JP7448944B2 - Anti-adhesion material and method for manufacturing the anti-adhesion material - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act January 23, 2020, Tokyo Denki University Graduate School of Science and Engineering, Department of Life Science and Engineering Master's Thesis Abstracts Volume 38-II (2019), Tokyo Denki University Science and Engineering Faculty Affairs Department Academic Affairs January 24, 2020, Master's Thesis Presentation, Department of Life Science and Engineering, Graduate School of Science and Engineering, Tokyo Denki University, Tokyo Denki University Saitama Hatoyama Campus (Ishizaka, Hatoyama-cho, Hiki-gun, Saitama Prefecture)

The present invention relates to an anti-adhesion material and a method for manufacturing the anti-adhesion material.

When open surgery is performed, there is a high probability that ``adhesions'' will occur, where organs and tissues that are not normally attached adhere to each other, which can cause 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 prevent adhesion by blocking direct friction and contact between the tissues. Furthermore, in recent years, laparoscopic surgery for the purpose of minimally invasive treatment has become popular, but even in laparoscopic surgery, it is necessary to prevent the formation of adhesions. In clinical practice, anti-adhesion materials such as Genzyme's Seprafilm (registered trademark, see, for example, Patent Documents 1 and 2) and Terumo's Adspray (registered trademark, see, for example, Patent Document 3) are used. It is being

Seprafilm is a film-like anti-adhesion material whose main ingredients are sodium hyaluronate and carboxymethylcellulose. However, it has low strength and high hydrophilicity, and once it has been adhered to a non-target area, it is physically impossible to reapply it. Furthermore, since Seprafilm when dry has poor plasticity, it is difficult to deliver or indwell Seprafilm into the abdominal cavity in laparoscopic surgery.

Adspray is a two-component in-situ gelling agent that consists of a solution of N-hydroxysuccinimide (NHS) carboxymethyl (CM) dextrin/trehalose hydrate and a solution of sodium carbonate/sodium hydrogen carbonate. It is an anti-adhesion material for molds. Therefore, Adspray can be used in laparoscopic surgery. However, Adspray is a two-component mixture and usually needs to be used within one hour after mixing. Therefore, it is not easy to handle because it requires complicated solution preparation immediately before or during surgery.

Special Publication No. 5-508161 Special Publication No. 6-508169 Patent No. 6285413

An object of the present invention is to provide an anti-adhesion material that is easy to handle during surgery and a method for manufacturing the anti-adhesion material.

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

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

In the above-mentioned anti-adhesion 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 its salt may be dissolved in the solution.

In the above-mentioned anti-adhesion material, the modified hyaluronic acid and/or its salt may be in the form of powder.

Further, according to an aspect of the present invention, modified hyaluronic acid and/or 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 has resistance to hyaluronidase degradation and/or Alternatively, a method for producing an anti-adhesion material containing a salt thereof is provided.

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

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

In reacting hyaluronic acid and polyglutamic acid in the above method for producing an anti-adhesion material, polyglutamic acid may be grafted in an amount of 1 mole or more per 50 moles of one structural unit of hyaluronic acid as a raw material.

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

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

According to the present invention, it is possible to provide an adhesion prevention material that is easy to handle during surgery and a method for manufacturing the adhesion prevention material.

It is a photograph showing the resistance to hyaluronidase degradation (degradation over time) of modified hyaluronic acid. It is a photograph showing the comparison results of resistance to hyaluronidase degradation of modified hyaluronic acid with different amounts of grafting. This is a photograph of a dissected rat. It is a graph showing the average value of the adhesion scores of three animals in each evaluated adhesion prevention material indwelling group. These are a hematoxylin-eosin (HE) stained photograph and a Masson's trichrome (MT) stained photograph of a tissue piece on the peritoneal cavity side of a normal rat group. These are a hematoxylin-eosin (HE) stained photograph and a Masson's trichrome (MT) stained photograph of tissue pieces on the peritoneal cavity side of the positive control group. These are a hematoxylin-eosin (HE) stained photograph and a Masson's trichrome (MT) stained photograph of tissue pieces on the peritoneal cavity side of the Seprafilm indwelling group. These are a hematoxylin-eosin (HE) stained photograph and a Masson's trichrome (MT) stained photograph of tissue pieces on the peritoneal cavity side of the Adspray indwelling group. These are a hematoxylin-eosin (HE) stained photograph and a Masson's trichrome (MT) stained photograph of tissue pieces on the peritoneal cavity side of the modified hyaluronic acid indwelling group.

Embodiments of the present invention will be described in detail below. However, the description and drawings that form part of this disclosure should not be understood as limiting the invention. Various alternative embodiments, implementations, and operational techniques will be apparent to those skilled in the art from this disclosure.

The anti-adhesion material according to this 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. The modified hyaluronic acid and/or its salt has resistance to hyaluronidase degradation. The anti-adhesion material according to this embodiment contains an effective amount of modified hyaluronic acid and/or a salt thereof to prevent adhesion. Hereinafter, modified hyaluronic acid and/or its salt may be simply referred to as "modified hyaluronic acid."

Hyaluronic acid is a linear polysaccharide consisting of a repeating structure of disaccharides of N-acetyl-D-glucosamine and D-glucuronic acid. The origin of hyaluronic acid is not particularly limited, but examples include those derived from lactic acid bacteria such as Streptococcus and Lactococcus, cockscomb, 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 or more. or more, or more than 800,000. Examples of the upper limit of the average molecular weight are 10,000,000 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 or less. The following may be mentioned. Note that two or more types of commercially available hyaluronic acids having different average molecular weights may be used in combination.

The average molecular weight of hyaluronic acid can be determined, for example, by a method combining size exclusion chromatography and a multi-angle light scattering detector (SEC/MALS), for example, "National Institute of Health Sciences Bulletin", 2003, Vol. 121, p. 30- 33) or a combination of the Morgan-Elson method and the Carbazol sulfuric acid method (see JP-A No. 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 modified hyaluronic acid, a polyglutamyl group such as a γ-polyglutamyl group (hereinafter also simply referred to as "γ-PGA") is bonded to hyaluronic acid, as shown in the following chemical formula (1). 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. Although not bound by theory, it is believed that the steric barrier of polyglutamyl groups inhibits decomposition enzymes such as hyaluronidase from decomposing hyaluronic acid.

Although polyglutamic acid is ultimately a safe substance that can be metabolized in the body, there may be sites within the body that do not have a direct degrading enzyme that uses polyglutamic acid as a substrate, and polypeptides may be subject to antigens. Modified hyaluronic acid The polyglutamic acid used for acid production is preferably of low molecular weight type. Therefore, the molecular weight of polyglutamic acid is 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.

As a water-soluble coupling agent and/or coupling aid when introducing polyglutamic acid into hyaluronic acid, for example, a water-soluble coupling agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC), etc. Carbodiimides can be used. Carbodiimide is capable of bonding a carboxyl group and an amine. Alternatively, an NHS ester may be introduced into a carboxyl group using N-hydroxysuccinimide (NHS) or a water-soluble analog thereof (sulfo-NHS), and then bonded to an amine.

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

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

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

In the modified hyaluronic acid and/or its salt, 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 modified hyaluronic acid and/or its salt has resistance to decomposition by degrading enzymes in the body such as hyaluronidase. and tends to maintain the physiological activity and viscoelastic properties originally possessed by hyaluronic acid.

Additionally, the greater the number of polyglutamyl groups contained in one structural unit of hyaluronic acid, the longer the adhesion prevention material will remain in the body. The less the adhesion prevention material is placed in the living body, the shorter the remaining time of the anti-adhesion material placed in the living body tends to be. Therefore, by adjusting the number of polyglutamyl groups contained in one constituent unit of hyaluronic acid depending on the purpose, it is possible to adjust the remaining time of the anti-adhesion material placed in the living body.

In the modified hyaluronic acid and/or its salt, the number of polyglutamyl groups contained in one constituent unit of hyaluronic acid can be identified by the bicinchoninic acid (BCA) method or ¹ H-NMR spectrum analysis.

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

The concentration of hyaluronic acid in the hyaluronic acid solution is not particularly limited as long as it can dissolve hyaluronic acid. 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 modified hyaluronic acid is not particularly limited, but for example, the viscosity of an aqueous solution of modified hyaluronic acid decreases as the molecular weight of modified hyaluronic acid decreases, so a method of measuring the viscosity of an aqueous solution of modified hyaluronic acid is a simple method. It can be used as a method. In addition, a method combining size exclusion chromatography and a multi-angle light scattering detector, and a method combining the Morgan-Elson method and Carbazol sulfuric acid method can also be used.

The kinematic viscosity of the aqueous solution of modified hyaluronic acid and/or its salt can be measured using an Ubbelohde viscometer (manufactured by Shibata Kagaku Kikai Kogyo Co., Ltd.). At this time, select an Ubbelohde viscometer with a coefficient such that the number of seconds of flow is 200 to 1,000 seconds. The kinematic viscosity (unit: mm ² /s) can be determined by multiplying the number of seconds of flow of the aqueous solution measured by the Ubbelohde viscometer and the coefficient of the Ubbelohde viscometer.

The anti-adhesion material according to this embodiment may contain other natural materials, preservatives, functional materials, etc. in addition to modified hyaluronic acid.

The anti-adhesion material according to the present embodiment further includes a wetting agent, an emulsifier, a lubricant such as sodium lauryl sulfate and magnesium stearate, a colorant, a release agent, a coating agent, a preservative, and an antioxidant. Good too.

Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium disulfite, sodium sulfite, and the like; (2) ascorbyl palmitate. , butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol, and (3) citric acid, ethylenediaminetetraacetic acid (EDTA). , sorbitol, tartaric acid, phosphoric acid, and the like.

The anti-adhesion material according to this embodiment may be provided as a powder or a solution. When the anti-adhesion 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 the anti-adhesion agent. When the anti-adhesion material according to this embodiment is provided as a solution, it can be used as is. When the anti-adhesion material according to this embodiment is a solution, it may be a one-component type. Therefore, unlike the two-part type, there is no need to mix the two solutions immediately before surgery.

The adhesion prevention material according to this embodiment can be used to prevent adhesions after open surgery, and can also be used to prevent adhesions after laparoscopic surgery. The method of applying the anti-adhesion material according to the present embodiment is not particularly limited, but the anti-adhesion material according to the present embodiment may be dropped, applied, or sprayed onto the area where adhesion is to be prevented. It can also be injected.

Hereinafter, this 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 in any way by the following examples.

(Example 1: Synthesis of modified hyaluronic acid and evaluation of hyaluronidase resistance)
Modified hyaluronic acids having different contents of γ-polyglutamyl groups were synthesized by changing the theoretical blending molar ratio of γ-polyglutamic acid and one structural unit of hyaluronic acid using the following procedure. When the theoretical blending molar ratio of γ-polyglutamic acid and one structural unit of hyaluronic acid is 1:5, add hyaluronic acid (HA200, molecular weight 2,000 kDa, manufactured by Kikkoman Biochemifa) to a 100 mL beaker at a final concentration of Dissolve the solution in 25 mL of 50 mmol/L phosphate buffer (pH 6.0) to give a final concentration of 4.2 mg/mL, and further add EDAC (manufactured by Dojindo Laboratories), a carbodiimide catalyst, to a final concentration of 0.4 mg/mL. Add the mixture while stirring. Furthermore, 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, and the mixture was maintained at 25° C. for 1440 minutes. Here, the "theoretical blending molar ratio" refers to the raw material ratio, in other words, the molar ratio assuming that the coupling efficiency is 100%.

A reaction solution for producing modified hyaluronic acid in which the theoretical blending molar ratio of γ-polyglutamic acid and one structural unit of hyaluronic acid is 1:10, a reaction for producing modified hyaluronic acid in which the theoretical blending molar ratio is 1:50. A reaction solution for producing modified hyaluronic acid with a ratio of 1:100 was also prepared. In the preparation method of these reaction solutions, the theoretical blending molar ratio of γ-polyglutamic acid and one structural 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 using the same method as a 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 using a dialysis membrane with a molecular weight cutoff (MWCO) of 8,000, and the dialysis was completed. Thereafter, 256 mg of modified hyaluronic acid containing a γ-polyglutamyl group represented by the above general formula (1) was obtained by freeze-drying.

PGA in Table 1 means γ-polyglutamic acid, and the actual amount of grafting was determined by the bicinchoninic acid method (reference: Smith, P.K., et al. (1985) Anal. Biochem. 150, 76-85). It was determined by measuring the protein content.

(Example 2: Hyaluronidase resistance evaluation of modified hyaluronic acid)
The obtained modified hyaluronic acid was dissolved in distilled water (Milli-Q water) to a concentration of 10 mg/mL. To 20 μL of this solution were added 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. Furthermore, 1.0 mg/mL of hyaluronidase (derived from sheep testis, manufactured by Seikagaku Corporation) was added to the solution to give a final concentration of 0.025 mg/mL solution (0.05 mol/L sodium phosphate buffer; pH 5.5). 5 μL of 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 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 taken, adjusted to pH 8 with NaOH, and heat-treated at 95° C. for 5 minutes to stop the hyaluronidase reaction. As a control, a group without enzyme addition was also conducted in the same manner.

Each sample was subjected to SDS-PAGE to observe changes in the molecular weight of modified hyaluronic acid and the formation pattern of degradation products (Stains All staining, manufactured by Cosmo Bio). 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 that of modified hyaluronic acid (theoretical blending molar ratio of γ-polyglutamic acid and one constituent unit of hyaluronic acid is 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, we compared the degradation resistance of γ-polyglutamic acid depending on the amount of grafting, and as shown in Figure 2, the resistance to degradation improved as the amount of grafting increased. It was revealed that the acid showed excellent resistance to hyaluronidase. In addition, considering coupling efficiency, the molar ratio of γ-polyglutamyl groups in modified hyaluronic acid produced by blending γ-polyglutamic acid at a raw material molar ratio of 1/50 is considered to be approximately 1/500. It will be done. Furthermore, the molar ratio of γ-polyglutamyl groups in modified hyaluronic acid produced by blending γ-polyglutamic acid at a raw material molar ratio of 1/100 is considered to be approximately 1/1000.

(Example 3: Preparation of adhesion prevention material for evaluation of peritoneal abrasion model)
In the peritoneal abrasion model, the adhesion prevention material to be evaluated is placed between the abraded peritoneum and the organ. Therefore, when comparing and evaluating the adhesion prevention materials to be evaluated that have various forms and operability, such as film and gel forms, it is easy to place the adhesion prevention materials to be evaluated and perform surgical procedures. This model makes it easy to evaluate the adhesion prevention effect.

(i: modified hyaluronic acid)
Hyaluronic acid with 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 by standing overnight at 4°C. 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 in the dark. After 30 minutes, γ-polyglutamic acid (molecular weight 1,500 to 5,000, manufactured by Sigma-Aldrich) was added, and the mixture was allowed to react in the dark for 24 hours. After the reaction was completed, dialysis was performed for 48 hours using a dialysis membrane with a molecular weight cut off of 300 kDa. At this time, dialysis was performed using 1.5 L of 0.1 mol/L NaPB for the first 24 hours, and dialysis was performed using 3 L of Milli-Q water for the next 24 hours. Each solution was changed three times during a 24 hour period. Thereafter, the modified hyaluronic acid obtained by freeze-drying was dissolved to 10 mg/mL with 0.45% NaCl and 0.7% _NaHCO3 , and filter sterilized with a 0.45 μm filter to obtain a gel-like modified hyaluronic acid solution. Obtained.

(ii: Seprafilm)
Seprafilm was purchased from Kaken Pharmaceutical Co., Ltd. The package was opened aseptically and the Sepra film was trimmed into a square approximately 2 cm x 2 cm.

(iii: Adspray)
Adspray was purchased from Terumo Corporation, and a gel-like solution was prepared according to the instruction manual. In addition, the spray amount for rats, which will be described later, was approximately 200 μL based on the body weight ratio, based on the fact that one bottle of the product is consumed when used in humans.

(Example 4: Placement of adhesion prevention material to be evaluated)
The following animal experiments were conducted with the approval of the Tokyo Denki University Animal Experiment Management and Operation Committee. Sprague-Dawley (SD) rats (4 weeks old, male) were first anesthetized using isoflurane. A U-shaped incision was made in the abdomen of the rat so that the skin on the upper body side of the rat was connected and each 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 abrasions were then rinsed with antibiotic-containing saline. The time from abdominal opening to abdominal closure was standardized to 15 minutes, and at the time of abdominal closure, each of the adhesion prevention materials to be evaluated was placed between the exposed digestive tract and peritoneum.

Seprafilm was placed on the gastrointestinal side. Adspray was sprayed into the peritoneum and gastrointestinal tract. About 100 μL of modified hyaluronic acid was applied to the peritoneal side and about 100 μL to the organ side using a syringe. After the sample was placed, the skin at the incision was sutured. During the rearing period, the rats were given 5 pieces of CE-2 (Nippon CLEA) as feed per day, and sterilized tap water containing antibiotics was given to the rats as drinking water.

During the rearing period after the adhesion prevention material to be evaluated was placed, rats in all evaluation groups ingested a constant amount of both feed and drinking water. In addition, we were able to visually observe that they were moving around normally. Furthermore, no major differences were observed in the weight changes of the animals during the breeding period among all groups. From this, it was inferred that none of the adhesion prevention materials evaluated had any effect on the health condition of the animals. The rats were 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)
The abdomen of the euthanized rat was incised, and the presence or absence of adhesions in the peritoneum and gastrointestinal tract was visually observed. The area to be evaluated for adhesions formed within the abdominal cavity was limited to the initially abraded intraperitoneal surface, and the formation of adhesions at sutures was excluded. Representative photographs of rats with abdominal incisions are shown in Figure 3.

In the positive control group, in which none of the adhesion prevention materials to be evaluated was placed, adhesions were observed between the peritoneum and organs. In the Seprafilm indwelling group and the Adspray indwelling group, the area of adhesion was smaller compared to the positive control group. Furthermore, in both groups, almost no adhesion was observed between the peritoneum and the gastrointestinal tract, and adhesion was observed only with the liver. These results revealed that when Seprafilm and Adspray were used, adhesions were likely to occur between the liver and the peritoneum. No adhesions were observed in the modified hyaluronic acid implantation group.

The degree of observed adhesion was scored using the following evaluation criteria.
No apparent adhesion formation observed: 0 points Adhesion formation observed in less than 25% of the evaluated area: 1 point Adhesion formation observed in less than 50% of the evaluated area: 2 points Adhesion formation observed in less than 75% of the evaluated area : 3 points Adhesion formation observed in 75% or more of the evaluated area: 4 points The average value of the adhesion scores of the three animals in each evaluation target adhesion prevention 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. Furthermore, the score of the Adspray group was lower than that of the Seprafilm 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 scores of the modified hyaluronic acid group were even lower than those of the Adspray group. Therefore, it was shown that modified hyaluronic acid has an excellent adhesion prevention effect.

The above results suggested that because the gel-like anti-adhesion material has an irregular shape, it is likely to spread between the gastrointestinal tract due to gastrointestinal peristalsis. On the other hand, it was suggested that the sheet-shaped anti-adhesion agent could only cover the surface of the organ and could not penetrate between the gastrointestinal tract.

(Example 6: Histological evaluation)
After observing the degree of adhesion formation, the full thickness of the skin was peeled off, and tissue pieces from the peritoneal cavity side were collected. The collected tissue pieces were fixed with 4% paraformaldehyde, washed with water, dehydrated with ethanol and xylene, and embedded in paraffin. The paraffin-embedded tissue pieces were sectioned into 8 μm thick sections using a microtome, and histological evaluation was performed 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 a single layer of squamous epithelium, were observed on the surface of the muscle layer, as shown in the left diagram of FIG. Furthermore, in the original color image, a blue-stained collagen layer was observed only between the mesothelial cells and the connective tissue immediately below them, and between the muscle fibers, as shown in the right figure of FIG.

In the positive control group, as shown in the left diagram of FIG. 6, the peritoneal tissue in the abrasion scar was thickened with inflammatory cells, fibroblasts, etc., and was observed to be connected to the small intestine. These histological data confirmed the presence of adhesion formation seen in the anatomical findings. Furthermore, from the right diagram of FIG. 6, matrix formation by collagen fibers was confirmed near the boundary between the regenerated tissue and the muscle layer. However, the area near 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 diagram of FIG. In addition, the regenerated tissue was observed to be porous, suggesting that Seprafilm is in the process of being degraded and absorbed, and that it is being sequentially replaced by inflammatory cells and fibroblasts that have invaded inside Seprafilm. . In addition, it was confirmed that inflammatory cells and fibroblasts had invaded deep into the muscle layer. This suggested that inflammation continued one week after the surgery. In the right image of FIG. 7, the regenerated tissue was not stained very blue in the original color drawing, suggesting that collagen-induced fibrosis had not progressed much at this point. On the other hand, in the muscle layer, invasion of fibroblasts was observed, and matrix formation by collagen fibers was observed.

In the Adspray indwelling group, thickening of the peritoneal tissue was observed, as shown in the left diagram of FIG. Furthermore, at the adhesion site with the liver, it was confirmed that inflammatory cells invaded into the liver tissue and were directly connected to the liver. Many large, cuboidal cells were observed on the surface layer of the adhesion site. It was suggested that these were macrophages that had migrated into the peritoneal cavity. The presence of macrophages was confirmed, suggesting that the inflammatory response was continuing. In addition, no trace of Adspray was observed in histological evaluation, suggesting that Adspray was degraded and absorbed one week after the surgery. Furthermore, as shown in the right image of Figure 8, the repaired area of the abrasion wound was stained blue by MT staining in the original color drawing, which confirmed that it was regenerating with fibrosis. Even in areas where no adhesions had occurred, there was considerable fibrosis overall. Therefore, it was suggested that the regeneration mechanism in the Adspray group was different from that in the Seprafilm group.

In the modified hyaluronic acid indwelling group, as shown in the left diagram of FIG. 9, thickening of the peritoneal tissue was observed compared to the normal tissue. However, the thickness of the regenerated tissue was thinner compared to the positive control group, Seprafilm indwelling group, and Adspray indwelling group, suggesting that modified hyaluronic acid may be able to suppress tissue thickening. Furthermore, as shown in the right diagram of FIG. 9, fibrosis due to collagen was observed in the regenerated tissue. Furthermore, traces of modified hyaluronic acid were confirmed as porous areas. These results showed that modified hyaluronic acid remained after the decomposition and absorption process one week after the surgery.

The results of histological evaluation showed that the tissue repair process differs depending on the adhesion prevention material being evaluated. Furthermore, the persistence of the adhesion prevention material to be evaluated may be related to tissue regeneration, and Seprafilm, which has slow bioabsorption and is difficult to decompose, had high persistence and slow tissue regeneration. In contrast, Adspray, which is bioabsorbable and easily degraded, had low persistence and rapid tissue regeneration. However, although Adspray was more effective in preventing adhesion than Seprafilm, adhesion formation was still observed. In contrast, modified hyaluronic acid remained even after one week after surgery, and tissue regeneration was rapid. Furthermore, modified hyaluronic acid showed excellent adhesion prevention effects.

Claims (9)

It contains a 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, the modified hyaluronic acid and/or its salt has resistance to decomposition by hyaluronidase, and is a constituent of hyaluronic acid. An anti-adhesion material in which the number of polyglutamyl groups contained in the unit is 0.0015 or more and 0.5 or less . The anti-adhesion material according to claim 1, wherein the polyglutamyl group is a γ-polyglutamyl group. The anti-adhesion material according to claim 1 or 2 , wherein the modified hyaluronic acid and/or its salt is dissolved in a solution. The anti-adhesion material according to claim 1 or 2 , wherein the modified hyaluronic acid and/or its salt is a powder. Production of an anti-adhesion material containing modified hyaluronic acid and/or a salt thereof modified with a polyglutamyl group and having resistance to hyaluronidase degradation, which comprises reacting hyaluronic acid with polyglutamic acid having a molecular weight of 750 or more and 20,000 or less. A method,
In reacting the hyaluronic acid and polyglutamic acid, grafting 1 mol or more of polyglutamic acid per 50 mol of one constituent unit of hyaluronic acid in raw material ratio;
A method for producing an anti-adhesion material . The method for producing an anti-adhesion material according to claim 5 , wherein the hyaluronic acid and the polyglutamic acid are reacted in the presence of a carbodiimide catalyst. The method for producing an anti-adhesion material according to claim 5 or 6 , wherein the polyglutamyl group is a γ-polyglutamyl group. The method for producing an anti-adhesion material according to any one of claims 5 to 7 , 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 5 to 7 , further comprising powdering the modified hyaluronic acid and/or its salt.

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