JP4902134B2 - Anti-adhesive material - Google Patents

Description

The present invention relates to an adhesion preventing material containing a hyaluronic acid compound that is a reaction product of hyaluronic acid and phosphatidylethanolamine.

  Post-surgical adhesion is an effective prevention method because it occurs when the peritoneum on the damaged organ surface regenerates by combining with the peritoneum of other tissues and may cause ileus, pain, infertility, etc. Is required. It is known that the process of peritoneal regeneration begins with infiltration of polynuclear leukocytes into the damaged tissue surface and is almost completed after 7 to 8 days when the damaged part is entirely covered with mesothelial cells. Therefore, during this period, it is considered that the formation of adhesions can be prevented by physically blocking the contact between the damaged tissue and the surrounding tissue.

  Under such circumstances, development of an adhesion preventing material using a polymer is being carried out all over the world. One particularly promising polymer is hyaluronic acid, a component of the extracellular matrix found throughout the human body. Hyaluronic acid solutions have been shown to reduce postoperative adhesion formation in animal models such as abdominal surgery (Non-Patent Document 1) and orthopedic surgery (Non-Patent Document 2). However, hyaluronic acid itself is characterized by a very rapid absorption time, which is incompatible with the residence time necessary to prevent adhesions. Actually, no statistically significant effect has been shown in clinical trials (Non-patent Document 3).

  Therefore, in order to strengthen the adhesion formation reducing effect, hyaluronic acid with improved residence time has been developed. Examples include bisepoxide crosslinked hyaluronic acid (Patent Document 1), divinylsulfone crosslinked hyaluronic acid (Patent Documents 2 and 3), formaldehyde crosslinked hyaluronic acid (Patent Document 4), and the like. However, in any case, since the crosslinking agent is a non-bioabsorbable substance, safety is a concern, and an anti-adhesion material with excellent safety is required. In fact, it has been shown that glycosaminoglycans bound with lipids suppress epithelial cell extension (Patent Document 5), but the effect of preventing adhesion in the living body is not clear.

  Sepla film (Kaken Pharmaceutical, Genzyme) has already been used clinically as an anti-adhesion material excellent in safety, and it has been suggested that it is effective in reducing adhesion (Non-Patent Documents 4 and 5). Sepra film gels in 24 to 48 hours after application, and is stored in the application site for approximately 7 days to physically isolate damaged tissue and surrounding tissue, thereby preventing adhesion. Demonstrate. Moreover, since the Sepra film is discharged to the outside of the body within 28 days after the application, there is an advantage that surgical removal is unnecessary. However, as a problem, since there is no flexibility in a dry state, it cannot be used if the use site has a complicated shape, and it is not suitable for a laparoscopic operation.

Japanese Patent Laid-Open No. 7-97401 US Pat. No. 4,582,865 US Pat. No. 4,605,691 JP-A-60-130601 JP 2003-160498 A Fertil Steril. 56, 563-567 (1991) J Hand Surg [Am]. 17, 935-941 (1992) J Hand Surg [Am]. 17, 132-136 (1992) Fertil Steril. 66, 904-910 (1996) J Am Coll Surg. 183, 297-306 (1996)

  An object of the present invention is to provide an anti-adhesion material excellent in biocompatibility and retention. More preferably, it is to provide an adhesion preventing material excellent in handleability.

  The inventors of the present invention have found that a hyaluronic acid compound, which is a reaction product of hyaluronic acid and phosphatidylethanolamine, is useful as an adhesion-preventing material, and have reached the present invention.

（１）
ここでは、Ｒは下記式（１）−ａ

（１）−ａ
で表される基、−ＯＨまたは−ＯＮａであり、Ｒ^１は炭素数１０〜２８のアルキル基またはアルケニル基であり、ｎは５０〜５０，０００の数である、但しＲの１０〜１００％が上記式（１）−ａで表される基であるものとする、で表される化合物を含有する癒着防止材。
２．ｎが１，０００〜１０，０００である上記に記載の癒着防止材。
３．Ｒの１０〜５０％が上記式（１）−ａで表される基である上記に記載の癒着防止材。
４．Ｒの３０〜５０％が上記式（１）−ａで表される基である上記に記載の癒着防止材。
５．式（１）−ａにおける２つの基Ｒ１ＣＯ−がいずれもオレオイル基である上記に記載の癒着防止材。 The present invention is as follows.
1. Following formula (1)

(1)
Here, R represents the following formula (1) -a

(1) -a
-OH or -ONa, R ¹ is an alkyl or alkenyl group having 10 to 28 carbon atoms, and n is a number of 50 to 50,000, provided that 10 to 100% of R An adhesion-preventing material containing a compound represented by the formula (1) -a:
2. The adhesion preventing material as described above, wherein n is 1,000 to 10,000.
3. 10-50% of R is the above formula (1) antiadhesive material described above is a group represented by -a.
4). 30-50% of R is the above formula (1) antiadhesive material described above is a group represented by -a.
5. The adhesion preventing material according to the above, wherein the two groups R1CO- in formula (1) -a are both oleoyl groups.

  The anti-adhesion material of the present invention is excellent in safety and retention in the body and exhibits an excellent anti-adhesion effect. In particular, when the hyaluronic acid compound is a paste-like gel, it has sufficient flexibility and is therefore easy to handle, can be applied to a complex-shaped part, and can also be applied to surgery using an endoscope.

  Hereinafter, the present invention will be described in detail. In addition, these Examples etc. and description illustrate this invention, and do not restrict | limit the scope of the present invention. It goes without saying that other embodiments may belong to the category of the present invention as long as they match the gist of the present invention.

で表される。上記式（１）中、Ｒは下記式（１）−ａ

で表されるホスファチジルエタノールアミノ基であるか、−ＯＨまたは−ＯＮａである。但し、Ｒの１０〜１００％がホスファチジルエタノールアミノ基である必要がある。ホスファチジルエタノールアミノ基の割合がＲの１０％未満であると、所望の癒着防止効果が達成されない。 The hyaluronic acid compound of the present invention has the following formula (1)

It is represented by In the above formula (1), R represents the following formula (1) -a

It is a phosphatidylethanolamino group represented by -OH or -ONa. However, 10 to 100% of R needs to be a phosphatidylethanolamino group. When the ratio of the phosphatidylethanolamino group is less than 10% of R, the desired adhesion preventing effect is not achieved.

  When the adhesion preventing material of the present invention is an irregular paste-like gel, the shape can be easily changed. Therefore, the adhesion preventing material of the present invention is preferably in the form of a gel from the viewpoint of handleability. If the proportion of phosphatidylethanolamino group in R exceeds 50%, a paste-like gel will not be formed. Therefore, for the purpose of forming a gel-like adhesion preventing material, the proportion of phosphatidylethanolamino group in R is 10 to 50%. Preferably there is.

  As the ratio of the phosphatidylethanolamino group in R increases, the retention is improved, and the anti-adhesion effect can be exhibited over a long period of time. In this respect, the proportion of phosphatidylethanolamino groups in R is preferably 30% or more.

  That is, in the adhesion preventing material of the present invention, the ratio of the phosphatidylethanolamino group in R is preferably 30 to 50%.

  R is an alkyl or alkenyl group having 10 to 28 carbon atoms, preferably 14 to 20 carbon atoms, and n is 50 to 50,000, preferably 300 to 30,000, more preferably 1,000 to 10, 000. Examples of the alkyl group having 10 to 28 carbon atoms of R include decyl, undecyl, lauryl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, stearyl, and eicosanyl.

Moreover, as a C10-C28 alkenyl group, it respond | corresponds to the said alkyl group illustrated. An alkenyl group having 1 to 3 carbon-carbon unsaturated bonds, such as an oleyl group, can be mentioned. As the compound represented by the above formula (1), those in which both of the two groups R ¹ CO— in the formula (1) -a are oleoyl groups are preferable.

  The hyaluronic acid compound represented by the above formula (1) can be produced, for example, by reacting hyaluronic acid with phosphatidylethanolamine.

The hyaluronic acid used in the present invention can be either extracted from animal tissue or produced by fermentation. The strain used in the fermentation method is preferably a microorganism having the ability to produce hyaluronic acid belonging to the genus Streptococcus. Gazette). Those cultured and purified using these mutant strains are used. The molecular weight of hyaluronic acid is preferably about 1 × 10 ⁵ to 1 × 10 ⁷ . The hyaluronic acid referred to in the present invention includes alkali metal salts thereof, for example, sodium, potassium and lithium salts.

  As the phosphatidylethanolamine used in the present invention, either phosphatidylethanolamine extracted from animal tissue or synthesized can be used. Examples of phosphatidylethanolamine and derivatives thereof include the following. Dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, diarachidyl phosphatidylethanolamine, dibehenoyl phosphatidylethanolamine, dilignocelloyl phosphatidylethanolamine, dicelloyl oil Phosphatidylethanolamine, dimontanoyl phosphatidylethanolamine, dilaurooleoyl phosphatidylethanolamine, dimyristooleoyl phosphatidylethanolamine, dipalmioleoyl phosphatidylethanolamine, dioleoylphosphatidylethanolamine, dinerbonoyl phosphatidylethanolamine, Jikimenoi Phosphatidylethanolamine, Jiri Noreno yl phosphatidylethanolamine, di Hirago noil phosphatidylethanolamine, Giara Kido noil phosphatidylethanolamine, and di docosahexaenoyl phosphatidylethanolamine. Among these, dioleoylphosphatidylethanolamine is preferable from the viewpoint of solubility.

Moreover, it is preferable that the usage-amount of phosphatidylethanolamine is 10-100 equivalent with respect to 100 equivalent of carboxyl groups of hyaluronic acid, 10-50 equivalent is more preferable, Furthermore, it is preferable that it is 30-50 equivalent. .
When the hyaluronic acid compound is solid, it is also preferable to add a biocompatible plasticizer for the purpose of imparting flexibility.

The details of the present invention will be described more specifically by the following examples. However, the present invention is not limited to these examples.
Sodium hyaluronate, tetrahydrofuran, 0.1 M HCl, 0.1 M NaOH, 1-Ethyl-3- [3- (dimethylamino) propyl] -carbodi-imide (EDC), 1-Hydroxybenzotriazole (HOBt) used in this example Is Wako Pure Chemical Industries, Ltd., L-α-dioleoylphosphatidylethanolamine (COATSOME ME-8181) is Nippon Oil & Fats, ethanol for disinfection (Wako Pure Chemical Industries), pentobarbital sodium (Nembutal Injection) Dainippon Pharmaceutical Co., Ltd.), isodine disinfectant (Meiji Seika), physiological saline (Otsuka Pharmaceutical), rat (Nippon Charles River Co., Ltd.).

[Example 1]
(Manufactured by compound tone)
220 mg (0.000066 mol) of L-α-dioleoylphosphatidylethanolamine (20 equivalents relative to 100 equivalents of the carboxyl group of hyaluronic acid) was dissolved in 200 ml of tetrahydrofuran / water = 1/1 (v / v). To this solution, 500 mg of sodium hyaluronate was added, and 0.1 M HCl / 0.1 M NaOH was added to adjust the pH to 6.8. 1-Ethyl-3- [3- (dimethylamino) propyl] -carbodiimide (EDC) 60 mg (0.000066 mol) and 1-hydroxybenzotriazole (HOBt) 50 mg (0.000066 mol) in 10 ml of an aqueous solution of tetrahydrofuran / water = 1/1 Dissolved and added to the reaction system and stirred overnight. After stirring, dialysis purification was performed and freeze-dried to obtain the desired product. Confirmation was performed by 1HNMR (JEOL JNM-alpha400) to confirm the production of the target product.
The lyophilized product 10mg dissolved in physiological saline 990 mg, was manufactured by adjusting the concentration 1 wt% of the hydrogel.

(Animal evaluation)
Rats were weighed on the day of intraperitoneal adhesion model preparation, and 40 individuals were selected by excluding heavy individuals (230.9 g or more) and light individuals (200.6 g or less), and the average weight of each test group Were divided into groups so that (1) Sushil Taparia, Ravindra Sharma, Suresh Narain Mathur and Ved Prakash Sharma: Intraperitoneal aprotinin (Antagosan) in the prevention of postoperative adhesions -An experimental study in rats-, Asian Med. J. 28 (1), 58-64 (1985), 2) HS Sachdeva, LV Gutierrez and AG Cox: Silicone fluid and steroids in the prevention of adhesions., Br. J Surg., 58 (5), 382-384 (1971)). That is, the rat was fixed in the dorsal position under anesthesia with intraperitoneal administration of sodium pentobarbital (30 mg / kg), shaved abdomen, and then disinfected with ethanol for disinfection. Further, after the surgical area was disinfected with isodine disinfectant, a 3-4 cm incision was made along the midline of the abdomen to expose the cecum. A certain area (1-2 cm ² ) of the exposed cecum was abraded with sterilized gauze until punctate bleeding occurred. After returning the cecum into the abdominal cavity, hyaluronic acid hydrogel (1 mL) was applied to the rubbing site, the muscle layer of the incision was continuously sutured, and the skin was sutured with 4 to 5 needles. The wound was disinfected with isodine disinfectant and then returned to the cage. 28 days after model preparation, the animals were laparotomized under pentobarbital sodium anesthesia, the degree of intraperitoneal adhesion was visually observed, and scored according to the criteria shown below.

(Grade classification)
Grade 0 (score 0): No adhesion observed Grade 1 (score 1): Adhesion grade 2 (score 2) that is thin and easily separable: Although it is a narrow range, it can withstand mild traction Weak adhesion grade 3 (score 3): fairly solid adhesion or at least 2 adhesions grade 4 (score 4): 3 or more adhesions

The degree of intraperitoneal adhesion of each animal was scored according to the grade classification, and the result was expressed as an average value ± standard error (mean ± SE). The test was performed using the Wilcoxon rank sum test as a test between the control group and the test substance group. The statistical software used was The SAS System Release 8.2 (TS2M0) for Windows (SAS Institute Inc.) and its linked system EXSAS Version 7.10 (Arm Corp.).
The effect of applying the test substance on the rat intraperitoneal adhesion model for 28 days was examined, and the results are shown in FIG.

[Example 2]
L-α-dioleoylphosphatidylethanolamine 440 mg (0.000132 mol) (40 equivalents to 100 equivalents of the carboxyl group of hyaluronic acid), 1-Ethyl-3- [3- (dimethylamino) propyl] -carbodiimide (EDC) 120 mg A hydrogel was prepared in the same manner as in Example 1 except that 100 mg (0.000132 mol) of 1-hydroxybenzotriazole (HOBt) (0.000132 mol) was used, and intraperitoneal adhesion evaluation of rats was performed. The results are shown in FIG.

[Comparative Example 1]
Rats were fixed in the dorsal position under anesthesia with intraperitoneal administration of sodium pentobarbital (30 mg / kg), shaved abdomen, and then disinfected with ethanol for disinfection. Further, after the surgical area was disinfected with isodine disinfectant, a 3-4 cm incision was made along the midline of the abdomen to expose the cecum. A certain area (1-2 cm ² ) of the exposed cecum was abraded with sterilized gauze until punctate bleeding occurred. The cecum was returned to the abdominal cavity, the muscle layer of the incision was continuously sutured, and the skin was sutured with 4 to 5 needles. The wound was disinfected with isodine disinfectant and then returned to the cage. 28 days after model preparation, the animals were laparotomized under pentobarbital sodium anesthesia, and the degree of intraperitoneal adhesion was visually observed and scored. The results are shown in FIG.

[Comparative Example 2]
55 mg (0.0000165 mol) of L-α-dioleoylphosphatidylethanolamine (5 equivalents relative to 100 equivalents of the carboxyl group of hyaluronic acid), 15 mg of 1-Ethyl-3- [3- (dimethylamino) propyl] -carbodiimide (EDC) A hydrogel was prepared in the same manner as in Example 1 except that (0.0000165 mol) and 12.5 mg (0.0000165 mol) of 1-hydroxybenzotriazole (HOBt) were used, and intraperitoneal adhesion evaluation of rats was performed. The results are shown in FIG.

  As shown in FIG. 1, the average adhesion score of Comparative Example 1 was 2.7 ± 0.4. In contrast, the hyaluronic acid compound of Example 1 showed an adhesion score of 1.3 ± 0.5, and although there was no statistically significant difference in the adhesion score compared to Comparative Example 1, the adhesion score Showed a decreasing trend. The hyaluronic acid compound of Example 2 showed an adhesion score of 1.2 ± 0.4 and also showed a statistically significant decrease in adhesion score compared to Comparative Example 1 (P <0.05). On the other hand, the average adhesion score with the hyaluronic acid compound of Comparative Example 2 was 2.4 ± 0.3, and no significant difference was observed in the adhesion score as compared with Comparative Example 1.

  From the above results, the hyaluronic acid compounds of Examples 1 and 2 showed a decrease in adhesion score when applied for 28 days, suggesting an adhesion prevention effect.

  The anti-adhesion material of the present invention has excellent retention stability in the body and is useful as an anti-adhesion material. In particular, when the anti-adhesion material is in a gel form, it can be applied to a part having a complicated shape, and can also be applied to an operation using an endoscope.

Adhesion score evaluation in Examples 1 and 2 and Comparative Examples 1 and 2.

Claims (5)

Following formula (1)

(1)
Here, R represents the following formula (1) -a

(1) -a
-OH or -ONa, R1 is an alkyl or alkenyl group having 10 to 28 carbon atoms, and n is a number of 50 to 50,000, provided that R is 10 to 100
% Is a group represented by the above formula (1) -a,
An adhesion-preventing material containing a compound represented by:   2. The adhesion preventing material according to claim 1, wherein n is 1,000 to 10,000. Antiadhesive material according to claim 1 or 2 10-50% of R is a group represented by the formula (1) -a. Antiadhesive material according to claim 3 30-50% of R is a group represented by the formula (1) -a. The adhesion preventing material according to any one of claims 1 to 3, wherein each of the two groups R ¹ CO- in formula (1) -a is an oleoyl group.

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