weight 2,000 kDa, manufactured by Kikkoman Biochemifa) is in a 100 mL beaker. Dissolve in 25 mL of 50 mmol / L phosphate buffer (pH 6.0) so as to be 4.2 mg / mL, and further add EDAC (manufactured by Dojin Chemical Research Institute), which is a carbodiimide catalyst, to a final concentration of 0.4 mg / mL. It was added with stirring so as to be. Further, γ-polyglutamic acid (molecular weight 1,500 to 5,000, manufactured by Sigma-Aldrich) was added to a final concentration of 7.3 mg / mL to prepare a reaction solution, which was held at 25 ° C. for 1440 minutes. Here, the "theoretical compounding molar ratio" is a raw material ratio, in other words, a molar ratio assuming that the coupling efficiency is 100%.

Reaction solution for producing modified hyaluronic acid having a theoretical compounding molar ratio of 1:10 of γ-polyglutamic acid and one constituent unit of hyaluronic acid, reaction for producing modified hyaluronic acid having a theoretical compounding ratio of 1:50. A reaction solution for producing a liquid, modified hyaluronic acid of 1: 100, was also prepared. In the method for preparing these reaction solutions, the theoretical compounding molar ratio of γ-polyglutamic acid and one constituent unit of hyaluronic acid was 1: 5, except that the concentration of the reaction reagent was changed as shown in Table 1. It was synthesized by the same method as the reaction solution for producing a certain modified hyaluronic acid.

Next, the reaction solution held at 25 ° C. for 1,440 minutes was dialyzed against a large excess amount of distilled water (milli-Q water) for 48 hours with a dialysis membrane having a fractional molecular weight (MWCO) of 8,000, and dialysis was completed. Then, by the freeze-drying method, 256 mg of modified hyaluronic acid containing a γ-polyglutamyl group represented by the above general formula (1) was obtained.

PGA in Table 1 means γ-polyglutamic acid, and the parenchymal graft amount is the bicinchoninic acid method (see Smith, PK, et al. (1985) Anal. Biochem. 150, 76-85.). It was determined by measuring the protein content according to the above.

(Example 2: Evaluation of hyaluronidase resistance of modified hyaluronic acid)
The obtained modified hyaluronic acid was dissolved in distilled water (milliQ water) at a concentration of 10 mg / mL. To 20 μL of this solution, 50 μL of 0.2 mol / L sodium phosphate buffer (pH 5.5), 1.0 mg / mL (20 μL) of bovine serum albumin and 105 μL of water were added. Further, in the solution, hyaluronidase (manufactured by Seikagaku Corporation derived from sheep testis) is added to a final concentration of 0.025 mg / mL solution (0.05 mol / L sodium phosphate buffer; pH 5.5) at 1.0 mg / mL. 5 μL of the solution was added. The final concentration of modified hyaluronic acid was 1.0 mg / mL, and the final concentration of bovine serum albumin was 0.1 mg / mL. After the addition of hyaluronidase, the cells were incubated at 37 ° C. for 0 minutes, 15 minutes, 30 minutes, and 1 hour.

100 μL of each sample was separated, the pH was adjusted to 8 with NaOH, and heat treatment was performed at 95 ° C. for 5 minutes to stop the hyaluronidase reaction. As a control, the enzyme-free group was also performed in the same manner.

They were subjected to SDS-PAGE respectively, and changes in the molecular weight of the modified hyaluronic acid and the formation pattern of decomposition products were observed (Stains All staining, manufactured by Cosmo Bio Co., Ltd.). The conditions for Stains All staining are as per the technical data attached to the product.

The decomposition resistance of unmodified hyaluronic acid was compared with the decomposition resistance of modified hyaluronic acid (theoretical compounding molar ratio of γ-polyglutamic acid and one constituent unit of hyaluronic acid was 1: 5). As shown in FIG. 1, unmodified hyaluronic acid was almost completely degraded when the incubation time with hyaluronidase exceeded 15 minutes. In contrast, modified hyaluronic acid was hardly degraded under the same conditions.

Next, when the decomposition resistance due to the difference in the graft amount of γ-polyglutamic acid was compared, as shown in FIG. 2, the decomposition resistance improved as the graft amount increased, and in particular, the modified hyaluronic acid having a graft amount of 1:50 or more. It was revealed that the acid shows excellent hyaluronidase resistance. Considering the coupling efficiency, it is considered that the molar ratio of the γ-polyglutamyl group in the modified hyaluronic acid produced by blending γ-polyglutamic acid with the raw material molar ratio of 1/50 is about 1/500. Be done. Further, it is considered that the molar ratio of the γ-polyglutamyl group in the modified hyaluronic acid produced by blending γ-polyglutamic acid with the raw material molar ratio of 1/100 is about 1/1000.

(Example 3: Preparation of adhesion-preventing material to be evaluated for peritoneal scraping model)
In the peritoneal scraping model, an anti-adhesion material to be evaluated is placed between the scraped peritoneum and the organ. Therefore, in comparing and evaluating the adhesion-preventing materials to be evaluated having various forms and operability such as film-like and gel-like, it is easy to indwell the adhesion-preventing material to be evaluated and to perform surgical treatment, and the adhesion-preventing material to be evaluated is easy to perform. It is a model that makes it easy to evaluate the adhesion prevention effect.

(I: Modified hyaluronic acid)
Hyaluronic acid having a molecular weight of 2,000 kDa was dissolved in 0.05 mol / L sodium phosphate buffer (NaPB) (pH = 5.8) and allowed to swell at 4 ° C. overnight. The next day, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and NHS were added, and the mixture was stirred at 37 ° C. for 30 minutes under shading. After 30 minutes, γ-polyglutamic acid (molecular weight 1,500 to 5,000, manufactured by Sigma-Aldrich) was added and reacted in the dark for 24 hours. After completion of the reaction, dialysis was performed for 48 hours using a dialysis membrane having a molecular weight cut off of 300 kDa. At this time, dialysis was performed with 1.5 L of 0.1 mol / L NaPB for the first 24 hours, and dialysis was performed with 3 L of Milli-Q water for the subsequent 24 hours. Each solution was changed 3 times in 24 hours. Then, the modified hyaluronic acid obtained by freeze-drying _{was dissolved in 10 mg / mL with 0.45% NaCl and 0.7% NaCl 3} and filtered and sterilized with a 0.45 μm filter to obtain a gelled modified hyaluronic acid solution. Obtained.

(Ii: Sepura film)
I purchased Sepura film from Kaken Pharmaceutical Co., Ltd. The package was aseptically opened and the sepra film was trimmed into squares of approximately 2 cm x 2 cm.

(Iii: Adspray)
Adspray was purchased from Terumo Corporation, and a gel solution was prepared according to the instruction manual. The amount of spray on rats, which will be described later, was set to about 200 μL converted from the body weight ratio with reference to the fact that one product is consumed when used in humans.

(Example 4: Placement of anti-adhesion material to be evaluated)
The following animal experiments were conducted with the approval of the Animal Experiment Management and Operation Committee of Tokyo Denki University. First, Sprague-Dawley (SD) rats (4 weeks old, male) were anesthetized with isoflurane. The abdomen of the rat was incised in a U-shape so that the skin on the upper body side of the rat was connected and the side was 2 cm. The exposed peritoneal surface was rubbed for 3 minutes, left for about 5 minutes, and then rubbed again for 3 minutes. The scratch was then rinsed with antibiotic-containing saline. The time from laparotomy to closure was unified to 15 minutes, and each of the adhesion-preventing materials to be evaluated was placed between the exposed gastrointestinal tract and the peritoneum at the time of abdominal closure.

The sepra film was placed on the gastrointestinal tract side. For ad spray, the peritoneum and gastrointestinal tract were sprayed. For modified hyaluronic acid, about 100 μL on the peritoneal side and about 100 μL on the organ side were applied using a syringe. After placing the sample, the skin at the incision was sutured. During the breeding period, CE-2 (Nippon Claire) was given to the rats as feed at 5 tablets / day, and sterilized tap water containing antibiotics was given to the rats as drinking water.

During the breeding period after indwelling the anti-adhesion material to be evaluated, all the rats in the evaluation group ingested a certain amount of both feed and drinking water. In addition, it was possible to visually observe how it moves around normally. In addition, there was no significant difference in the body weight changes of the animals during the breeding period in all groups. From this, it was inferred that none of the evaluation target anti-adhesion materials affected the health condition of the animals. The rat was dissected on the 7th day after the anti-adhesion material to be evaluated was placed. Each experimental group was performed using 3 animals.

(Example 5: Anatomical evaluation)
An incision was made in the abdomen of the euthanized rat, and the presence or absence of adhesions between the peritoneum and the gastrointestinal tract was visually observed. The area to be evaluated for adhesions formed in the abdominal cavity was limited to the initially scraped intra-abdominal surface, and adhesion formation at the sutured portion was excluded. A representative photograph of a rat with an incised abdomen is shown in FIG.

Adhesions were observed between the peritoneum and organs in the positive control group in which no anti-adhesion material was placed. In the Sepura film indwelling group and the Adspray indwelling group, the adhesion range was smaller than that in the positive control group. Furthermore, adhesions between the peritoneum and the gastrointestinal tract were scarcely observed in both groups, and adhesions were observed only with the liver. From this result, it was clarified that adhesion between the liver and the peritoneum is likely to occur when the sepra film and the ad spray are used. No adhesions were confirmed in the modified hyaluronic acid indwelling group.

The degree of adhesion observed was scored according to the following evaluation criteria.
No apparent adhesion formation: 0 points Adhesion formation is observed in less than 25% of the evaluation area: 1 point Adhesion formation is observed in less than 50% of the evaluation area: 2 points Adhesion formation is observed in less than 75% of the evaluation area : 3 points Adhesion formation is observed in 75% or more of the evaluation area: 4 points The average value of the scores of the three adhesions in each evaluation target adhesion-preventing material indwelling group, which was scored according to the above evaluation criteria, is shown in FIG.

The scores of the Seprafilm indwelling group and the Adspray indwelling group were lower than those of the positive control group. In addition, the score of the ad spray indwelling group was lower than that of the sepra film indwelling group. Therefore, it was suggested that the gel-like adhesion-preventing material has a higher adhesion-preventing effect than the film-like adhesion-preventing material. The score of the modified hyaluronic acid indwelling group was even lower than that of the adspray indwelling group. Therefore, it was shown that the modified hyaluronic acid has an excellent anti-adhesion effect.

From the above results, it was suggested that the gel-like anti-adhesion material has an irregular shape and therefore easily spreads between the gastrointestinal tracts due to peristalsis of the gastrointestinal tract. On the other hand, it was suggested that the sheet-shaped anti-adhesion agent can cover only the surface of the organ and cannot enter between the digestive tracts.

(Example 6: Histological evaluation)
After observing the degree of adhesion formation, the entire skin layer was peeled off, and a piece of tissue on the peritoneal lumen side was collected. The collected tissue pieces were fixed with 4% paraformaldehyde, dehydrated with ethanol and xylene through a washing step, and embedded in paraffin. Paraffin-embedded tissue pieces were sliced into 8 μm thick slices with a microtome and histologically evaluated by hematoxylin-eosin (HE) staining and Masson's trichrome (MT) staining.

In the peritoneum of the normal rat group in which the peritoneum was not scraped, mesothelial cells, which are simple squamous epithelium, were confirmed on the surface of the muscular layer, as shown in the left figure of FIG. In addition, a blue-stained collagen layer was observed only between the mesothelial cells and the connective tissue immediately below them and between the muscle fibers shown in the right figure of FIG. 5 in the original color drawing.

In the positive control group, as shown in the left figure of FIG. 6, it was observed that the peritoneal tissue was thickened by inflammatory cells, fibroblasts and the like in the scratched scar and bound to the small intestine. These histological data confirmed the presence of adhesion formation seen in the anatomical findings. Furthermore, from the right figure of FIG. 6, it was confirmed that collagen fibers were used as a substrate near the boundary between the regenerated tissue and the muscle layer. However, the area around the junction with the small intestine was not stained very blue, suggesting that collagen-induced fibrosis was delayed and the inflammatory response was sustained.

In the seprafilm indwelling group, thickening of the peritoneal tissue was observed as shown in the left figure of FIG. 7. In addition, the observation of the regenerated tissue in a porous form suggests that the sepra film is in the process of decomposition and absorption, and is sequentially replaced with the tissue by inflammatory cells and fibroblasts that have invaded the inside of the sepra film. .. In addition, it was confirmed that inflammatory cells and fibroblasts invaded deeply in the muscular layer. This suggests that inflammation continues one week after surgery. In the right figure of FIG. 7, the regenerated tissue was not stained very blue in the original color drawing, suggesting that fibrosis by collagen was not so advanced at this point. On the other hand, invasion of fibroblasts was observed in the muscle layer, and substrate formation by collagen fibers was observed.

In the Adspray indwelling group, thickening of the peritoneal tissue was observed as shown in the left figure of FIG. In addition, it was confirmed that inflammatory cells invaded the liver tissue and were directly connected to the liver at the adhesion site with the liver. Many large, cubic cells were observed on the surface of the adhesion site. It was suggested that this is a macrophage that has migrated into the abdominal cavity. The presence of macrophages was confirmed, suggesting that the inflammatory response is continuing. In addition, no trace of adspray was confirmed in histological evaluation, suggesting that adspray was decomposed and absorbed one week after the operation. Further, as shown in the right figure of FIG. 8, since the repaired portion of the scratch wound was dyed blue by MT staining in the original color drawing, it was confirmed that the restoration was accompanied by fibrosis. Even in areas where adhesions did not occur, there was considerable fibrosis overall. Therefore, it was suggested that the regeneration mechanism of the ad spray indwelling group was different from that of the sepra film indwelling group.

In the modified hyaluronic acid indwelling group, as shown in the left figure of FIG. 9, thickening of the peritoneal tissue was observed as compared with the normal tissue. However, the thickness of the regenerated tissue was thinner than that of the positive control group, the seprafilm indwelling group, and the adspray indwelling group, suggesting that modified hyaluronic acid may suppress tissue thickening. Further, as shown in the right figure of FIG. 9, fibrosis due to collagen was observed in the regenerated tissue part. In addition, traces of modified hyaluronic acid were identified as porous portions. From these results, it was shown that the modified hyaluronic acid remained in the decomposition and absorption process one week after the operation.

The results of histological evaluation showed that the tissue repair process was different for each anti-adhesion material to be evaluated. In addition, the residual property of the adhesion-preventing material to be evaluated may be related to tissue regeneration, and the sepra film, which has slow bioabsorbability and is not easily decomposed, has high residual property and slow tissue regeneration. On the other hand, the adspray, which has a high bioabsorbability and is easily decomposed, has a low residual property and a fast tissue regeneration. However, although Adspray was more effective in preventing adhesions than Seprafilm, adhesion formation was still observed. On the other hand, the modified hyaluronic acid remained even one week after the operation, and the tissue regeneration was quick. In addition, modified hyaluronic acid showed an excellent anti-adhesion effect.

Claims (11)

An anti-adhesion material comprising modified hyaluronic acid and / or a salt thereof modified with a polyglutamyl group having a molecular weight of 750 or more and 20,000 or less, wherein the modified hyaluronic acid and / or a salt thereof has resistance to hyaluronidase decomposition. The adhesion-preventing material according to claim 1, wherein the polyglutamyl group is a γ-polyglutamyl group. The adhesion-preventing material according to claim 1 or 2, wherein the number of polyglutamyl groups contained in one structural unit of hyaluronic acid is 0.0015 or more and 0.5 or less. The adhesion-preventing material according to any one of claims 1 to 3, wherein the modified hyaluronic acid and / or a salt thereof is dissolved in the solution. The adhesion-preventing material according to any one of claims 1 to 3, wherein the modified hyaluronic acid and / or a salt thereof is a powder. Production of anti-adhesion material containing modified hyaluronidase resistant to hyaluronidase degradation and / or a salt thereof modified with a polyglutamyl group, which comprises reacting hyaluronic acid with polyglutamic acid having a molecular weight of 750 or more and 20,000 or less. Method. The method for producing an adhesion-preventing material according to claim 6, wherein the hyaluronic acid is reacted with the polyglutamic acid in the presence of a carbodiimide catalyst. The method for producing an adhesion-preventing material according to claim 6 or 7, wherein the polyglutamyl group is a γ-polyglutamyl group. The adhesion-preventing material according to any one of claims 6 to 8, wherein in reacting the hyaluronic acid with polyglutamic acid, 1 mol or more of polyglutamic acid is grafted per 50 mol of one constituent unit of hyaluronic acid in terms of raw material ratio. Manufacturing method. The method for producing an anti-adhesion material according to any one of claims 6 to 9, further comprising preparing a solution containing modified hyaluronic acid and / or a salt thereof. The method for producing an anti-adhesion material according to any one of claims 6 to 9, further comprising powdering modified hyaluronic acid and / or a salt thereof.

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