Anti-adhesion composition

Abstract

To provide an adhesion prevention material with high adhesion prevention effect. [Solution] The present invention relates to a biocompatible sponge-like laminate comprising a first and second sponge-like layer of monovalent metal salt of low-endotoxin alginate, at least a portion of which is crosslinked with a curing agent, wherein the weight-average molecular weight of the monovalent metal salt of alginate in the first layer is 30,000 to 300,000, and the weight-average molecular weight of the monovalent metal salt of alginate in the second layer is 1,000 to 200,000, and the weight-average molecular weights are measured by the GPC-MALS method after decrosslinking treatment, and the weight-average molecular weight of the monovalent metal salt of alginate in the first layer is higher than that of the monovalent metal salt of alginate in the second layer.

Description

[Technical field]

[0001] The present invention relates to an adhesion preventing material, a method for producing the same, and a sponge-like laminate. [Background technology]

[0002] Adhesion refers to a state in which the surfaces of tissues that should be separated from each other are connected or fused by fibrous tissue. Adhesion occurs when an exudate containing fibrin is produced on the surface of tissue due to trauma or inflammation, and this exudate becomes organized and connects or fuses the tissue surfaces. Trauma to the surface of tissue during surgery, inflammation caused by trauma, and inflammation caused by drying of the tissue surface during surgery are causes of adhesion.

[0003] Adhesion can sometimes cause infertility, intestinal obstruction, and chronic pelvic pain. In addition, a second surgery may be necessary to remove adhesions that have developed after surgery. For example, multiple surgeries are effective for recurrent cases of liver cancer, but the decision on whether or not to perform a second surgery, the risk of treatment, the amount of bleeding during surgery, and the duration of surgery all depend heavily on the prevention of adhesions after the previous surgery. For these reasons, it is necessary to prevent adhesions, and various measures have been taken to prevent adhesions.

[0004] Some of the means for preventing adhesions include providing a physical barrier between the site of trauma or inflammation and the adjacent tissue to prevent the joining or fusing of the tissues, such as a sheet-like barrier.

[0005] Specifically, sheet-type products include polytetrafluoroethylene (PTFE) film (Preclude (product name) (WL Gore and Associates, Inc.)), a sheet containing hyaluronic acid (HA) and carboxymethylcellulose (CMC) (Seprafilm (product name) (Genzyme GmbH)), and a regenerated oxidized cellulose sheet (INTERCEED (product name) (Johnson & Johnson)). Of these, PTFE film is not biodegradable and has the problem of remaining in the body. Although the sheets containing HA and CMC and the regenerated oxidized cellulose sheets are biodegradable, they cannot completely prevent severe adhesions such as those that occur after liver resection, and there is room for improvement in terms of the effectiveness of adhesion prevention.

[0006] Here, it is known that biocompatible materials selected from proteins such as collagen and polysaccharides such as carboxymethylcellulose, hyaluronic acid, and alginic acid are made into sheets or particles and used as medical absorbents, medical patches, adhesion prevention materials, biological tissue reinforcement materials, and the like (Patent Documents 1 to 6). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Unexamined Patent Publication No. 48-79870 [Patent Document 2] JP 2003-126235 A [Patent Document 3] International Publication No. 2005 / 26214 [Patent Document 4] JP 2011-25013 A [Patent Document 5] JP 2013-165884 A [Patent Document 6] Special Publication No. 2016-502874 Summary of the Invention [Problem to be solved by the invention]

[0008] Under these circumstances, there has been a demand for an adhesion prevention material that has at least one of the following properties: high adhesion prevention effect, ability to suppress both wound adhesion and de novo adhesion, no adverse effects on the living body to which it is applied, no impediment to wound healing, usability for intestinal anastomosis, etc., ease of application via a trocar in endoscopic surgery, and ability to adjust the application position and reapplication. [Means for solving the problem]

[0009] The present inventors have conducted extensive research into adhesion anti-adhesion materials that combine the advantages of film (sheet)-type adhesion anti-adhesion materials with the advantages of spray (liquid / gel)-type adhesion anti-adhesion materials, using animal adhesion models simulating various types of clinical surgery. As a result, they have found that an adhesion anti-adhesion material that is applicable to the body and in which the first and second layers have different dissolution rates, specifically, an adhesion anti-adhesion material that comprises a sponge-like laminate that is applicable to the body and comprises a spongy first layer containing a monovalent metal salt of low-endotoxin alginic acid having a relatively high weight-average molecular weight, and a spongy second layer containing a monovalent metal salt of low-endotoxin alginic acid having a relatively low weight-average molecular weight, is effective not only in preventing adhesions at the surgical site, but also in preventing adhesions over a wide range of applicable areas, thereby completing the present invention.

[0010] The present invention is as follows. [1-1] An adhesion prevention material comprising a sterilized, biocompatible sponge-like laminate comprising first and second layers of sponge-like low-endotoxin monovalent metal salt of alginic acid, at least a portion of which is cross-linked with a hardener, wherein the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is 10,000 to 2,000,000 and the weight-average molecular weight of the monovalent metal salt of alginic acid in the second layer is 1,000 to 1,000,000, the weight-average molecular weights being measured by a GPC-MALS method after a de-cross-linking treatment, and the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than the weight-average molecular weight of the monovalent metal salt of alginic acid in the second layer. [1-1a] An adhesion prevention material comprising a sponge-like laminate applicable to a living body, comprising a first sponge-like layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 10,000 to 2,000,000, and a second sponge-like layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 1,000 to 1,000,000, wherein the weight-average molecular weights are measured by a GPC-MALS method, and the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that of the second layer. [1-1b] An adhesion prevention material comprising a sponge-like laminate applicable to a living body, comprising first and second layers of sponge-like low-endotoxin monovalent metal salt of alginic acid, at least a portion of which is crosslinked with a hardener, wherein the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is 30,000 to 300,000 and the weight-average molecular weight of the monovalent metal salt of alginic acid in the second layer is 1,000 to 200,000, the weight-average molecular weights being measured by a GPC-MALS method after a de-crosslinking treatment, and the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than the weight-average molecular weight of the monovalent metal salt of alginic acid in the second layer.

[0011] [1-2] The adhesion preventing material according to the above [1-1], [1-1a] or [1-1b], wherein either the first layer or the second layer contains a hardener. [1-3] The adhesion preventing material according to any one of the above [1-1] to [1-2], wherein both the first layer and the second layer contain a curing agent. [1-4] The total amount of low-endotoxin monovalent metal salt of alginic acid used in the first and second layers is 0.1 mg / cm 2 ~3mg / cm 2 The adhesion preventing material according to any one of the above [1-1] to [1-3], wherein the adhesion preventing material has a viscosity within the range of 0.1 to 1.0 μm. [1-5] The adhesion preventing material according to any one of the above [1-1] to [1-4], wherein the endotoxin content of the monovalent metal salt of alginic acid in the first layer and the second layer is 500 EU / g or less.

[0012] [1-6] The adhesion barrier according to any one of the above [1-1] to [1-5], wherein the monovalent metal salt of alginic acid in the first layer and the second layer is sodium alginate or potassium alginate. [1-7] The hardener for the first and second layers is CaCl 2 , CaSO 4 , ZnCl 2 , SrCl 2 , FeCl 3 , BaCl 2 , CaHPO 4 The adhesion preventing material according to any one of the above [1-1] to [1-6], which is at least one metal ion compound selected from the group consisting of calcium gluconate, calcium oxalate, and calcium lactate. [1-8] The adhesion barrier according to any one of the above [1-1] to [1-7], for application with the first layer facing the surface of the wound site. [1-9] The adhesion preventing material according to any one of the above [1-1] to [1-8], wherein the sponge-like laminate has been sterilized by electron beam and / or gamma ray irradiation and / or ethylene oxide gas at an absorbed dose of 10 kGy to 150 kGy.

[0013] [1-10] An adhesion prevention material comprising a sponge-like laminate applicable to a living body, the sponge-like laminate including a first layer and a second layer, each of which comprises a monovalent metal salt of low-endotoxin alginic acid at least partially crosslinked with a hardener, wherein the dissolution rate of the first layer is slower than that of the second layer. [1-10a] An adhesion prevention material comprising a sponge-like laminate applicable to a living body, the sponge-like laminate including a first layer and a second layer, each of which contains a monovalent metal salt of low-endotoxin alginic acid, wherein the dissolution rate of the first layer is slower than that of the second layer. [1-11] An anti-adhesion material according to [1-10] or [1-10a] above, in which, in a dissolution test using the dissolution of a monovalent metal salt of alginic acid in a phosphate buffer solution of pH 7.5 as an index, the proportion of the amount of dissolution of a monovalent metal salt of alginic acid in the first layer is less than 50% at 1 hour and less than 70% at 2 hours from the start of measurement, when the amount of dissolution of the monovalent metal salt of alginic acid in the second layer is taken as 100%. [1-12] The adhesion prevention material of [1-10] or [1-10a] above, in which, in a dissolution test using the dissolution of a monovalent metal salt of alginic acid in a phosphate buffer solution of pH 7.5 as an index, 25±10% by weight of the monovalent metal salt of alginic acid is dissolved in the first layer within 1 hour, and 80±10% by weight is dissolved within 4 hours, and 70±10% by weight of the monovalent metal salt of alginic acid is dissolved in the second layer within 1 hour, and 90±10% by weight is dissolved within 4 hours.

[0014] [1-13] An adhesion prevention material according to [1-10] or [1-10a] above, in which, in a dissolution test using the dissolution of a monovalent metal salt of alginic acid in a phosphate buffer solution of pH 7.5 through agarose gel as an index, the ratio of the amount of dissolution of a monovalent metal salt of alginic acid from the first layer to the amount of dissolution of a monovalent metal salt of alginic acid from the second layer, taken as 100%, is 30 to 70% from the second day after the start of measurement. [1-14] The anti-adhesion material of [1-10] or [1-10a] above, in which, in a dissolution test using the dissolution of a monovalent metal salt of alginic acid into a phosphate buffer solution of pH 7.5 through agarose gel as an index, about 15±5% by weight of the monovalent metal salt of alginic acid is dissolved within 2 days and about 25±10% by weight is dissolved within 8 days in the first layer, and about 30±8% by weight of the monovalent metal salt of alginic acid is dissolved within 2 days and about 60±10% by weight is dissolved within 8 days in the second layer.

[0015] [1-15] The adhesion preventing material according to any one of the above [1-1] to [1-14], which is obtained by pressing a sponge-like laminate. [1-16] The adhesion preventing material according to any one of the above [1-1] to [1-15], which has one or more of the following characteristics: (1) A piece of JIS K6251 tensile tester No. 8 is subjected to a tensile test, and the Young's modulus determined based on the obtained stress-strain curve is 0.3 to 300 MPa. (2) A piece of JIS K6251 tensile tester No. 8 is subjected to a tensile test, and the breaking strength determined based on the obtained stress-strain curve is 5 to 5,000 kPa. (3) A piece of JIS K6251 tensile tester No. 8 is subjected to a tensile test, and the ratio of breaking strength to Young's modulus of the sponge-like laminate determined based on the obtained stress-strain curve is 2 to 50. (4) When the sponge-like laminate is contacted with an agarose gel soaked in phosphate-buffered saline for 2 to 6 hours, the weight increase rate of the sponge-like laminate is 200 to 50,000%, assuming that the weight of the sponge-like laminate before contact with phosphate-buffered saline is 100%. (5) In a dissolution test using the dissolution of a monovalent metal salt of alginic acid into a phosphate buffer solution of pH 7.5 through agarose gel as an indicator, the ratio of the amount of dissolution of the monovalent metal salt of alginic acid from the first layer to the amount of dissolution of the monovalent metal salt of alginic acid from the second layer, taken as 100%, is 30 to 70% from the second day after the start of the measurement. (6) In a dissolution test using the dissolution of a monovalent metal salt of alginic acid into a phosphate buffer solution of pH 7.5 through agarose gel as an indicator, in the first layer, about 15±5% by weight of the monovalent metal salt of alginic acid is dissolved within 2 days, and about 25±10% by weight is dissolved within 8 days, and in the second layer, about 30±8% by weight of the monovalent metal salt of alginic acid is dissolved within 2 days, and about 60±10% by weight is dissolved within 8 days.

[0016] [2-1] A method for preventing adhesions, comprising applying a sterilized, biocompatible sponge-like laminate to a subject in need of adhesion prevention, the sponge-like laminate comprising first and second layers of low-endotoxin monovalent metal salt of alginic acid, at least a portion of which is crosslinked with a hardener, the monovalent metal salt of alginic acid in the first layer having a weight-average molecular weight of 10,000 to 2,000,000 and the monovalent metal salt of alginic acid in the second layer having a weight-average molecular weight of 1,000 to 1,000,000, the weight-average molecular weights being measured by a GPC-MALS method after a de-crosslinking treatment, and the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer being higher than the weight-average molecular weight of the monovalent metal salt of alginic acid in the second layer, with the first layer facing the wound surface. [2-1a] A method for preventing adhesions, comprising applying a sponge-like laminate applicable to a living body to a subject in need of adhesion prevention, the sponge-like laminate comprising a first sponge-like layer containing a low-endotoxin monovalent metal salt of alginic acid having a weight-average molecular weight of 10,000 to 2,000,000 and a second sponge-like layer containing a low-endotoxin monovalent metal salt of alginic acid having a weight-average molecular weight of 1,000 to 1,000,000, wherein the molecular weights are measured by a GPC-MALS method and the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that of the second layer. [2-1b] A method for preventing adhesions, comprising applying a sponge-like laminate applicable to a living body, comprising first and second layers of sponge-like low-endotoxin monovalent metal salt of alginic acid at least a portion of which is crosslinked with a hardener, the monovalent metal salt of alginic acid in the first layer having a weight-average molecular weight of 30,000 to 300,000 and the monovalent metal salt of alginic acid in the second layer having a weight-average molecular weight of 1,000 to 200,000, the weight-average molecular weights being measured by a GPC-MALS method after a de-crosslinking treatment, and the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer being higher than the weight-average molecular weight of the monovalent metal salt of alginic acid in the second layer, to a subject in need of adhesion prevention, with the first layer facing the surface of the wound.

[0017] [2-2] The adhesion prevention method according to the above [2-1], [2-1a] or [2-1b], wherein either the first layer or the second layer contains a hardener. [2-3] The adhesion preventing method according to any one of the above items [2-1] to [2-2], wherein both the first layer and the second layer contain a hardening agent. [2-4] The total amount of low-endotoxin monovalent metal salt of alginic acid used in the first and second layers is 0.1 mg / cm 2 ~3mg / cm 2 The adhesion preventing method according to any one of the above items [2-1] to [2-3], wherein the range is [2-5] The adhesion prevention method according to any one of the above [2-1] to [2-4], wherein the endotoxin content of the monovalent metal salt of alginic acid in the first layer and the second layer is 500 EU / g or less. [2-6] The adhesion prevention method according to any one of the above [2-1] to [2-5], wherein the monovalent metal salt of alginic acid in the first layer and the second layer is sodium alginate or potassium alginate. [2-7] The hardener for the first and second layers is CaCl 2 , CaSO 4 , ZnCl 2 , SrCl 2 , FeCl 3 , BaCl 2 , CaHPO 4 The adhesion preventing method according to any one of the above [2-1] to [2-6], wherein the metal ion compound is at least one selected from the group consisting of calcium gluconate, calcium oxalate and calcium lactate. [2-8] The adhesion prevention method according to any one of the above [2-1] to [2-7], wherein the sponge-like laminate has been sterilized by electron beam and / or gamma irradiation and / or ethylene oxide gas at an absorbed dose of 10 kGy to 150 kGy.

[0018] [2-9] A method for preventing adhesions, comprising applying a sponge-like laminate applicable to a living body, the sponge-like laminate comprising a first layer and a second layer, each of which comprises a monovalent metal salt of low-endotoxin alginic acid at least partially crosslinked with a hardener, wherein the dissolution rate of the first layer is slower than that of the second layer, to a subject in need of adhesion prevention. [2-9a] A method for preventing adhesions, comprising applying a sponge-like laminate applicable to a living body, the sponge-like laminate comprising a first layer and a second layer, each of which contains a monovalent metal salt of low-endotoxin alginic acid, wherein the dissolution rate of the first layer is slower than that of the second layer, to a subject in need of adhesion prevention. [2-10] The adhesion prevention method according to [2-9] or [2-9a] above, in which, in a dissolution test using the dissolution of a monovalent metal salt of alginic acid in a phosphate buffer solution of pH 7.5 as an indicator, the proportion of the amount of dissolution of a monovalent metal salt of alginic acid in the first layer, when the amount of dissolution of the monovalent metal salt of alginic acid in the second layer is taken as 100%, is less than 50% at 1 hour and less than 70% at 2 hours from the start of measurement. [2-11] The adhesion prevention method according to [2-9] or [2-9a] above, wherein, in a dissolution test using the dissolution of a monovalent metal salt of alginic acid into a phosphate buffer solution of pH 7.5 as an indicator, the first layer is one in which 25±10% by weight of the monovalent metal salt of alginic acid dissolves within 1 hour, and 80±10% by weight of the monovalent metal salt of alginic acid dissolves within 4 hours, and the second layer is one in which 70±10% by weight of the monovalent metal salt of alginic acid dissolves within 1 hour, and 90±10% by weight of the monovalent metal salt of alginic acid dissolves within 4 hours.

[0019] [2-12] The adhesion prevention method according to [2-9] or [2-9a] above, in which, in a dissolution test using the dissolution of a monovalent metal salt of alginic acid in a phosphate buffer solution of pH 7.5 through agarose gel as an indicator, the ratio of the amount of dissolution of a monovalent metal salt of alginic acid from the first layer to the amount of dissolution of a monovalent metal salt of alginic acid from the second layer, taken as 100%, is 30 to 70% on or after the second day from the start of measurement. [2-13] The adhesion prevention method according to [2-9] or [2-9a] above, wherein in a dissolution test using the dissolution of a monovalent metal salt of alginic acid into a phosphate buffer solution of pH 7.5 through agarose gel as an indicator, the first layer is one in which about 15±5 weight% of the monovalent metal salt of alginic acid dissolves within 2 days, and about 25±10 weight% dissolves within 8 days, and the second layer is one in which about 30±8 weight% of the monovalent metal salt of alginic acid dissolves within 2 days, and about 60±10 weight% dissolves within 8 days.

[0020] [2-14] The adhesion preventing method according to any one of the above [2-1] to [2-13], wherein the sponge-like laminate is a pressed product. [2-15] The adhesion preventing method according to any one of the above [2-1] to [2-14], wherein the sponge-like laminate has one or more of the following properties: (1) A piece of JIS K6251 tensile tester No. 8 is subjected to a tensile test, and the Young's modulus determined based on the obtained stress-strain curve is 0.3 to 300 MPa. (2) A piece of JIS K6251 tensile tester No. 8 is subjected to a tensile test, and the breaking strength determined based on the obtained stress-strain curve is 5 to 5,000 kPa. (3) A piece of JIS K6251 tensile tester No. 8 is subjected to a tensile test, and the ratio of breaking strength to Young's modulus of the sponge-like laminate determined based on the obtained stress-strain curve is 2 to 50. (4) When the sponge-like laminate is contacted with an agarose gel soaked in phosphate-buffered saline for 2 to 6 hours, the weight increase rate of the sponge-like laminate is 200 to 50,000%, assuming that the weight of the sponge-like laminate before contact with phosphate-buffered saline is 100%. (5) In a dissolution test using the dissolution of a monovalent metal salt of alginic acid into a phosphate buffer solution of pH 7.5 through agarose gel as an indicator, the ratio of the amount of dissolution of the monovalent metal salt of alginic acid from the first layer to the amount of dissolution of the monovalent metal salt of alginic acid from the second layer, taken as 100%, is 30 to 70% from the second day after the start of the measurement. (6) In a dissolution test using the dissolution of a monovalent metal salt of alginic acid into a phosphate buffer solution of pH 7.5 through agarose gel as an indicator, in the first layer, about 15±5% by weight of the monovalent metal salt of alginic acid is dissolved within 2 days, and about 25±10% by weight is dissolved within 8 days, and in the second layer, about 30±8% by weight of the monovalent metal salt of alginic acid is dissolved within 2 days, and about 60±10% by weight is dissolved within 8 days.

[0021] [3-1] A method for producing an adhesion prevention material comprising a sponge-like laminate applicable to a living body, comprising the following steps: (1) hardening a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 10,000 to 2,000,000 with a hardener; (2) freezing the hardened monovalent metal salt of alginic acid; (3) A step of hardening a monovalent metal salt of low-endotoxin alginic acid having a weight average molecular weight of 1,000 to 1,000,000 with a hardener on the monovalent metal salt of alginic acid obtained in (2) to obtain a laminate; (4) freeze-drying the obtained laminate to obtain a sponge-like laminate; Here, the molecular weight is measured by a GPC-MALS method, The sponge-like laminate comprises a spongy first layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 10,000 to 2,000,000, and a spongy second layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 1,000 to 1,000,000, wherein the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that in the second layer.

[0022] [3-1a] A method for producing an adhesion prevention material comprising a sponge-like laminate applicable to a living body, comprising the following steps: (1) hardening a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 10,000 to 2,000,000 with a hardener; (2) A step of curing a monovalent metal salt of low-endotoxin alginic acid having a weight average molecular weight of 1,000 to 1,000,000 with a curing agent on the monovalent metal salt of alginic acid obtained in (1) to obtain a laminate; (3) freeze-drying the obtained laminate to obtain a sponge-like laminate; Here, the molecular weight is measured by a GPC-MALS method, The sponge-like laminate comprises a spongy first layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 10,000 to 2,000,000, and a spongy second layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 1,000 to 1,000,000, wherein the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that in the second layer.

[0023] [3-1b] A method for producing an adhesion prevention material comprising a sponge-like laminate applicable to a living body, comprising the following steps: (1) hardening a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 30,000 to 300,000 with a hardener; (2) freezing the hardened monovalent metal salt of alginic acid; (3) A step of curing a monovalent metal salt of low-endotoxin alginic acid having a weight average molecular weight of 1,000 to 200,000 with a curing agent on the monovalent metal salt of alginic acid obtained in (2) to obtain a laminate; (4) freeze-drying the obtained laminate to obtain a sponge-like laminate; Here, the molecular weight is measured by a GPC-MALS method, The sponge-like laminate comprises a spongy first layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 30,000 to 300,000, and a spongy second layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 1,000 to 200,000, wherein the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that in the second layer.

[0024] [3-1c] A method for producing an adhesion prevention material comprising a sponge-like laminate applicable to a living body, comprising the following steps: (1) hardening a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 30,000 to 300,000 with a hardener; (2) A step of hardening a monovalent metal salt of low-endotoxin alginic acid having a weight average molecular weight of 1,000 to 200,000 with a hardener on the monovalent metal salt of alginic acid obtained in (1) to obtain a laminate; (3) freeze-drying the obtained laminate to obtain a sponge-like laminate; Here, the molecular weight is measured by a GPC-MALS method, The sponge-like laminate comprises a spongy first layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 30,000 to 300,000, and a spongy second layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 1,000 to 200,000, wherein the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that in the second layer.

[0025] [3-2] A method for producing an adhesion prevention material according to any one of the above [3-1] to [3-1c], wherein the sponge-like laminate has been sterilized by electron beam and / or gamma irradiation and / or ethylene oxide gas at an absorbed dose of 10 kGy to 150 kGy. [3-3] A method for producing an adhesion preventing material according to any one of the above [3-1] to [3-2], further comprising a step of pressing the sponge-like laminate obtained in (3) or (4). [3-4] The hardener is CaCl 2 , CaSO 4 , ZnCl 2 , SrCl 2 , FeCl 3 , BaCl 2 , CaHPO 4 The method for producing an adhesion preventing material according to any one of the above [3-1] to [3-3], wherein the metal ion compound is at least one selected from the group consisting of calcium gluconate, calcium oxalate, and calcium lactate.

[0026] [3-5] A method for producing an adhesion preventing material according to any one of the above [3-1] to [3-4], wherein the sponge-like laminate has one or more of the following properties: (1) A piece of JIS K6251 tensile tester No. 8 is subjected to a tensile test, and the Young's modulus determined based on the obtained stress-strain curve is 0.3 to 300 MPa. (2) A piece of JIS K6251 tensile tester No. 8 is subjected to a tensile test, and the breaking strength determined based on the obtained stress-strain curve is 5 to 5,000 kPa. (3) A piece of JIS K6251 tensile tester No. 8 is subjected to a tensile test, and the ratio of breaking strength to Young's modulus of the sponge-like laminate determined based on the obtained stress-strain curve is 2 to 50. (4) When the sponge-like laminate is contacted with an agarose gel soaked in phosphate-buffered saline for 2 to 6 hours, the weight increase rate of the sponge-like laminate is 200 to 50,000%, assuming that the weight of the sponge-like laminate before contact with phosphate-buffered saline is 100%. (5) In a dissolution test using the dissolution of a monovalent metal salt of alginic acid into a phosphate buffer solution of pH 7.5 through agarose gel as an indicator, the ratio of the amount of dissolution of the monovalent metal salt of alginic acid from the first layer to the amount of dissolution of the monovalent metal salt of alginic acid from the second layer, taken as 100%, is 30 to 70% from the second day after the start of the measurement. (6) In a dissolution test using the dissolution of a monovalent metal salt of alginic acid into a phosphate buffer solution of pH 7.5 through agarose gel as an indicator, in the first layer, about 15±5% by weight of the monovalent metal salt of alginic acid is dissolved within 2 days, and about 25±10% by weight is dissolved within 8 days, and in the second layer, about 30±8% by weight of the monovalent metal salt of alginic acid is dissolved within 2 days, and about 60±10% by weight is dissolved within 8 days.

[0027] [4-1] A sponge-like laminate applicable to a living body, obtained by the following steps (1) to (4): (1) hardening a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 10,000 to 2,000,000 with a hardener; (2) freezing the hardened monovalent metal salt of alginic acid; (3) A step of hardening a monovalent metal salt of low-endotoxin alginic acid having a weight average molecular weight of 1,000 to 1,000,000 with a hardener on the monovalent metal salt of alginic acid obtained in (2) to obtain a laminate; (4) freeze-drying the obtained laminate to obtain a sponge-like laminate; Here, the molecular weight is measured by a GPC-MALS method, The sponge-like laminate comprises a spongy first layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 10,000 to 2,000,000, and a spongy second layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 1,000 to 1,000,000, wherein the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that in the second layer.

[0028] [4-1a] A sponge-like laminate applicable to a living body, obtained by the following steps (1) to (3): (1) hardening a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 10,000 to 2,000,000 with a hardener; (2) A step of curing a monovalent metal salt of low-endotoxin alginic acid having a weight average molecular weight of 1,000 to 1,000,000 with a curing agent on the monovalent metal salt of alginic acid obtained in (1) to obtain a laminate; (3) freeze-drying the obtained laminate to obtain a sponge-like laminate; Here, the molecular weight is measured by a GPC-MALS method, The sponge-like laminate comprises a spongy first layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 10,000 to 2,000,000, and a spongy second layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 1,000 to 1,000,000, wherein the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that in the second layer.

[0029] [4-1b] A sponge-like laminate applicable to a living body, obtained by the following steps (1) to (4): (1) hardening a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 30,000 to 300,000 with a hardener; (2) freezing the hardened monovalent metal salt of alginic acid; (3) A step of curing a monovalent metal salt of low-endotoxin alginic acid having a weight average molecular weight of 1,000 to 200,000 with a curing agent on the monovalent metal salt of alginic acid obtained in (2) to obtain a laminate; (4) freeze-drying the obtained laminate to obtain a sponge-like laminate; Here, the molecular weight is measured by a GPC-MALS method, The sponge-like laminate comprises a spongy first layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 30,000 to 300,000, and a spongy second layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 1,000 to 200,000, wherein the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that in the second layer.

[0030] [4-1c] A sponge-like laminate applicable to a living body, obtained by the following steps (1) to (3): (1) hardening a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 30,000 to 300,000 with a hardener; (2) A step of hardening a monovalent metal salt of low-endotoxin alginic acid having a weight average molecular weight of 1,000 to 200,000 with a hardener on the monovalent metal salt of alginic acid obtained in (1) to obtain a laminate; (3) freeze-drying the obtained laminate to obtain a sponge-like laminate; Here, the molecular weight is measured by a GPC-MALS method, The sponge-like laminate comprises a spongy first layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 30,000 to 300,000, and a spongy second layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 1,000 to 200,000, wherein the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that in the second layer.

[0031] [4-2] The sponge-like laminate according to any one of the above [4-1] to [4-1c], which is used as an adhesion barrier. [4-3] A sponge-like laminate described in any one of the above [4-1] to [4-2], which has been sterilized by electron beam and / or gamma irradiation and / or ethylene oxide gas at an absorbed dose of 10 kGy to 150 kGy. [4-4] The sponge-like laminate according to any one of the above [4-1] to [4-3], further comprising a step of pressing the sponge-like laminate obtained in (3) or (4). [4-5] The hardener is CaCl 2 , CaSO 4 , ZnCl 2 , SrCl 2 , FeCl 3 , BaCl 2 , CaHPO 4 The sponge-like laminate according to any one of the above [4-1] to [4-4], wherein the metal ion compound is at least one selected from the group consisting of calcium gluconate, calcium oxalate and calcium lactate.

[0032] [4-6] The sponge-like laminate according to any one of the above [4-1] to [4-5], which has one or more of the following properties: (1) A piece of JIS K6251 tensile tester No. 8 is subjected to a tensile test, and the Young's modulus determined based on the obtained stress-strain curve is 0.3 to 300 MPa. (2) A piece of JIS K6251 tensile tester No. 8 is subjected to a tensile test, and the breaking strength determined based on the obtained stress-strain curve is 5 to 5,000 kPa. (3) A piece of JIS K6251 tensile tester No. 8 is subjected to a tensile test, and the ratio of breaking strength to Young's modulus of the sponge-like laminate determined based on the obtained stress-strain curve is 2 to 50. (4) When the sponge-like laminate is contacted with an agarose gel soaked in phosphate-buffered saline for 2 to 6 hours, the weight increase rate of the sponge-like laminate is 200 to 50,000%, assuming that the weight of the sponge-like laminate before contact with phosphate-buffered saline is 100%. (5) In a dissolution test using the dissolution of a monovalent metal salt of alginic acid into a phosphate buffer solution of pH 7.5 through agarose gel as an indicator, the ratio of the amount of dissolution of the monovalent metal salt of alginic acid from the first layer to the amount of dissolution of the monovalent metal salt of alginic acid from the second layer, taken as 100%, is 30 to 70% from the second day after the start of the measurement. (6) In a dissolution test using the dissolution of a monovalent metal salt of alginic acid into a phosphate buffer solution of pH 7.5 through agarose gel as an indicator, in the first layer, about 15±5% by weight of the monovalent metal salt of alginic acid is dissolved within 2 days, and about 25±10% by weight is dissolved within 8 days, and in the second layer, about 30±8% by weight of the monovalent metal salt of alginic acid is dissolved within 2 days, and about 60±10% by weight is dissolved within 8 days.

[0033] [5-1] A combination of raw materials for producing an adhesion prevention material, comprising a first raw material containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 10,000 to 2,000,000, and a second raw material containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 1,000 to 1,000,000, wherein the weight-average molecular weight of the first raw material is higher than that of the second raw material. [5-1a] A combination of raw materials for producing an adhesion prevention material, comprising a first raw material containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 30,000 to 300,000 and a second raw material containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 1,000 to 200,000, wherein the weight-average molecular weight of the first raw material is higher than that of the second raw material. [5-2] A combination of raw materials according to [5-1] or [5-1a] above, further comprising a curing agent. [5-3] The hardener is CaCl 2 , CaSO 4 , ZnCl 2 , SrCl 2 , FeCl 3 , BaCl 2 , CaHPO 4 The combination of raw materials described in [5-2] above, wherein the metal ion compound is at least one selected from the group consisting of calcium gluconate, calcium oxalate and calcium lactate. [5-4] A combination of raw materials according to any one of the above [5-1] to [5-3], which is for use in producing a sponge-like laminate. [6-1] A method for producing a sponge-like laminate applicable to a living body, comprising the following steps: (1) hardening a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 10,000 to 2,000,000 with a hardener; (2) freezing the hardened monovalent metal salt of alginic acid; (3) A step of hardening a monovalent metal salt of low-endotoxin alginic acid having a weight average molecular weight of 1,000 to 1,000,000 with a hardener on the monovalent metal salt of alginic acid obtained in (2) to obtain a laminate; (4) freeze-drying the obtained laminate to obtain a sponge-like laminate; Here, the molecular weight is measured by a GPC-MALS method, The sponge-like laminate comprises a spongy first layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 10,000 to 2,000,000, and a spongy second layer containing a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 1,000 to 1,000,000, wherein the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that in the second layer. Effect of the Invention

[0034] According to the present invention, it is possible to provide an adhesion prevention material that has at least one of the following advantages: it has a high adhesion prevention effect, it can suppress both wound adhesion and de novo adhesion, it has no adverse effects on the living body to which it is applied, it does not hinder the healing of wounds, it can be used for intestinal anastomosis, etc., it can be easily applied via a trocar in endoscopic surgery, it can be reapplied by adjusting the application position, it has excellent manufacturing efficiency, and it is suitable for mass production. [Brief description of the drawings]

[0035] [Figure 1] FIG. 1 is a diagram showing an example of an anti-adhesion material. [Diagram 2] FIG. 1 shows the dissolution rate of each layer of an adhesion preventing material evaluated by an immersion method. [Diagram 3] FIG. 1 shows an evaluation of the dissolution rate of each layer of an adhesion preventing material using a patch method. [Figure 4] Evaluation of adhesion formation in a partial liver resection model. (A) Number of animals with adhesion formation on the dissected surface, (B) Dissected surface grade, (C) Dissected surface extent (mm). ** p<0.01, * p<0.05. [Diagram 5] A figure showing the evaluation of adhesion formation in a partial liver resection model. (A) Number of animals that formed adhesions on non-detached sections, (B) grade of non-detached sections, (C) extent of non-detached sections (mm). ** p<0.01, * p<0.05. [Figure 6]Figure 1 shows the evaluation of body weight change and spleen weight in a partial liver resection model, (A) body weight change, (B) spleen weight. [Figure 7] Figure 1 shows the evaluation of adhesion formation in a Pean liver resection model. (A) Number of animals with adhesion formation on the dissected surface, (B) Dissected surface grade, (C) Dissected surface extent (mm). ** p<0.01, * p<0.05. [Figure 8] Figure 1 shows the evaluation of adhesion formation in a Pean liver resection model. (A) Number of animals that formed adhesions on the non-detached section, (B) grade of the non-detached section, (C) extent (mm) of the non-detached section. ** p<0.01, * p<0.05. [Figure 9] 1 shows the evaluation of body weight change and spleen weight in a Péan hepatectomy model, (A) body weight change, (B) spleen weight. [Figure 10] FIG. 13 shows the results of a sponge swelling test before and after pressing. [Figure 11] FIG. 1 shows the change over time in the height (A) of the tip of each test piece after spraying and the angle (B) from the test stand. [Figure 12] FIG. 13 shows the results of a rehydration test of a laminated sponge after sterilization. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The present invention will be described in detail below. The following embodiments are merely illustrative for explaining the present invention, and the present invention can be implemented in various forms without departing from the gist of the present invention.

[0037] 1. Prevention of adhesions "Adhesion" refers to a state in which the surfaces of tissues that should be separated from each other are connected or fused by fibrous tissue. The causes of adhesion include trauma to the tissue surface during surgery, inflammation caused by trauma, and inflammation caused by drying of the tissue surface during surgery. These traumas and inflammations cause exudates containing fibrin to form on the tissue surface, and adhesions are formed when the exudates become organized and connect or fuse the tissue surfaces.

[0038] "Adhesion prevention" refers to reducing adhesion formation. Adhesion prevention does not necessarily require complete prevention of adhesion formation, and it is sufficient that adhesion formation is prevented compared to the state when the adhesion inhibitor of the present invention is not applied. In other words, "adhesion prevention" may be said as reduction of adhesion, and for example, it is sufficient that at least one selected from the frequency, range, and degree of adhesion is reduced. "Adhesion prevention" may be, for example, when the adhesion grade evaluation described in the Examples is performed, the average adhesion grade may be lower compared to the average adhesion grade when the adhesion inhibitor of the present invention is not applied. Alternatively, "adhesion prevention" may be, for example, when the adhesion extent evaluation described in the Examples is performed, the average adhesion extent may be lower compared to the average adhesion extent when the adhesion inhibitor of the present invention is not applied. "Adhesion prevention" is preferably the prevention of adhesion caused by surgery, more preferably peritoneal adhesion caused by surgery. In other words, "adhesion prevention" is preferably the prevention of postoperative adhesion. Furthermore, as shown in the examples, the adhesions of interest include adhesions at the site where the target organ was removed during surgery and de novo adhesions (adhesion to the surrounding area other than the surgical site, and to body cavities such as the abdominal cavity, and to a wide range of sites within the body).

[0039] 2. Anti-adhesion materials The present invention provides an adhesion barrier (hereinafter, sometimes referred to as "adhesion barrier A") that includes a sponge-like laminate that can be applied to a living body and includes a sponge-like first layer and a sponge-like second layer that includes a monovalent metal salt of low endotoxin alginic acid at least partially crosslinked with a curing agent, the sponge-like first layer including a monovalent metal salt of low endotoxin alginic acid having a relatively high weight-average molecular weight, and a sponge-like second layer including a monovalent metal salt of low endotoxin alginic acid having a relatively low weight-average molecular weight. The weight-average molecular weights of the monovalent metal salt of low endotoxin alginic acid used in the first layer and the second layer are, for example, 10,000 to 2,000,000 and 1,000 to 1,000,000, respectively. Such weight-average molecular weights are measured by a GPC-MALS method after decrosslinking treatment, for example, dissolving in a chelating agent solution.

[0040] The monovalent metal salt of alginic acid used in each layer may be a combination of multiple monovalent metal salts of alginic acid with different average molecular weights. For example, in the first layer, multiple monovalent metal salts of alginic acid having different weight average molecular weights within the range of 10,000 to 2,000,000 may be used in combination, and / or in the second layer, multiple monovalent metal salts of alginic acid having different weight average molecular weights within the range of 1,000 to 1,000,000 may be used in combination. The number of monovalent metal salts of alginic acid to be combined is not particularly limited, and may be, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more per layer. The monovalent metal salt of alginic acid may be a combination of different types of salts with different weight average molecular weights, or a combination of the same type of salts with different weight average molecular weights. The combination ratio is not particularly limited, and for example, when two types of salts are combined, the ratio may be 1:100 to 100:1, 1:50 to 50:1, 1:25 to 25:1, 1:10 to 10:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, 1:1, etc. In this specification, when the symbol "~" is used to indicate a numerical range, it means "not less than the lower limit and not more than the upper limit," and the numerical values ​​on both ends of the symbol are included in the range.

[0041] The adhesion barrier A contains low-endotoxin monovalent metal salts of alginic acid with different molecular weights in the first and second layers of the sponge-like laminate. Specifically, the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that in the second layer. The layer containing the monovalent metal salt of alginic acid has a slower dissolution rate if the weight-average molecular weight of the monovalent metal salt of alginic acid is larger, while the dissolution rate is faster if the weight-average molecular weight is smaller. For this reason, for example, when applied to a wound in the abdominal cavity, the adhesion barrier A of the present invention is applied with the first layer facing the wound side and the second layer facing the abdominal cavity side, so that the first layer remains in the wound and the second layer dissolves relatively quickly, which is expected to suppress adhesion throughout the abdominal cavity.

[0042] The present invention also provides an adhesion barrier ("adhesion barrier B") that includes a sponge-like laminate applicable to a living body, which includes a first layer and a second layer, each of which includes a monovalent metal salt of low-endotoxin alginic acid at least partially crosslinked with a curing agent, and in which the first layer and the second layer have different dissolution rates. Specifically, the dissolution rate of the first layer is slower than that of the second layer. To make the dissolution rate of the first layer slower than that of the second layer, for example, the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer may be made higher than that of the second layer, as in adhesion barrier A, or the degree of crosslinking of the monovalent metal salt of alginic acid in the first layer may be made higher than that of the second layer by changing the type of crosslinker or the concentration of the crosslinker, etc.

[0043] Preferably, in a dissolution test using the dissolution of a monovalent metal salt of alginic acid as an index, the proportion of the dissolution amount of the monovalent metal salt of alginic acid in the first layer is less than 50% at 1 hour and less than 70% at 2 hours from the start of measurement, relative to the dissolution amount of the monovalent metal salt of alginic acid in the second layer, which is taken as 100%. The dissolution test is specifically as described in Example 2 below. As another preferred example, in a dissolution test using the dissolution of a monovalent metal salt of alginic acid as an index, the first layer dissolves 25±10% by weight of the monovalent metal salt of alginic acid within 1 hour and 80±10% by weight within 4 hours, and the second layer dissolves 70±10% by weight of the monovalent metal salt of alginic acid within 1 hour and 90±10% by weight within 4 hours. The dissolution test is specifically as described in Example 2 below.

[0044] In another preferred example, in a dissolution test using the dissolution of a monovalent metal salt of alginic acid through agarose gel as an index, the proportion of the amount of dissolution of a monovalent metal salt of alginic acid in the first layer is about 30% to about 70%, preferably about 40% to about 60%, and more preferably about 44% to about 55%, when the amount of dissolution of a monovalent metal salt of alginic acid in the second layer is taken as 100% on or after the second day from the start of measurement. The dissolution test is specifically as described in Example 2-2 below. As another preferred example, in a dissolution test using the dissolution of a monovalent metal salt of alginic acid through agarose gel as an index, the first layer dissolves about 15±5% by weight, particularly about 16±2% by weight, of the monovalent metal salt of alginic acid within 2 days, and about 25±10% by weight, particularly about 33±7% by weight, within 8 days, while the second layer dissolves about 30±8% by weight, particularly about 31±6% by weight, of the monovalent metal salt of alginic acid within 2 days, and about 60±10% by weight, particularly about 60±7% by weight, within 8 days. The dissolution test is specifically as described in Example 2-2 below.

[0045] Here, in this specification, "anti-adhesion material A" and "anti-adhesion material B" may be collectively referred to as "anti-adhesion material". The "first layer" is the layer that becomes the lower layer when the sponge-like laminate is applied to a subject, that is, the layer that comes into contact with the surface of the tissue to which the subject is to be applied. The "second layer" is the layer that becomes the upper layer when the sponge-like laminate is applied to a subject, that is, the layer that does not come into contact with the surface of the tissue to which the subject is to be applied. "Applicable to a living body" means that it can be placed on the surface of the tissue to which it is to be applied as a medical material.

[0046] In the sponge-like laminate applicable to the living body used in the adhesion prevention material, a clear boundary surface may or may not be formed between the first layer and the second layer. For example, in the laminate, the components contained in the first layer and the components contained in the second layer may be mixed near the boundary surface between the first layer and the second layer during the manufacturing process, and a clear boundary surface may not be formed. Furthermore, the laminate may have a third layer containing an arbitrary component in addition to the first layer and the second layer, and may have a multilayer structure. Also included are sponge-like laminates in which each layer does not have a clear boundary surface and has a structure in which the molecular weight increases or decreases continuously.

[0047] An example of an adhesion preventing material is shown in Figure 1. The adhesion preventing material 1 comprises a sponge-like laminate 4 including a first layer 2 and a second layer 3. The first layer 2 and the second layer 3 are each sponge-like. "Sponge-like" refers to a porous state.

[0048] The shape of the sponge-like laminate applicable to a living body is not particularly limited, and can be appropriately selected in consideration of the surface area, shape, unevenness, etc. to be applied. The shape of the sponge-like laminate may be, for example, a flat plate as shown in FIG. 1, or a disk, cylinder, rectangular parallelepiped, etc. It is preferably a flat plate or disk. When it is a flat plate or disk, the adhesion inhibitor can be further cut according to the surface area, shape, unevenness, etc. to be applied to the surface, so the size of the flat plate or disk is not particularly limited. For example, when the shape of a flat plate is expressed as length x width x height (thickness), the length and width are not particularly limited, and the height (thickness) is preferably 0.2 mm to 30 mm, more preferably 0.3 mm to 15 mm, and even more preferably 0.5 mm to 10 mm. More preferably, in addition to such height (thickness), the length and width are each 1 mm to 300 mm x 1 mm to 300 mm, particularly preferably 3 mm to 200 mm x 3 mm to 200 mm, and further preferably 5 mm to 150 mm x 5 mm to 150 mm. Note that the thickness does not have to be uniform, and may be a gradient structure in which one side is thick and the other is thin.

[0049] The sponge-like laminate of the adhesion preventing material of the present invention is more flexible and less likely to crack than Seprafilm (trade name).

[0050] In some embodiments, the sponge-like laminate is pressed. "Pressed" is as described below. When the sponge-like laminate is pressed, its height (thickness) is preferably 0.01 mm to 5 mm, more preferably 0.02 mm to 3 mm, and even more preferably 0.03 mm to 1.5 mm. More preferably, in addition to such height (thickness), the length and width are each 1 mm to 300 mm x 1 mm to 300 mm, particularly preferably 3 mm to 200 mm x 3 mm to 200 mm, and even more preferably 5 mm to 150 mm x 5 mm to 150 mm. In some embodiments, the thickness after pressing is uniform.

[0051] The sponge-like laminate of the adhesion preventing material of the present invention can have the following physical properties. (1) Young’s modulus A JIS K6251 tensile No. 8 piece is placed in a tensile tester, and the Young's modulus of the sponge-like laminate determined based on the obtained stress-strain curve may be, for example, 0.3 to 300 MPa, preferably 0.4 to 250 MPa, more preferably 0.5 to 200 MPa, and particularly preferably 1 to 180 MPa. The Young's modulus can be increased by pressing the laminate.

[0052] (2) Breaking strength The breaking strength of the sponge-like laminate, determined based on a stress-strain curve obtained by subjecting a JIS K6251 tensile No. 8 piece to a tensile tester, is, for example, 5 to 5000 kPa, preferably 10 to 4800 kPa, more preferably 15 to 4500 kPa, and particularly preferably 20 to 4000 kPa. The breaking strength can be increased by pressing the laminate.

[0053] (3) Breaking strength / Young's modulus ratio A JIS K6251 tensile No. 8 piece is placed in a tensile tester, and the ratio of breaking strength to Young's modulus of the sponge-like laminate (breaking strength (kPa) ÷ Young's modulus (MPa)) determined based on the obtained stress-strain curve is, for example, 2 to 50, preferably 3 to 45, more preferably 5 to 40, and particularly preferably 10 to 35.

[0054] (4) Water absorption amount When a sponge-like laminate (freeze-dried) is contacted with agarose gel soaked in phosphate-buffered saline for 2 to 6 hours, the weight increase rate of the sponge-like laminate is, for example, 200 to 50,000%, preferably 500 to 30,000%, more preferably 1,000 to 20,000%, and particularly preferably 1,500 to 15,000%, relative to the weight of the sponge-like laminate before contact with phosphate-buffered saline, taken as 100%.

[0055] 3. Monovalent metal salts of alginic acid "Monovalent metal salts of alginic acid" are salts of alginic acid in which the hydrogen atom of the 6th carboxylate is replaced by Na + Or K + Alginic acid is a water-soluble salt produced by ion exchange with monovalent metal ions such as sodium alginate and potassium alginate. Specific examples of monovalent metal salts of alginic acid include sodium alginate and potassium alginate, with sodium alginate being particularly preferred, which is commercially available. A solution of a monovalent metal salt of alginic acid forms a gel when mixed with a hardener.

[0056] The "alginic acid" used in the present invention is a biodegradable polymeric polysaccharide, which is a polymer formed by linear polymerization of two types of uronic acid, D-mannuronic acid (M) and L-guluronic acid (G). More specifically, it is a block copolymer in which a homopolymer fraction of D-mannuronic acid (MM fraction), a homopolymer fraction of L-guluronic acid (GG fraction), and a fraction in which D-mannuronic acid and L-guluronic acid are randomly arranged (MG fraction) are arbitrarily bonded. The composition ratio (M / G ratio) of D-mannuronic acid and L-guluronic acid in alginic acid varies mainly depending on the type of organism from which seaweed and the like are derived, and is also influenced by the growing place and season of the organism, and ranges widely from a high G type with an M / G ratio of about 0.4 to a high M type with an M / G ratio of about 5.

[0057] Monovalent metal salts of alginic acid are high molecular weight polysaccharides, and it is difficult to accurately determine their molecular weight. It is known that when measuring the molecular weight of polymeric substances derived from natural products, the values ​​can vary depending on the measurement method.

[0058] According to the GPC-MALS method, the absolute weight average molecular weight can be measured. The weight average molecular weight of the monovalent metal salt of alginic acid used as a raw material, measured by the GPC-MALS method, is, for example, 10,000 to 2,000,000, preferably 15,000 to 1,500,000, more preferably 20,000 to 1,000,000, and particularly preferably 25,000 to 500,000, in the first layer of the sponge-like laminate. In addition to being such a first layer, the weight average molecular weight measured by the GPC-MALS method, in the second layer, is, for example, 1,000 to 1,000,000, preferably 1,000 to 500,000, more preferably 2,000 to 250,000, and particularly preferably 3,000 to 100,000.

[0059] In some embodiments, for example, after irradiation with electron beam and / or gamma ray sterilization, the weight average molecular weight measured by the GPC-MALS method is, for example, 10,000 to 300,000, preferably 10,000 to 200,000, more preferably 10,000 to 100,000, and particularly preferably 10,000 to 80,000 in the first layer of the sponge-like laminate. In addition to such a first layer, the weight average molecular weight measured by the GPC-MALS method is, for example, 1,000 to 100,000, preferably 1,000 to 80,000, more preferably 2,000 to 60,000, and particularly preferably 3,000 to 60,000 in the second layer.

[0060] In a specific embodiment, the weight average molecular weight of the monovalent metal salt of alginic acid used as a raw material in the first layer of the sponge-like laminate, as measured by the GPC-MALS method, is, for example, 30,000 to 300,000, preferably 80,000 to 280,000, more preferably 100,000 to 270,000, even more preferably 150,000 to 260,000, and particularly preferably 170,000 to 250,000. In addition to being such a first layer, the weight average molecular weight of the second layer measured by a GPC-MALS method is, for example, 1,000 to 200,000, preferably 1,500 to 180,000, more preferably 2,000 to 150,000, still more preferably 2,500 to 120,000, and particularly preferably 3,000 to 100,000. By using a monovalent metal salt of alginic acid having a weight average molecular weight measured by the GPC-MALS method in the above-mentioned range as the raw material for the first layer of the sponge-like laminate, the gelation rate of the monovalent metal salt of alginic acid in the production of the first layer of the sponge-like laminate can be optimized, and the quality and production time of the first layer can be optimized.

[0061] In some specific embodiments, for example, after irradiation with electron beam and / or γ-ray sterilization, the weight average molecular weight measured by the GPC-MALS method is, for example, 30,000 to 80,000, preferably 40,000 to 75,000, more preferably 42,000 to 73,000, even more preferably 43,000 to 72,000, and particularly preferably 44,000 to 71,000 in the first layer of the sponge-like laminate. In addition to such a first layer, the weight average molecular weight measured by the GPC-MALS method is, for example, 1,000 to 70,000, preferably 1,000 to 65,000, more preferably 2,000 to 60,000, even more preferably 2,500 to 55,000, and particularly preferably 3,000 to 50,000 in the second layer.

[0062] For the monovalent metal salt of alginic acid at least partly crosslinked with a curing agent, the weight average molecular weight as the monovalent metal salt of uncrosslinked alginic acid can be measured by GPC-MALS method after any decrosslinking treatment. For example, the decrosslinking treatment can be dissolved in a solution of any chelating agent such as EDTA (ethylenediaminetetraacetic acid) or phytic acid. EDTA is preferably used as the chelating agent.

[0063] The weight average molecular weight of the monovalent metal salt of alginic acid in the first layer of the sponge-like laminate is higher than that in the second layer. The weight average molecular weight of the monovalent metal salt of alginic acid in the raw material of the sponge-like laminate or the first layer contained in the sponge-like laminate is, for example, 1,000 to 1,000,000 higher, preferably 2,000 to 500,000 higher, and more preferably 3,000 to 300,000 higher than that in the second layer.

[0064] Usually, when the molecular weight of a polymeric polysaccharide is calculated by the above-mentioned method, a measurement error of 10 to 20% by weight may occur. For example, the value may vary within the range of 8,000 to 12,000 for 10,000, 80,000 to 120,000 for 100,000, 160,000 to 240,000 for 200,000, 320,000 to 480,000 for 400,000, and 400,000 to 600,000 for 500,000. The molecular weight of alginic acids can be measured according to a conventional method. Representative conditions for using GPC-MALS for molecular weight measurement are as described in Example 1 of the present specification. As a detector, for example, an RI detector and a light scattering detector (MALS) can be used.

[0065] Alginic acids generally have a large molecular weight when first extracted from brown algae, but the molecular weight gradually decreases during processes such as drying by heat and purification. Alginic acids with different molecular weights can be produced by controlling conditions such as temperature during the production process, selecting brown algae as raw material, and molecular weight fractionation during the production process. Furthermore, it is possible to produce alginic acids with the desired molecular weight by mixing with alginic acids from different lots having different molecular weights.

[0066] The monovalent metal salt of alginic acid used in the present invention has been treated to reduce endotoxins. The treatment to reduce endotoxins can be carried out by a known method or a method similar thereto. For example, the purification of sodium hyaluronate can be carried out by the method of Suga et al. (see, for example, Japanese Patent Application Laid-Open No. 9-324001, etc.), the purification of β1,3-glucan by Yoshida et al. (see, for example, Japanese Patent Application Laid-Open No. 8-269102, etc.), the purification of biopolymer salts such as alginate and gellan gum by Williams et al. (see, for example, Japanese Translation of PCT International Publication No. 2002-530440, etc.), the purification of polysaccharides by James et al. (see, for example, the pamphlet of International Publication No. 93 / 13136, etc.), the method of Lewis et al. (see, for example, the specification of U.S. Patent No. 5,589,591, etc.), the purification of alginate by Herman Frank et al. (see, for example, Appl Microbiol Biotechnol (1994) 40:638-643, etc.), etc., or methods similar thereto. The low endotoxin treatment of the present invention is not limited to these, and may be carried out by known methods such as washing, filtration with a filter (endotoxin removal filter, charged filter, etc.), ultrafiltration, purification using a column (endotoxin adsorption affinity column, gel filtration column, column with ion exchange resin, etc.), adsorption to hydrophobic substances, resins or activated carbon, organic solvent treatment (extraction with organic solvent, precipitation / sedimentation by adding organic solvent, etc.), surfactant treatment (see, for example, JP-A-2005-036036, etc.), or by appropriately combining these. These treatment steps may be appropriately combined with known methods such as centrifugation. It is desirable to appropriately select a method according to the type of alginic acid.

[0067] The endotoxin level can be confirmed by known methods, for example, a method using Limulus Alkaline Lympholysate (LAL) or a method using Endospecy (registered trademark) ES-24S set (Seikagaku Corporation).

[0068] The method for treating endotoxins in the monovalent metal salt of alginic acid used in the present invention is not particularly limited, but as a result, when endotoxin is measured using a Limulus reagent (LAL), the endotoxin content of the bioabsorbable polysaccharide is preferably 500 endotoxin units (EU) / g or less, more preferably 100 EU / g or less, particularly preferably 50 EU / g or less, and particularly preferably 30 EU / g or less. Low-endotoxin treated sodium alginate is available, for example, under the trademark Sea Matrix (Mochida Pharmaceutical Co., Ltd.), PRONOVA TM It is available as a commercially available product such as UP LVG (FMCBioPolymer).

[0069] The amount of the monovalent metal salt of alginic acid used in the sponge-like laminate can be appropriately selected in consideration of the adhesion prevention effect. The amount of the monovalent metal salt of alginic acid used is, for example, 0.1 mg / cm in total for the first layer and the second layer of the sponge-like laminate. 2 ~10.0mg / cm 2 and preferably 0.1 mg / cm 2 ~3.0mg / cm 2 and more preferably 0.5 mg / cm 2 ~2.5mg / cm 2 and more preferably 1.8 mg / cm 2 ~2.2mg / cm 2 and particularly preferably 2.0 mg / cm 2 The amount of monovalent metal salt of alginic acid used in the first layer and the second layer of the sponge-like laminate is 1.0 mg / cm. 2 ~3.0mg / cm 2 This is expected to have a higher adhesion prevention effect. 2If the dose is less than 0.1 mg / cm, there is little risk of adverse events such as accumulation in the body or hypertrophy of specific organs. 2 If the amount is more than this, a sufficient adhesion prevention effect can be expected.

[0070] The ratio (weight ratio) of the amount of monovalent metal salt of alginic acid used in the first layer to the second layer is preferably 1:20 to 20:1, more preferably 1:5 to 5:1, even more preferably 1:3 to 3:1, and particularly preferably 1:2 to 2:1.

[0071] 4. Hardener (crosslinking agent) In the adhesion prevention material, either the first layer or the second layer may contain a sclerosing agent (i.e., either the first layer or the second layer may not contain a sclerosing agent), or both the first layer and the second layer may contain a sclerosing agent. Alternatively, neither the first layer nor the second layer of the adhesion preventing material may contain a hardening agent.

[0072] Here, in some embodiments, the first layer and the second layer are at least partially crosslinked with a curing agent.

[0073] The hardener hardens by crosslinking the solution of monovalent metal salt of alginic acid. 2+ , Mg 2+ , B.A. 2+ , Sr 2+ , Zn 2+ , Fe 3+ Examples of the divalent or higher metal ion compounds include CaCl 2 , MgCl 2 , CaSO 4 , ZnCl 2 , FeCl 3 , BaCl 2 , SrCl 2 , Dicalcium Phosphate (CaHPO 4), inorganic metal salts such as calcium gluconate, calcium oxalate, calcium lactate, and organic acid metal salts (preferably CaCl 2 , CaSO 4 , ZnCl 2 , SrCl 2 , FeCl 3 , BaCl 2 , Dicalcium Phosphate (CaHPO 4 ), calcium gluconate, etc.) as a crosslinking agent having 2 to 4 amino groups in the molecule, and a lysyl group (-COCH(NH 2 )-(CH 2 ) 4 -NH 2 ), that is, diaminoalkanes and derivatives in which the amino group is substituted with a lysyl group to form a lysylamino group, and specific examples include diaminoethane, diaminopropane, and N-(lysyl)-diaminoethane.

[0074] The amount of the hardener used in the first and second layers is desirably adjusted as appropriate depending on the amount of the monovalent metal salt of alginic acid used, the molecular weight, the desired hardening time, etc. When a hardener is used, the amount of the hardener used in the first layer is, for example, 0.1 μmol / cm 2 ~100μmol / cm 2 and preferably 0.5 μmol / cm 2 ~2.0μmol / cm 2 When a hardener is used, the amount of the hardener used in the second layer is, for example, 0.1 μmol / cm 2 ~10μmol / cm 2 and preferably 0.6 μmol / cm 2 ~2.4μmol / cm 2 In addition, by reducing the amount of curing agent used (concentration), the curing time of each layer can be extended, and conversely, by increasing the amount of curing agent used (concentration), the curing time of each layer can be shortened.

[0075] 5. Method for preparing adhesion prevention material The adhesion preventing material containing the biocompatible sponge-like laminate and the biocompatible sponge-like laminate can be produced, for example, through the following steps.

[0076] (1) hardening a monovalent metal salt of low-endotoxin alginic acid having a weight-average molecular weight of 10,000 to 2,000,000 with a hardener; (2) freezing the hardened monovalent metal salt of alginic acid; (3) A step of hardening a monovalent metal salt of low-endotoxin alginic acid having a weight average molecular weight of 1,000 to 1,000,000 with a hardener on the monovalent metal salt of alginic acid obtained in (2) to obtain a laminate; (4) A step of freeze-drying the obtained laminate to obtain a sponge-like laminate.

[0077] In the above step (1), first, a solution of a monovalent metal salt of low endotoxin alginic acid having a weight average molecular weight of 10,000 to 2,000,000 (hereinafter referred to as "first alginate") and a solution of a hardener are prepared. The solution of the first alginate and the solution of the hardener can be prepared by a known method or a method similar thereto. The solvent is not particularly limited as long as it is a solvent that can be applied to a living body, but is preferably an aqueous solvent, such as purified water, pure water (e.g., distilled water, ion-exchanged water), Milli-Q water, physiological saline, phosphate buffered saline, DMSO, etc., and more preferably pure water. These are preferably sterilized, and are preferably treated to reduce endotoxins.

[0078] The first alginate can then be hardened by mixing the solution of the first alginate with the solution of the hardener.

[0079] In the above step (2), the first alginate hardened in step (1) is frozen by a conventional method. By freezing once before step (3), the proportion of the first layer and the second layer mixed can be reduced. The freezing temperature and time are, for example, -20°C for 4 hours. This step (2) can also be omitted. By omitting it, the manufacturing time of the sponge-like laminate can be shortened, and production efficiency can be improved.

[0080] In the above step (3), first, a solution of a monovalent metal salt of low endotoxin alginic acid having a weight average molecular weight of 1,000 to 1,000,000 (hereinafter referred to as "second alginate") and a solution of a hardener are prepared. The solution of the second alginate and the solution of the hardener can be prepared by a known method or a method equivalent thereto. The solvent is the same as that described in the above step (1).

[0081] The second alginate can then be hardened by mixing the solution of the second alginate with the solution of the hardener.

[0082] The hardened second alginate may be further frozen prior to step (4), for example at a temperature of -20°C for 4 hours.

[0083] In the above step (4), the laminate obtained in step (3) is freeze-dried to obtain a sponge-like laminate. The freeze-drying can be performed by a known method. The freeze-drying conditions can be appropriately adjusted, and a primary drying step, a secondary drying step, etc. may be provided.

[0084] Through these steps, it is possible to obtain a biocompatible sponge-like laminate comprising a first spongy layer containing a first alginate and a hardener, and a second spongy layer containing a second alginate and a hardener, in which the weight-average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that in the second layer, as well as an adhesion prevention material comprising the sponge-like laminate.

[0085] In the above method, a sponge-like first layer containing a first alginate and a hardener is first prepared, and a sponge-like second layer containing a second alginate and a hardener is then prepared thereon, but it is also possible to prepare the second layer first, and then prepare the first layer thereon. In this case, the sponge-like laminate can be prepared, for example, through the following steps. (1') hardening the second alginate with a hardener; (2') freezing the hardened second alginate (optional); (3') a step of hardening the first alginate on the second alginate obtained in (2') with a hardener to obtain a laminate; (4') A step of freeze-drying the obtained laminate to obtain a sponge-like laminate. The specific explanation of each step is the same as in the above method.

[0086] Alternatively, a sponge-like laminate can be obtained by hardening and freeze-drying a first alginate to produce a first sponge-like layer, and separately hardening and freeze-drying a second alginate to produce a second sponge-like layer, and then bonding together the resulting sponge-like layers. The specific explanation of each step is the same as in the above method.

[0087] When hardening the first alginate and the second alginate, a container, mold, substrate, porous membrane, nonwoven fabric, woven fabric, etc. of the desired size, height and shape can be used to obtain an adhesion prevention material including a sponge-like laminate of the desired size, height and shape.

[0088] The curing time of the first alginate and the second alginate can be extended by reducing the molecular weight of the alginate used, reducing the concentration of the curing agent, or reducing the concentration of the alginate, or can be shortened by increasing the molecular weight of the alginate used, increasing the concentration of the curing agent, or increasing the concentration of the alginate. If the curing time is long, the operability of the alginate during curing, such as filling it into a container or mold, is improved, but if the curing time is too long, the production efficiency is affected. For example, the weight average molecular weight of the monovalent metal salt of alginic acid used as a raw material, measured by the GPC-MALS method, is, for example, 5,000 to 300,000, preferably 10,000 to 280,000, more preferably 20,000 to 270,000, even more preferably 30,000 to 260,000, and particularly preferably 40,000 to 250,000, to optimize the curing time of the alginate.

[0089] The anti-adhesion sponge-like laminate is preferably further sterilized. Examples of sterilization include, but are not limited to, gamma ray sterilization, electron beam sterilization, ethylene oxide gas sterilization, ethanol sterilization, hydrogen peroxide low-temperature gas plasma sterilization, hydrogen peroxide vapor sterilization, and formaldehyde gas sterilization. More preferably, the anti-adhesion material is sterilized by electron beam and / or gamma ray irradiation. By irradiating a polymeric material with gamma rays, electron beam, or the like, a medical material with high biocompatibility and controlled retention in the living body can be obtained (see, for example, JP 2000-237294 A). Irradiation conditions for electron beam and / or gamma ray sterilization include, for example, an absorbed dose of 10 kGy to 150 kGy, more preferably 20 kGy to 100 kGy, and even more preferably 40 kGy to 80 kGy. Other preferred embodiments of irradiation conditions for electron beam and / or gamma ray sterilization include, for example, an absorbed dose of 20 kGy to 80 kGy, 20 kGy to 60 kGy, 40 kGy to 60 kGy, etc. Electron beam sterilization is more preferable than gamma ray sterilization. In another preferred embodiment, the sponge-like laminate of the adhesion barrier is sterilized by a conventional method using ethylene oxide gas (EOG). In yet another embodiment, the sponge-like laminate of the adhesion preventing material may be produced under aseptic conditions using sterilized or aseptic materials. The sponge-like laminate produced in this manner can be provided in an aseptic condition without sterilization.

[0090] In some embodiments, the method further includes a step of pressing the laminate obtained in the above step (4) or the like. The pressing is performed manually or by using a press machine to sandwich and press the laminate. In addition, commonly used steps such as compression and thinning are also included in the pressing in the present invention. The pressing pressure is, for example, 1 kPa to 100 MPa, more preferably 10 kPa to 80 MPa, and even more preferably 100 kPa to 60 MPa. The manual pressing is performed using something that can be pressed by hand so that pressure is applied uniformly to the laminate, such as an acrylic ruler, an acrylic plate, a glass plate, a metal plate, or the like. In addition, an example of the pressing machine used is a hot press machine (AH-1T manufactured by AS ONE Corporation).

[0091] The physical properties of the obtained sponge-like laminate, such as tensile strength, tear strength, decomposition (solubility), water absorption rate (amount of water absorbed), shear (viscoelasticity), breathability, and slipperiness, can be measured by standard methods (e.g., methods specified in JIS).

[0092] 6.How to use Anti-adhesion materials are used by applying them to a subject requiring adhesion prevention. Anti-adhesion materials are preferably absorbed and decomposed after remaining at the applied site for about one week, which is usually required for the adhesion prevention effect to be exerted, and are ultimately metabolized and excreted within about one to two months, and therefore have excellent safety.

[0093] The adhesion barrier may be applied to a wound surface, for example to the surface of tissue associated with surgery.

[0094] "Tissues related to surgery" refers to tissues whose surface has been traumatized during surgery, or tissues whose surface has become inflamed or may become inflamed due to drying during surgery. Tissues related to surgery are preferably organs covered by the peritoneum (e.g., stomach, jejunum, ileum, appendix, colon, liver, gallbladder, spleen, duodenum, uterus, fallopian tubes, ovaries, abdominal wall, and pancreas), organs covered by the pleura (lungs, chest wall), organs covered by the pericardium (heart, pericardium), etc. The adhesion barrier of a preferred embodiment of the present invention can effectively prevent severe adhesions such as those that occur after liver resection.

[0095] Moreover, "applying" refers to placing the adhesion barrier on the surface of the wound (e.g., the surface of a tissue related to a surgical procedure). Specifically, for example, the adhesion barrier can be placed on the surface of the wound (e.g., the surface of a tissue related to a surgical procedure) so that the surface of the first layer of the sponge-like laminate contacts the surface of the wound (e.g., the surface of a tissue) and the surface of the second layer faces the opposite side (e.g., the serous cavity side) from the surface of the wound (e.g., the surface of the tissue). The above tissues include, but are not limited to, tissues covered with a serous membrane (e.g., stomach, jejunum, ileum, appendix, colon, liver, gallbladder, spleen, duodenum, pancreas, uterus, fallopian tube, ovary, omentum, mesentery, abdominal wall, lungs, heart, pericardium, chest wall, etc. in the abdominal cavity). Therefore, the adhesion preventing material can be arranged so that the surface of the first layer of the sponge-like laminate contacts the wound side surface of the tissue that is the direct target of surgery, etc., or can be arranged so that it contacts the wound side surface of the body wall, etc., where an invasion has been made to access the tissue. For example, in a surgical operation involving a large invasion of a skin incision, such as an abdominal operation, the surface of the first layer of the sponge-like laminate can be used so that it contacts the skin incision side (body wall side) when the incision is closed.

[0096] Depending on the size of the incision and the degree of invasiveness, multiple sponge-like laminates can be used in one operation. When multiple sponge-like laminates are used, all sponge-like laminates can be placed so that the surface of the first layer of each sponge-like laminate contacts the surface of the wound side (visceral side) of the target tissue, or all sponge-like laminates can be placed so that the surface of the first layer of each sponge-like laminate contacts the surface of the skin incision side (body wall side), or some sponge-like laminates can be placed so that the surface of the first layer of each sponge-like laminate contacts the surface of the wound side (visceral side) of the target tissue, and other sponge-like laminates can be placed so that the surface of the first layer of each sponge-like laminate contacts the surface of the skin incision side (body wall side). Since the first layer of the sponge-like laminate has a relatively high weight-average molecular weight, it remains on the surface of the tissue with a wound without being decomposed for a sufficient time to prevent adhesion, and acts as a physical barrier on the wound surface. On the other hand, since the second layer of the sponge-like laminate has a relatively low weight-average molecular weight, it dissolves and spreads quickly, and plays a role in preventing adhesion on the non-wound surface.

[0097] Preferably, the anti-adhesion sponge laminate is more flexible and less likely to break than Seprafilm (trade name). Therefore, in a preferred embodiment, the anti-adhesion material is not limited to the surface of the tissue to which it is applied, and can be used, for example, by wrapping it around the intestine during intestinal anastomosis. In another preferred embodiment, the anti-adhesion material can be easily inserted from the passage through which surgical instruments are inserted and removed from the subject during endoscopic surgery. In yet another preferred embodiment, the anti-adhesion material can be reapplied. In addition, the sponge laminate of the adhesion preventing material preferably has a wider range of targets for adhesion prevention compared to INTERCEED (product name).

[0098] Preferably, the adhesion barrier is prepared in an appropriate size depending on the area, shape, irregularities, etc. of the surface to which it is applied, and applied to the surface of the tissue related to the surgery to which adhesions should be prevented. A "subject" is a human or a non-human organism, for example, a bird or a non-human mammal (e.g., cow, monkey, cat, mouse, rat, guinea pig, hamster, ferret, pig, dog, rabbit, sheep, goat, and horse).

[0099] A sponge laminate of an adhesion preventing material, particularly a pressed sponge laminate, can be folded up into a compact shape, so that, for example, in endoscopic surgery, the adhesion preventing material can be applied relatively easily to the affected area via a trocar, etc. The adhesion preventing material applied to the affected area preferably absorbs moisture present in the affected area or moisture applied to the affected area to restore its thickness.

[0100] Preferably, the adhesion barrier is safe for use in subjects, similar to Seprafilm® and INTERCEED®.

[0101] After application to the tissue surface relevant to the surgery, suturing between the adhesion barrier and the tissue surface relevant to the surgery is not usually necessary, but if desired, the adhesion barrier and the tissue surface relevant to the surgery may be sutured together.

[0102] Also provided is a method for preventing adhesion, which comprises applying the sponge-like laminate to a subject in need of adhesion prevention. Specific methods are as described above.

[0103] Also provided is the use of the sponge-like laminate for producing an adhesion barrier. Specific uses are as described above.

[0104] Further, a sponge-like laminate for preventing adhesion is provided. Specific examples of the sponge-like laminate are as described above.

[0105] 7. Concomitant medications Concomitant drugs such as antibiotics, such as streptomycin, penicillin, tobramycin, amikacin, gentamicin, neomycin, and amphotericin B, aspirin, nonsteroidal antipyretic analgesics (NSAIDs), and anti-inflammatory drugs, such as acetaminophen, may be administered before, simultaneously with, or after application of the adhesion barrier of the present invention to tissues involved in surgery. These drugs may be mixed into the adhesion barrier of the present invention. The sponge-like laminate is porous and absorbent, so it is easier to load drugs on the sponge by preparation at the time of use, compared to nonporous Seprafilm (trade name). By impregnating the sponge with a drug solution and administering it, adhesion prevention and local sustained release of the drug can be achieved simultaneously in the abdominal cavity, thoracic cavity, cardiac cavity, subarachnoid cavity, serous cavity (peritoneal cavity, pleural cavity, pericardial cavity), joint cavity, etc. Furthermore, by loading the drug on layers with different dissolution rates, it is possible to release the drug at fast and slow sustained release rates.

[0106] All publications cited in this specification, including prior art documents, unexamined patent publications, patent publications and other patent documents, are hereby incorporated by reference in their entirety.

[0107] The present invention will be described in more detail with reference to the following examples, but the present invention should not be understood as being limited to these examples. EXAMPLES

[0108] Example 1: Preparation of an alginate-laminated sponge An alginate layered sponge was prepared as follows.

[0109] [reagent] The reagents used in the preparation of the alginate layered sponge are as follows: Low-endotoxin sodium alginate was obtained from Mochida Pharmaceutical Co., Ltd. · AL10: (Lot No. 5K12202), endotoxin content 4EU / g. AL500: (Lot No. BL150713-500), endotoxin content 19EU / g. Calcium chloride was obtained from Wako Pure Chemical Industries, Ltd. (product code: 036-00485).

[0110] [Equipment used] 35mm untreated dish (IWAKI product code 1000-035) Micropipette (Gilson Pipetman (product name)) Pure water production device (Merck Millipore Elix Essential UV5 (product name)) Freezer (SHARP SJ-56S (product name)) Freeze dryer (TAITEC VD-550R (product name))

[0111] [Preparation procedure] (1) Preparation of solutions AL500 was dissolved in pure water at a concentration of 1.0 wt% to prepare an AL500 solution. Similarly, AL10 was dissolved in pure water at a concentration of 1.0 wt% to prepare an AL10 solution. Furthermore, calcium chloride was dissolved in pure water to prepare 10 mM and 15 mM calcium chloride aqueous solutions.

[0112] (2) Preparation of AL500 layer (lower layer) 1.0 mL of AL500 solution and 1.0 mL of 10 mM calcium chloride aqueous solution were added to a 35 mm untreated dish using a micropipette and mixed uniformly by pipetting. This was left to stand overnight to gel. The dish was transferred to a freezer and frozen at -20°C for 4 hours.

[0113] (3) Lamination of AL10 layer (upper layer) The dish was removed from the freezer, and 1.0 mL of AL10 solution and 1.0 mL of 15 mM calcium chloride solution were added to the frozen AL500 layer with a micropipette and mixed uniformly by pipetting. The dish was then transferred back to the freezer and frozen at -20°C for 4 hours.

[0114] (4) Preparation of sponge After freezing, the dish was placed in a freeze-dryer and freeze-dried for two nights to obtain the desired alginate layered sponge.

[0115] The target alginate layered sponge contained a sponge-like lower layer (i.e., the first layer) containing AL500 and calcium chloride, and a sponge-like upper layer (i.e., the second layer) containing AL10 and calcium chloride. The alginate layered sponge was approximately circular with a diameter of 35 mm and a thickness of 1.83 ± 0.13 cm (n = 4). The total amount of sodium alginate used in the upper and lower layers was approximately 2.0 mg / cm. 2 The ratio (weight ratio) of the amount of sodium alginate used in the upper layer to that in the lower layer was 1:1. The amount of calcium chloride used in the upper layer was about 1.0 μmol / cm 2 and in the lower layer, about 1.5 μmol / cm 2 It was.

[0116] (5) Measurement of weight average molecular weight The weight average molecular weight of the alginic acid used as the raw material was measured by the following GPC-MALS method.

[0117] [Pretreatment method] The sample was dissolved in an eluent, and the solution was filtered through a 0.45 μm membrane filter to prepare a measurement solution.

[0118] [Measurement conditions (refractive index increment (dn / dc) measurement)] Differential refractometer: Optilab T-rEX Measurement wavelength: 658nm Measurement temperature: 40℃ Solvent: 200mM sodium nitrate aqueous solution Sample concentration: 0.5-2.5mg / mL (5 concentrations)

[0119] [Measurement conditions (absolute molecular weight distribution measurement)] Column: TSKgel GMPW-XL x 2 + G2500PW-XL (7.8 mm ID x 300 mm x 3) Eluent: 200mM sodium nitrate aqueous solution Flow rate: 1.0mL / min. Concentration: 0.05% Detector: RI detector, light scattering detector (MALS) Column temperature: 40℃ Injection volume: 200μL

[0120] [result] AL10: 55,000 AL500: 280,000

[0121] In addition, the single-layer sponge containing AL10 and the single-layer sponge containing AL500, which were produced according to the methods of steps (1), (2) and (4) above, were sterilized by electron beam, dissolved in EDTA (ethylenediaminetetraacetic acid) solution, and their molecular weights were measured by GPC-MALS method. The results are shown below.

[0122] [result] (When the electron beam sterilization dose is 20 kGy) AL10: 36,000 AL500: 75,000 (When the electron beam sterilization dose is 40 kGy) AL10: 27,000 AL500: 45,000

[0123] In Examples 3, 3-2, 4, and 9 described later, the obtained laminated sponge was sterilized with electron beam (20 kGy) and used as the laminated sponge. In Examples 6 and 7 described below, a non-sterile laminated sponge was used.

[0124] Example 1-2: Preparation of alginate laminated sponge (1) Preparation of sponge Using the alginic acid listed under [Reagents] below, AL100 or AL500 was used as the raw material for the lower layer, and AL10 or AL20 was used as the raw material for the upper layer, alginate laminated sponges were prepared in the following combinations: AL10 (upper layer)-AL100 (lower layer), AL20 (upper layer)-AL100 (lower layer), and AL20 (upper layer)-AL500 (lower layer) in accordance with the method described in Example 1. In the following, the combination of the upper layer and the lower layer may be written as "lower layer / upper layer" for convenience. According to this notation, for example, the combination of AL10 (upper layer)-AL100 (lower layer) is written as "AL100 / AL10", and the combination of AL20 (upper layer)-AL500 (lower layer) is written as "AL500 / AL20".

[0125] [reagent] AL10: Same as Example 1 · AL20: (Lot No. BL150713-20), endotoxin content 13EU / g. · AL100: (Lot No. 5G17201), endotoxin content 6EU / g. AL500: Same as Example 1

[0126] (2) Measurement of weight average molecular weight Among the alginic acids used to prepare the sponge, the weight-average molecular weights of AL20 and AL100 were measured by the GPC-MALS method using the method described in Example 1. [result] AL20: 82,000 AL100: 170,000

[0127] In addition, the molecular weights of the single-layer sponge containing AL20 and the single-layer sponge containing AL100 produced according to the method described in Example 1 after electron beam sterilization were measured using the method described in Example 1. The results are shown below.

[0128] [result] (When the electron beam sterilization dose is 20 kGy) AL20: 46,000 AL100:63,000 (When the electron beam sterilization dose is 40 kGy) AL20: 33,000 AL100: 40,000

[0129] Example 1-3: Preparation of alginate laminated sponge (hardened with calcium gluconate) An alginate laminated sponge was prepared in accordance with the method of Example 1, except that 10 mM calcium gluconate (manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of 10 mM calcium chloride when gelling the AL500 layer. The prepared sponge was used in Example 3-3, Experimental Group 1 described later.

[0130] Example 1-4: Preparation of alginate laminated sponge (EOG sterilization) An alginic acid laminated sponge of AL10 (upper layer) - AL100 (lower layer) was prepared by the method of Example 1, steps (1) to (4), except that the alginic acid used in Example 1-2 was used as the lower layer. The sponge obtained was sterilized by a conventional method using ethylene oxide gas. The sponge obtained was used in Example 3-3, experimental group 4 described later.

[0131] Example 1-5: Preparation of alginate laminated sponge (press after first freezing) Except for using AL100 as the alginic acid for the lower layer, which was the same as in Example 1-2, a solution was prepared by the method of Example 1, step (1), and then an alginic acid layered sponge was produced by the following steps from (2) onwards. (2) Preparation of AL100 layer (lower layer) 1.0 mL of the AL100 solution and 1.0 mL of a 10 mM calcium chloride aqueous solution were added to a 35 mm untreated dish using a micropipette and mixed uniformly by pipetting. This was left to stand for 1 to 2 hours to gel. (3) Lamination of AL10 layer (upper layer) On the gelled AL100 layer, 1.0 mL of the AL10 solution and 1.0 mL of a 15 mM calcium chloride aqueous solution were added with a micropipette and mixed uniformly by pipetting. The dish was transferred to a freezer and frozen at -20°C for 4 hours. (4) Preparation of sponge After freezing, the dish was placed in a freeze-dryer and freeze-dried for two nights to obtain the desired alginate layered sponge. (5) Pressing the sponge The sponge obtained in (4) above was set in a press (manufactured by AS ONE Corporation, product name AH-1T). The sponge was pressed at room temperature under a pressure of 10 MPa and held for 5 minutes. The sponge after pressing was sterilized by a conventional method using an electron beam. The sponge obtained was used in Example 3-3 and experimental group 5 described later.

[0132] Example 2: Measurement of the dissolution rate of each layer of an alginate laminated sponge (immersion method) We prepared laminated sponges with fluorescently modified upper and lower layers, and measured the dissolution rate. The specific method is shown below. FTSC (Fluorescein-5-Thiosemicarbazide) was used as the fluorescent labeling reagent, and alginic acid was labeled by the usual method. A layered sponge was prepared according to the method described in Example 1 using fluorescently labeled alginic acid.

[0133] [material] Low endotoxin sodium alginate was as described in Example 1. Phosphate buffer was prepared using sodium dihydrogen phosphate (Wako Pure Chemical Industries, Ltd., 197-09705 (trade name)), potassium dihydrogen phosphate (Wako Pure Chemical Industries, Ltd., 166-04255 (trade name)), sodium chloride (Wako Pure Chemical Industries, Ltd., 191-01665 (trade name)), and potassium chloride (Wako Pure Chemical Industries, Ltd., 166-17945 (trade name)). Sodium ethylenediaminetetraacetate (N001) was purchased from Dojindo.

[0134] [Equipment used] 8mm diameter biopsy trephine (Kai medical BP-80F (product name)) 96-well black microplate (Nunc 137101 (product name)) Fluorescence plate reader (Perkin Elmer ARVO X3 (product name))

[0135] [procedure] First, the fluorescently modified laminated sponge was punched with a biopsy trephine (Kai Medical BP-60F (trade name)) with a diameter of 8 mm. This was immersed in 10 mL of 150 mM phosphate buffer (pH 7.5), and 200 μL of the immersion solution was collected at regular intervals. The collected solution was transferred to a 96-well plate, and the amount of dissolved alginic acid was quantified by measuring the fluorescence intensity using a fluorescent plate reader.

[0136] [result] The measurement results of the dissolution behavior of each layer of the laminated sponge are shown in Figure 2. As shown in Figure 2, in the lower layer, 25 ± 10 wt% of the monovalent metal salt of alginic acid was dissolved within 1 hour, and 80 ± 10 wt% was dissolved within 4 hours. On the other hand, in the upper layer, 70 ± 10 wt% of the monovalent metal salt of alginic acid was dissolved within 1 hour, and 90 ± 10 wt% was dissolved within 4 hours. In addition, when the amount of eluted monovalent metal salt of alginic acid in the upper layer is taken as 100%, the ratio of the amount of eluted monovalent metal salt of alginic acid in the lower layer was 36% (less than 50%) at 1 hour from the start of measurement and 55% (less than 70%) at 2 hours. Thus, it was confirmed that the dissolution rate of the upper layer was faster than that of the lower layer. For example, when applying to wounds of intraperitoneal organs, by applying the alginate sponge with the lower layer facing the wound and the upper layer facing the abdominal cavity, the lower layer will remain on the wound and prevent adhesion at the wound, while the upper layer will dissolve relatively quickly and suppress de novo adhesions that form in large numbers even in places far away from the wound, such as adhesions throughout the abdominal cavity.

[0137] The dissolution rate was also measured in the same manner for each of the laminated sponges prepared in Example 1-2 (AL10 (upper layer)-AL100 (lower layer); AL20 (upper layer)-AL100 (lower layer); AL20 (upper layer)-AL500 (lower layer)). As a result, the dissolution rate of the upper layer was faster than that of the lower layer, similar to the laminated sponge prepared in Example 1 (AL10 (upper layer)-AL500 (lower layer)).

[0138] Example 2-2: Measurement of the dissolution rate of each layer of an alginate laminated sponge (adhesive method) According to the method of Example 2, laminated sponges in which the upper or lower layer of AL500 / AL10 was fluorescently modified were prepared, and the dissolution rate was measured according to the following procedure. [procedure] 1) The fluorescently modified laminated sponge was punched out using a biopsy trephine with a diameter of 8 mm (Kai Medical BP-60F (product name)). 2) Agarose (Wako Pure Chemical Industries, Ltd., product code: 010-08725) was dissolved in boiling water at 2% by weight and cooled to room temperature to prepare an agarose gel. The agarose gel was cut into 2 cm x 2 cm squares and immersed in phosphate buffer on a glass petri dish to moisten it. Phosphate buffer (pH 7.5) was added to the glass petri dish so that the liquid level was slightly below the top surface of the agarose gel. 3) The sponge from 1) was placed on an agarose gel, and the phosphate buffer solution was sampled at regular intervals and the fluorescence intensity was measured. [result] The measurement results of the dissolution behavior of each layer of the laminated sponge are shown in Table 1 and Figure 3. The values ​​indicate the weight percentage of eluted alginic acid at each time point, with the weight of alginic acid before the start of the test taken as 100 weight %.

[0139] [Table 1]

[0140] This method is a test system in which moisture is supplied only from the application surface, and the dissolution behavior of the laminated sponge in an environment closer to that in the body can be confirmed. In this test system, it is considered that the alginic acid in the upper layer passes through the side and the lower layer to reach the agarose gel, and then dissolves into the phosphate buffer solution in the petri dish. As shown in Table 1 and FIG. 3, in the lower layer, about 16±2% by weight of the monovalent metal salt of alginic acid was dissolved within 2 days, and about 33±7% by weight was dissolved within 8 days. On the other hand, in the upper layer, about 31±6% by weight of the monovalent metal salt of alginic acid was dissolved within 2 days, and about 60±7% by weight was dissolved within 8 days. In addition, when the amount of dissolution of the monovalent metal salt of alginic acid in the upper layer is taken as 100%, the ratio of the amount of dissolution of the monovalent metal salt of alginic acid in the lower layer was about 50±5% (about 44% to about 55%) on the second day after the start of measurement. Thus, it was confirmed that the dissolution rate of the upper layer is faster than that of the lower layer even under conditions closer to that in the body.

[0141] Example 3: Rat partial hepatectomy model Adhesion formation was evaluated using a rat partial hepatectomy model. The rat partial hepatectomy model induces severe inflammation and allows for highly reproducible observation of the formation of strong adhesions (Shimizu A et al., (2014) Surg Today. (44): 314-323). Specifically, adhesion formation was evaluated as follows.

[0142] [material] Low endotoxin sodium alginate is as described in Example 1. Seprafilm (trade name) is a sheet-shaped material made of a mixture of carboxymethylcellulose (CMC) and hyaluronic acid, and was obtained from Genzyme GmbH. Interceed (trade name) is a regenerated oxidized cellulose sheet, obtained from Johnson & Johnson.

[0143] [Experimental group] Control group (n=8): The edge of the left lateral lobe was measured to 3 cm, excised, and coagulated to stop bleeding (untreated control group). AL500 / AL10 laminated sponge group (n=8): The alginate sponge prepared in Example 1 was used as an adhesion barrier. Seprafilm group (n=8): 2×3 cm of Seprafilm was applied as an adhesion barrier. Interceed group (n=8): 2×3 cm Interceed was applied as an adhesion barrier.

[0144] [procedure] The rats were anesthetized by intraperitoneal administration of 35 mg / kg equivalent pentobarbital, and their weights were measured using an electronic balance. The rats were then opened through a midline incision. Next, the abdominal wall was pinched and lifted with tweezers, and the abdominal wall was cut. In preparation for liver resection, the left lateral lobe was pulled out from the back of the abdominal cavity, and gauze was placed underneath it. Next, the actual liver resection was performed. Specifically, a ruler was placed on the liver, and the position where the dissected surface was 3 cm was found, and both ends were cauterized with a bipolar and marked. The area between the two marked points was resected in a straight line. In the control group, the abdominal wall was immediately closed and the procedure was completed. In the group in which adhesion inhibitors were applied, the gauze was removed and the adhesion inhibitor was applied. Next, the abdominal wall and skin were sutured twice and the abdomen was closed. Biodegradable thread was used to suture the abdominal wall, and non-absorbable thread was used to suture the skin. One week after abdominal closure, the rats were euthanized by an overdose of about 2 mL of pentobarbital as anesthesia, and their body weight was measured using an electronic balance. The abdomen was then opened again, and adhesions were evaluated as follows: the spleen weight was measured using an electronic balance after the spleen was removed from the abdominal cavity.

[0145] [Evaluation of adhesions] Adhesion was assessed as follows.

[0146] (1) Dissection surface The liver sections described in the above [Procedure] were evaluated for the following (a) and (b). (a) Adhesion grade Adhesion was evaluated by visual inspection. Adhesion scores were given to the cut surfaces of the liver according to the following scoring method. Adhesion score: Grade 0: No adhesion is observed. Grade 1: Adhesion that can be torn off by its own weight (physiological adhesion) Grade 2: Adhesion that can be peeled off with tweezers (blunt adhesion) Grade 3: Adhesion that cannot be removed without using scissors or a scalpel (sharp adhesion)

[0147] (b) Extent of adhesion The width of the 3 cm dissected liver section where adhesions had formed was measured with a ruler and expressed as length (unit: mm) (therefore, the maximum extent of the dissected liver section was 30 mm).

[0148] (2) Non-dissection surface The following (a) and (b) were evaluated for areas other than the dissected liver surface, specifically the liver surface, omentum, peritoneum, small intestine, and immediately below the midline wound. Adhesion on non-dissected sections is an indicator of de novo adhesion. (a) Adhesion grade Adhesion was evaluated visually. Adhesion scores were given to sites other than the cut surface of the liver according to the scoring method described below. The site was not specified, and the maximum adhesion score observed was recorded as the adhesion score of the test animal. Adhesion score: Grade 0: No adhesion is observed. Grade 1: Adhesion that can be torn off by its own weight (physiological adhesion) Grade 2: Adhesion that can be peeled off with tweezers (blunt adhesion) Grade 3: Adhesion that cannot be removed without using scissors or a scalpel (sharp adhesion)

[0149] (b) Extent of adhesion For tissue areas where adhesions had formed, except for the cut surface of the liver, the width of the adhesion was measured with a ruler and expressed as length (unit: mm). As in (2)(a) above, the site was not specified, and the maximum width of adhesion formation was recorded as the adhesion extent of the test animal.

[0150] [result] The results of adhesion evaluation are shown in Figure 4 (dissected section) and Figure 5 (non-dissected section). The results of body weight measurement and spleen weight measurement are shown in Figure 6. On the detached cross section, a tendency for adhesion to be suppressed in each group was observed compared to the control group (FIGS. 4(A) to (C)). On the non-detached sections, a significant adhesion prevention effect was confirmed in the AL500 / AL10 laminated sponge group (Figures 5(A)-(C)). No adhesion prevention effect was observed with Seprafilm (trade name) and Interceed (trade name), which were used as positive controls, and a tendency for adhesions to worsen was observed compared to the control group. On the other hand, a significant adhesion prevention effect was confirmed in the AL500 / AL10 laminated sponge group. There were no significant differences in body weight and spleen weight among the control group, Seprafilm group, Interceed group, and AL500 / AL10 laminated sponge group, confirming that application of the AL500 / AL10 laminated sponge had no adverse effects on the living body (Figures 6(A) and (B)). Here, unless otherwise specified, the significant difference test in the examples was performed by Student's t-test, and only the evaluation of Grade was performed by Mann-Whitney U test.

[0151] Example 3-2: Rat partial hepatectomy model using Pean forceps Except for the use of Pean forceps during liver transection, the adhesion grade and adhesion extent on the transected and non-transected sections of the liver were evaluated using the same materials, experimental groups, procedures, and adhesion evaluation methods as in Example 3.

[0152] Specifically, the liver transection using Pean forceps was performed as follows: That is, the "linear resection between the two marked points" in the [Procedure] of Example 3 was performed by crushing the liver parenchyma with Pean forceps and cauterizing the exposed blood vessels with a bipolar.

[0153] [result] The results of adhesion evaluation are shown in Figure 7 (dissected section) and Figure 8 (non-dissected section), and the results of body weight measurement and spleen weight measurement are shown in Figure 9. On the detached sections, each group (n = 8) showed a tendency for adhesion to be suppressed compared to the control group (n = 8), and a statistically significant difference was observed between the AL500 / AL10 layered sponge group and the control group (Figures 7(A) to (C)). On the non-detached sections, a significant adhesion prevention effect was confirmed in the AL500 / AL10 laminated sponge group (Figures 8(A)-(C)). No adhesion prevention effect was observed for Seprafilm (trade name), used as a positive control, and a tendency for adhesion to worsen was observed for Interceed (trade name) compared to the control group. On the other hand, a significant adhesion prevention effect was confirmed in the AL500 / AL10 laminated sponge group. There were no significant differences in body weight and spleen weight among the control group, Seprafilm group, Interceed group, and AL500 / AL10 laminated sponge group, confirming that application of the AL500 / AL10 laminated sponge had no adverse effects on the living body (Figures 9(A) and (B)).

[0154] Example 3-3: Rat partial hepatectomy model using Pean forceps Using the same method as in Example 3-2, the adhesion grade and adhesion extent on the separated and non-separated liver sections were evaluated for the following experimental groups. Experimental group 1: AL500 / AL10 hardened with calcium gluconate [prepared in Examples 1-3] Experimental group 2: AL500 / AL10 press [prepared in Example 6 (1-2)] Experimental group 3: AL100 / AL10 Electron beam sterilization [prepared in Example 1-2] Experimental group 4: AL100 / AL10 EOG sterilization [prepared in Examples 1-4] Experimental group 5: AL100 / AL10 1-stage freezing press [prepared in Examples 1-5] Separate experiments were also carried out for an untreated control group, a Seprafilm group, and an AL500 / AL10 group [prepared in Example 1], and the results of these groups were compared with those of the experimental groups. [result] In all of the experimental groups 1 to 5, the same effects as those of the laminated sponge produced in Example 1 were obtained.

[0155] Example 4: Visualization of each layer of an alginate-laminated sponge by fluorescent labeling A test for visualization of each layer of the alginate laminated sponge using fluorescent labeling was carried out as follows.

[0156] [material] Low endotoxin sodium alginate is as described in Example 1.

[0157] [Equipment used] Handy UV lamp (UVP UVGL-58 (product name))

[0158] [procedure] The rat liver resection procedure was as described in Example 3. An alginate sponge with a fluorescently labeled first or second layer was attached to the prepared dissected surface of the liver. The sponge was attached at a concentration of 4.0 mg / cm, which is higher than that in Example 1, in order to make it easier to observe the remaining material. 2 A laminated sponge using this alginic acid was prepared and used according to the method of Example 1. The abdomen was then closed according to the procedure described in Example 3, and one week later, the abdomen was opened. The exposed abdominal cavity was irradiated with ultraviolet light from a lamp to visualize the intraperitoneal distribution of fluorescently labeled alginic acid.

[0159] As a result, it was confirmed that the AL10 layer was widely distributed not only on the dissected surface but also on the surface of the peritoneum, suggesting that the second layer of the sponge-like laminate dissolves and spreads rapidly in the peritoneal cavity because the weight-average molecular weight of the monovalent metal salt of alginic acid is relatively low. Although the AL500 layer was also observed in some areas of the abdominal wall, the fluorescence from the dissected surface was more prominent. This suggests that the first layer of the sponge-like laminate is retained on the dissected surface and acts as a physical barrier, since the weight-average molecular weight of the monovalent metal salt of alginic acid is relatively high.

[0160] Example 5: Winding test In order to demonstrate the ability of the alginate laminated sponge to conform to a curved surface, a wrapping test was carried out as follows.

[0161] [material] Low endotoxin sodium alginate was as described in Example 1. Agar (010-08725) was purchased from Wako Pure Chemical Industries.

[0162] [procedure] After dissolving agar in hot water, the mixture was poured into a cylindrical mold and cooled to prepare an agarose gel cylinder with a diameter of 20 mm. This was used as a model tubular organ, and the sponge prepared in Example 1 was wrapped around it to verify the followability of the wrapping.

[0163] As a result, it was confirmed that the flexibility of the alginate laminated sponge made it possible to wrap it around a cylinder that mimicked the intestinal tract, suggesting that anti-adhesion materials containing sponge-like laminates could also be used for intestinal anastomosis.

[0164] Example 6: Sponge pressing and swelling test The pressing of the alginate laminated sponge, the measurement of the thickness after pressing, and the swelling test were carried out as follows.

[0165] [material] Alginate layered sponges (AL10 (top layer)-AL500 (bottom layer)) were as described in Example 1.

[0166] [procedure] (1) Pressing and measuring thickness after pressing (1-1) Manual press The alginate laminated sponge was placed on a flat surface and pressed with the palm of the hand using an acrylic ruler so that pressure was applied evenly across the sponge. The thickness of the sponge before and after pressing was measured with an electronic caliper, and the average value was calculated (n=4).

[0167] (1-2) Pressing with a press machine The alginate laminated sponge was set in a press (product name AH-1T, manufactured by AS ONE Corporation). The alginate laminated sponge was pressed at room temperature with a pressure of 10 MPa and held for 5 minutes. The thickness of the sponge after pressing was measured with an electronic caliper, and the average value was calculated (n=4).

[0168] (2) Swelling test Agarose gel / glass petri dish was prepared in the same manner as in Example 2-2. The agarose gel was moistened with pure water. The alginate laminated sponge cut into 1 cm x 1 cm squares before and after pressing was placed on the agarose gel. Photographs were taken from the side at regular intervals, and the thickness of the sponge was calculated from the photographed images to confirm the influence of pressing on swelling (before pressing: n=3; after pressing: n=3. In the swelling test, the pressed alginate laminated sponge was one pressed with a press machine.

[0169] [result] The measurement results of the thickness after pressing are shown in Table 2.

[0170] [Table 2]

[0171] The average thickness of the sponge was about 1.5 mm before pressing, about 0.33 mm after manual pressing, and about 0.16 mm after mechanical pressing. The pressed sponge did not increase in thickness over time and maintained the above thickness.

[0172] The change in sponge thickness over time during the swelling test is shown in Figure 10. The results of the swelling test confirmed that the pressed laminated alginate sponge absorbed water and became almost the same thickness as the unpressed alginate sponge. This suggests that since the sponge can be compressed into a compact shape, it may be relatively easy to apply the sponge, which acts as an adhesion prevention material, to the affected area via a trocar or the like during endoscopic surgery. It was also suggested that the pressed sponge applied to the affected area absorbs moisture present in the affected area or that is applied to the affected area, and regains its thickness. By regaining its thickness, the sponge can function as a laminated sponge.

[0173] Example 7: Misting test The following test was carried out on the alginate laminated sponge and Seprafilm (trade name) to evaluate their fragility when absorbing water.

[0174] [material] The alginic acid laminated sponge was as described in Example 1, and Seprafilm (product name) was as described in Example 3. In addition, as a pressed alginic acid laminated sponge, one pressed with the press machine described in Example 6 was used.

[0175] [procedure] A 1 cm x 2 cm test piece was prepared from an alginate laminated sponge and Seprafilm (trade name). Double-sided tape was attached to one end of the test piece at 1 cm x 1 cm, and the test piece was held at the edge of a test stand. This fixed the test piece so that the other end, 1 cm x 1 cm, was exposed in the air. Each test piece was sprayed with pure water five times using a spray bottle, and a video was taken of the process in which the test piece bent downward as it became wet. By analyzing the images of the obtained video, both the height and angle of the tip of the test piece from the test stand were calculated and the changes over time were plotted.

[0176] [result] The results are shown in Figure 11. Regarding height, the alginate laminated sponge showed a drop in height of less than 2 mm up to 50 seconds after spraying, and even after 90 seconds, it was only about 3 mm (Figure 11(A)). The pressed alginate laminated sponge also showed a drop in height of about 9 mm even after 90 seconds after spraying. On the other hand, Seprafilm (product name) showed a significant drop in height immediately after spraying (Figure 11(A)). The angle results were similar to those for height (Figure 11(B)). This suggests that the alginate laminated sponge maintains its shape and strength for a while even when it is in a water-absorbed state, regardless of whether it is pressed or not. Therefore, when it is applied to the affected area as an adhesion inhibitor, it is possible to adjust the application position and reapply it, or when applying the sponge as an adhesion inhibitor to the affected area through a trocar during endoscopic surgery, it is possible to avoid situations where it cannot be spread properly due to absorption of moisture in the trocar. It has been confirmed in Examples 3 and 5 that the alginate laminated sponge has suitable pressure adhesion to the affected area or its model system.

[0177] Example 8: Young's modulus / break strength The mechanical properties of the alginate-laminated sponge were evaluated using a tensile tester. [material] The unpressed alginic acid laminated sponge used was that produced in Example 1, while the pressed alginic acid laminated sponge used was that pressed with the press described in Example 6. [procedure] A JIS K6251 tensile No. 8 dumbbell-shaped piece (width 10 mm) was prepared from each sponge. The piece was attached to a tensile tester (CR-3000EX-S, manufactured by Sun Scientific Co., Ltd.) to obtain a stress-strain curve. Young's modulus and breaking strength were calculated from the obtained stress-strain curve.

[0178] [result] The measurement results were as follows (mean ± standard error). Young's Modulus: AL500 / AL10 sponge (unpressed) 0.79±0.164 MPa AL500 / AL10 sponge (pressed) 14.88±1.434 MPa Breaking Strength: AL500 / AL10 sponge (unpressed) 51.55±6.391 kPa AL500 / AL10 sponge (pressed) 272.80±61.892 kPa As the above results show, by pressing the sponge, the Young's modulus and breaking strength increased, and the sponge was toughened.

[0179] Example 8-1: Young's modulus / breaking strength (2) [material] An alginate layered sponge was prepared having the following composition: [Table 3]

[0180] Formulation 1 was prepared by preparing an alginic acid laminated sponge according to the method described in Example 1, and pressing it according to the method described in Example 6 (1-2). Formulations 2-3 were prepared by preparing an alginic acid laminated sponge according to the method described in Example 1-3, and pressing it according to the method described in Example 6 (1-2). Formulations 4-6 were prepared by preparing an alginic acid laminated sponge according to the method described in Example 1, except that AL500 (same as in Example 1) and AL100 (same as in Example 1-2) were mixed to a predetermined weight ratio as the sodium alginate in the lower layer, and pressing it according to the method described in Example 6 (1-2). Formulation 7 was prepared according to the method described in Example 1-5. Formulation 8 was prepared according to the method described in Example 1. Parts of formulations 7 and 8 were pressed according to the method described in Example 6 (1-2).

[0181] [procedure] According to the method described in Example 8, the Young's modulus and breaking strength of each formulation were determined (n=4). [result] The measurement results were as follows: All of the prepared alginate laminated sponges had strength and flexibility suitable for use as an adhesion barrier. [Table 4]

[0182] Example 9: Condensation test The following test was carried out to evaluate the water absorbency of the alginate-laminated sponge. [material] The agarose gel / glass dish was the same as in Example 2-2. The sponges used for the measurement were AL500 / AL10 (Example 1), AL100 / AL10 (Example 1-2), and AL500G / AL10 (Example 1-3). All sponges used for the measurement were sterilized with electron beam (20 kGy). Each sponge was punched out with a biopsy trephine (Kai medical BP-60F (product name)) with a diameter of 8 mm and used for the test. [procedure] The mesh was placed on agarose gel placed on a petri dish filled with phosphate-buffered saline, and the sponge to be measured was then placed on top of the mesh. The change in sponge weight (%) over 6 hours was measured. [result] The measurement results are shown in Figure 12. All sponges showed good water absorption. The weight gain rate of AL100 / AL10 was lower than that of the other sponges because the water resorption and sponge dissolution proceeded simultaneously. [Explanation of symbols]

[0183] 1. Anti-adhesion materials 2. First Layer 3. Second Layer 4 Sponge-like laminate

Claims

1. The present invention comprises a first layer and a second layer of sponge-like low endotoxin monovalent metal salt of alginic acid, at least a portion of which is crosslinked with a curing agent, the weight average molecular weight of the monovalent metal salt of alginic acid in the first layer is 30,000 to 300,000, the weight average molecular weight of the monovalent metal salt of alginic acid in the second layer is 1,000 to 200,000, the weight average molecular weights being measured by a GPC-MALS method after a decrosslinking treatment, the weight average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than the weight average molecular weight of the monovalent metal salt of alginic acid in the second layer, the amount of the curing agent used in the first layer is 0.5 μmol / cm 2 to 2.0 μmol / cm 2 , and the amount of the curing agent used in the second layer is 0.6 μmol / cm 2 to 2.4 μmol / cm 2 . The adhesion preventing material comprises a sponge-like laminate that is applicable to a living body, wherein the endotoxin content of the monovalent metal salt of alginic acid in the first layer and the second layer is 500 EU / g or less.

2. The total amount of low endotoxin monovalent metal salt of alginic acid used in the first and second layers is 0.1 mg / cm ２ ~3mg / cm ２ The adhesion preventing material according to claim 1, wherein the adhesion preventing material has a viscosity in the range of 0.1 to 1000 MPa.

3. 3. The adhesion preventing material according to claim 1 or 2, wherein the monovalent metal salt of alginic acid in the first layer and the second layer is sodium alginate or potassium alginate.

4. The hardener for the first and second layers is CaCl ２ , CaSO ４ , ZnCl ２ , SrCl ２ , FeCl ３ , BaCl ２ , CaHPO ４ The adhesion preventing material according to any one of claims 1 to 3, which is at least one metal ion compound selected from the group consisting of calcium gluconate, calcium oxalate and calcium lactate.

5. The adhesion preventing material according to any one of claims 1 to 4, which is intended to be applied with the first layer facing the surface of the wound.

6. The adhesion preventing material according to any one of claims 1 to 5, having one or more of the following characteristics: (1) A piece of JIS K6251 tensile test No. 8 is subjected to a tensile tester, and the Young's modulus determined based on the obtained stress-strain curve is 0.3 to 300 MPa. (2) A piece of JIS K6251 tensile test No. 8 is placed in a tensile tester, and the breaking strength determined based on the resulting stress-strain curve is 5 to 5,000 kPa. (3) A piece of JIS K6251 tensile tester No. 8 is subjected to a tensile test, and the ratio of breaking strength to Young's modulus of the sponge-like laminate determined based on the resulting stress-strain curve is 2 to 50. (4) When the sponge-like laminate is contacted with an agarose gel soaked in phosphate-buffered saline for 2 to 6 hours, the weight increase rate of the sponge-like laminate is 200 to 50,000%, assuming that the weight of the sponge-like laminate before contact with phosphate-buffered saline is 100%. (5) In a dissolution test using the elution of a monovalent metal salt of alginic acid into a phosphate buffer solution of pH 7.5 through agarose gel as an index, the ratio of the elution amount of the monovalent metal salt of alginic acid from the first layer to the elution amount of the monovalent metal salt of alginic acid from the second layer, taken as 100%, is 30 to 70% on or after the second day from the start of the measurement. (6) In a dissolution test using the dissolution of a monovalent metal salt of alginic acid into a phosphate buffer solution of pH 7.5 through agarose gel as an indicator, about 15±5% by weight of the monovalent metal salt of alginic acid is dissolved in the first layer within 2 days, and about 25±10% by weight is dissolved in the second layer within 2 days, and about 30±8% by weight of the monovalent metal salt of alginic acid is dissolved in the second layer within 2 days, and about 60±10% by weight is dissolved in the second layer within 8 days.

7. The adhesion preventing material according to any one of claims 1 to 6, wherein the sponge-like laminate is pressed.

8. The adhesion preventing material according to any one of claims 1 to 7, wherein the sponge-like laminate has been subjected to one or more treatments selected from electron beam sterilization, gamma ray sterilization, and ethylene oxide gas sterilization.

9. The adhesion barrier is adapted to be applied to tissues associated with a surgical procedure of a subject, The adhesion preventing material according to any one of claims 1 to 8, wherein the tissue associated with the surgery is selected from the group consisting of organs covered by the peritoneum selected from the stomach, jejunum, ileum, appendix, colon, liver, gallbladder, spleen, duodenum, uterus, fallopian tubes, ovaries, abdominal wall and pancreas; organs covered by the pleura selected from the lung and chest wall; and organs covered by the pericardium selected from the heart and pericardium.

10. A method for producing an adhesion preventing material comprising a sponge-like laminate applicable to a living body, comprising the following steps: (1) hardening a monovalent metal salt of low-endotoxin alginic acid having a weight average molecular weight of 30,000 to 300,000 with a hardener; (2) A step of curing a monovalent metal salt of low-endotoxin alginic acid having a weight average molecular weight of 1,000 to 200,000 with a curing agent on the monovalent metal salt of alginic acid obtained in (1) to obtain a laminate; (3) freeze-drying the obtained laminate to obtain a sponge-like laminate; Here, the molecular weight is measured by a GPC-MALS method, The sponge-like laminate comprises a spongy first layer containing a low endotoxin monovalent metal salt of alginic acid having a weight average molecular weight of 30,000 to 300,000, and a spongy second layer containing a low endotoxin monovalent metal salt of alginic acid having a weight average molecular weight of 1,000 to 200,000, wherein the weight average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that of the second layer, the amount of hardener used in the first layer is 0.5 μmol / cm 2 to 2.0 μmol / cm 2 , the amount of hardener used in the second layer is 0.6 μmol / cm 2 to 2.4 μmol / cm 2 , and the endotoxin content of the monovalent metal salt of alginic acid in the first and second layers is 500 EU / g or less.

11. The hardener is CaCl ２ , CaSO ４ , ZnCl ２ , SrCl ２ , FeCl ３ , BaCl ２ , CaHPO ４ 11. The method of claim 10, wherein the at least one metal ion compound is selected from the group consisting of calcium gluconate, calcium oxalate and calcium lactate.

12. The method according to claim 10 or 11, further comprising a step of subjecting the sponge-like laminate obtained in (3) to one or more treatments selected from electron beam sterilization, gamma ray sterilization, and ethylene oxide gas sterilization.

13. A method for producing a sponge-like laminate applicable to a living body, comprising the following steps (1) to (3): (1) hardening a monovalent metal salt of low-endotoxin alginic acid having a weight average molecular weight of 30,000 to 300,000 with a hardener; (2) A step of curing a monovalent metal salt of low-endotoxin alginic acid having a weight average molecular weight of 1,000 to 200,000 with a curing agent on the monovalent metal salt of alginic acid obtained in (1) to obtain a laminate; (3) freeze-drying the obtained laminate to obtain a sponge-like laminate; Here, the molecular weight is measured by a GPC-MALS method, The sponge-like laminate comprises a spongy first layer containing a low endotoxin monovalent metal salt of alginic acid having a weight average molecular weight of 30,000 to 300,000, and a spongy second layer containing a low endotoxin monovalent metal salt of alginic acid having a weight average molecular weight of 1,000 to 200,000, wherein the weight average molecular weight of the monovalent metal salt of alginic acid in the first layer is higher than that of the second layer, the amount of hardener used in the first layer is 0.5 μmol / cm 2 to 2.0 μmol / cm 2 , the amount of hardener used in the second layer is 0.6 μmol / cm 2 to 2.4 μmol / cm 2 , and the endotoxin content of the monovalent metal salt of alginic acid in the first and second layers is 500 EU / g or less.

14. A method for producing a sponge-like laminate according to claim 13, wherein the sponge-like laminate is used as an anti-adhesion material.

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