WO2019172261A1

WO2019172261A1 - Esophageal stricture preventing material

Info

Publication number: WO2019172261A1
Application number: PCT/JP2019/008651
Authority: WO
Inventors: 大知伊藤; 誠一太田; 蟠戚; 光弘藤城; 陽介辻
Original assignee: 国立大学法人東京大学
Priority date: 2018-03-05
Filing date: 2019-03-05
Publication date: 2019-09-12

Abstract

The present invention provides a composition for coating an injured portion of an esophagus which comprises ionically crosslinkable polysaccharides that are crosslinked with a curing agent, and which is used to coat an injured portion of the esophagus. Thus, a novel composition for coating an injured portion of the esophagus is provided.

Description

Esophageal stricture prevention material

The present invention relates to a composition for covering an esophageal damaged part.

The esophagus is a tube that connects the pharynx and stomach, and serves to send food from the pharynx to the stomach. The wall of the esophagus is divided into four layers from the inside toward the outside: the mucosa, the submucosa, the intrinsic muscle layer, and the outer membrane. The mucosa is divided into three layers from the inside to the outside: the stratified squamous epithelium, the lamina propria, and the mucosal lamina.

Esophageal cancer is a cancer that kills about 11,000 people annually in Japan and is difficult to detect, and is a cancer that is more likely to progress, metastasize, recur, and is difficult to treat than other gastrointestinal organs. It is. Esophageal cancer treatment methods can be broadly divided into four categories: surgery, endoscopic treatment, chemotherapy, and radiation therapy. Endoscopic mucosal resection (EMR), which is an endoscopic treatment method, is a standard treatment method particularly for early esophageal cancer in which cancer is confined to the mucosa. There are important organs such as the heart and lungs around the esophagus, and surgery is difficult and highly invasive. Therefore, it is expected to expand the indication of endoscopic treatment that is the least invasive and resectable. Yes.

In recent years, endoscopic submucosal dissection (ESD) has been developed that can remove a wide range of cancers at a time compared with conventional EMR methods, and is becoming popular for esophageal cancer following gastric cancer. ESD is a treatment method for resecting cancer using an endoscope. Specifically, physiological saline, hyaluronic acid (HA), etc. were injected into the submucosal layer under the lesion, and it was raised by the injection. This is a treatment method in which the lesion is removed by removing the part with a scalpel or the like. However, the ulcer that occurs after ESD of a wide range of lesions is large, and stenosis caused by the ulcer is a problem. In patients with extensive resection of esophagus 3/4 or more (sub-total circumference), postoperative stenosis occurs frequently, and postoperative stenosis is very difficult to treat. Frequent endoscopic treatment for postoperative stenosis Patients must undergo balloon dilatation, causing a reduction in quality of life (QOL).

While steroid local injection is used as a preventive method for esophageal stricture, and balloon dilation and stent placement are used as treatment methods, all of them still have the following problems.

Steroid local injection requires multiple treatments, and injection of steroids into the muscle layer during treatment may cause the muscle layer to dissolve and may cause delayed perforation. In addition, since balloon dilatation requires a plurality of treatments, tearing not only to the surface of the esophagus but also deeper than the surface of the esophagus may cause pain and perforation of the esophagus. In stent placement, existing esophageal stents are made of metal or plastic, and recently made of biodegradable materials. Previous studies have reported metal stent perforations, ulcers, formation of new stenosis, and recurrence rate of stenosis after removal as high as 50%, and plastic stents have a migration rate of 30% to 80%. In recent years, biodegradable stents that degrade in about 8 to 10 weeks are often used, but due to the high prevalence of inflammation and granulation tissue overgrowth, it has been pointed out that their effectiveness and safety are still insufficient. Yes.

Furthermore, when using a material for preventing stenosis after ESD, it requires a high level of technology and a long operation time to deliver the sheet-like material to the affected area through the endoscope channel. .

Here, Patent Document 1 describes a balloon having a drug coating. Examples of excipients for drug coating include alginate, and examples of applying a balloon include treatment of esophageal stricture. . In addition, it is known that alginic acid or a derivative thereof or a salt thereof is used as a wound covering prevention material or an adhesion prevention material (Patent Documents 2 and 3). Non-Patent Document 1 describes maleimide-modified alginic acid derivatives.

US Patent Application Publication No. 2016/0213890 JP 2007-75425 A International Publication No. 2016/114355 Pamphlet

Under such circumstances, a new composition for covering the damaged part of the esophagus has been demanded.

As a result of extensive studies, the present inventors have cross-linked with an ion-crosslinkable polysaccharide (for example, a monovalent metal salt of alginic acid or a derivative thereof) cross-linked with a curing agent, and optionally with transglutaminase. The present inventors have found that an esophageal lesion covering material containing a protein or a polysaccharide having an amino group is suitable for coating an esophageal lesion, and thus completed the present invention.

The present invention is as follows. [1-1] containing an ionically crosslinkable polysaccharide,
The ion-crosslinkable polysaccharide is cross-linked with a curing agent and used to coat the esophageal lesion with a protein or amino group cross-linked using transglutaminase.
A composition for covering an esophageal lesion.
[1-2] The composition according to [1-1] above, wherein the ion-crosslinkable polysaccharide is at least one selected from the group consisting of alginic acid, a derivative thereof, and a salt thereof.
[1-3] The composition according to [1-2] above, wherein the alginic acid derivative is alginic acid modified with a maleimide group, alginic acid modified with a thiol group, or alginic acid modified with an acrylate group.
[1-4] The composition according to [1-3] above, wherein the modification is performed via a spacer.
[1-5] Any one of the above [1-2] to [1-4], wherein the salt of alginic acid or a derivative thereof is sodium alginate, potassium alginate, sodium salt of alginic acid derivative, or potassium salt of alginic acid derivative. The composition according to item.
[1-6] The above [1-1] to [1], wherein the curing agent is at least one metal ion compound selected from the group consisting of Ca ²⁺ , Mg ²⁺ , Ba ²⁺ , Sr ²⁺ , Zn ²⁺ and Fe ^3+. The composition according to any one of [5].
[1-7] The composition according to any one of [1-1] to [1-6] above, wherein the protein or polysaccharide having an amino group is gelatin.
[1-8] The composition according to any one of the above [1-1] to [1-7], wherein the composition is in the form of fluid liquid or powder.
[1-9] The composition according to any one of [1-1] to [1-8] above, wherein the composition is in a liquid form having fluidity and is applied to the surface of the esophageal lesion by spraying. .
[1-10] The composition according to any one of [1-1] to [1-8] above, wherein the composition is in a powder form and is applied to the surface of an esophageal injury site by spraying.
[1-11] The composition according to [1-8] or [1-10] above, wherein the particle diameter of the powder is in the range of 10 μm to 500 μm.
[1-12] The composition according to any one of [1-1] to [1-11] above, which is used in combination with another drug.
[1-13] The composition according to any one of [1-1] to [1-12] above, which is used for preventing esophageal stricture.

[2-1] (a) a polysaccharide capable of ion crosslinking, and (b) a polysaccharide having a protein or an amino group,
The ion-crosslinkable polysaccharide is crosslinked with a curing agent, and the polysaccharide having a protein or amino group is crosslinked using transglutaminase, the crosslinked ion-crosslinkable polysaccharide and the polysaccharide having a crosslinked protein or amino group. Used to cover esophageal lesions with sugars,
Kit for covering the damaged part of the esophagus.
[2-2] The kit according to [2-1] above, wherein the ion-crosslinkable polysaccharide is at least one selected from the group consisting of alginic acid, a derivative thereof, and a salt thereof.
[2-3] The kit according to [2-2] above, wherein the alginic acid derivative is alginic acid modified with a maleimide group, alginic acid modified with a thiol group, or alginic acid modified with an acrylate group.
[2-4] The kit according to [2-3] above, wherein the modification is performed via a spacer.
[2-5] Any one of the above [2-2] to [2-4], wherein the salt of alginic acid or a derivative thereof is sodium alginate, potassium alginate, sodium salt of alginic acid derivative, or potassium salt of alginic acid derivative. Kit according to item.
[2-6] The above [2-1] to [2], wherein the curing agent is at least one metal ion compound selected from the group consisting of Ca ²⁺ , Mg ²⁺ , Ba ²⁺ , Sr ²⁺ , Zn ²⁺ and Fe ^3+. The kit according to any one of -5].
[2-7] The kit according to any one of [2-1] to [2-6] above, wherein the protein or polysaccharide having an amino group is gelatin.
[2-8] The above [2-1] to [2], wherein (a) the ion-crosslinkable polysaccharide and (b) the protein or the polysaccharide having an amino group are in a liquid or powder form having fluidity. The kit according to any one of [-7].
[2-9] (a) an ion-crosslinkable polysaccharide and (b) a protein or polysaccharide having an amino group is in a liquid form having fluidity, and (a) an ion-crosslinkable polysaccharide and (b) a protein Alternatively, the kit according to any one of [2-1] to [2-8] above, wherein the polysaccharide having an amino group is applied to the surface of the esophageal lesion by spraying.
[2-10] (a) An ion-crosslinkable polysaccharide and (b) a protein or polysaccharide having an amino group are in powder form, (a) an ion-crosslinkable polysaccharide and (b) a protein or amino group The kit according to any one of [2-1] to [2-8] above, wherein the possessed polysaccharide is applied to the surface of the esophageal lesion by spraying.
[2-11] The kit according to [2-8] or [2-10] above, wherein the particle diameter of the powder is in the range of 10 μm to 500 μm.
[2-12] The kit according to any one of [2-1] to [2-11], further comprising another drug.
[2-13] The kit according to any one of [2-1] to [2-12] above, which is used for preventing esophageal stricture.

[3-1] Applying ion-crosslinkable polysaccharide and protein or amino group-containing polysaccharide to the target esophageal lesion,
The ion-crosslinkable polysaccharide is crosslinked with a curing agent, and the polysaccharide having a protein or amino group is crosslinked using transglutaminase, the crosslinked ion-crosslinkable polysaccharide and the polysaccharide having a crosslinked protein or amino group. Cover the damaged part of the esophagus with sugars,
How to cover esophageal lesions.
[3-2] The method according to [3-1] above, wherein the ion-crosslinkable polysaccharide is at least one selected from the group consisting of alginic acid, a derivative thereof, and a salt thereof.
[3-3] The method according to [3-2] above, wherein the alginic acid derivative is alginic acid modified with a maleimide group, alginic acid modified with a thiol group, or alginic acid modified with an acrylate group.
[3-4] The method according to [3-3] above, wherein the modification is performed via a spacer.
[3-5] Any one of [3-2] to [3-4] above, wherein the salt of alginic acid or a derivative thereof is sodium alginate, potassium alginate, sodium salt of alginic acid derivative, or potassium salt of alginic acid derivative. The method according to item.
[3-6] The above [3-1] to [3], wherein the curing agent is at least one metal ion compound selected from the group consisting of Ca ²⁺ , Mg ²⁺ , Ba ²⁺ , Sr ²⁺ , Zn ²⁺ and Fe ^3+. The method according to any one of -5].
[3-7] The method according to any one of [3-1] to [3-6] above, wherein the protein or polysaccharide having an amino group is gelatin.
[3-8] The above [3-1] to [3], wherein (a) the ion-crosslinkable polysaccharide and (b) the protein or the polysaccharide having an amino group are in a liquid or powder form having fluidity. The method according to any one of [-7].
[3-9] (a) an ion-crosslinkable polysaccharide and (b) a protein or polysaccharide having an amino group is in a fluid liquid state, (a) an ion-crosslinkable polysaccharide and (b) a protein Alternatively, the method according to any one of [3-1] to [3-8] above, wherein the polysaccharide having an amino group is applied to the surface of the esophageal lesion by spraying.
[3-10] (a) An ion-crosslinkable polysaccharide and (b) a protein or a polysaccharide having an amino group are in powder form, (a) an ion-crosslinkable polysaccharide and (b) a protein or an amino group The method according to any one of [3-1] to [3-8] above, wherein the polysaccharide possessed is applied to the surface of the esophageal lesion by spraying.
[3-11] The method according to [3-8] or [3-10] above, wherein the particle diameter of the powder is in the range of 10 μm to 500 μm.
[3-12] The method according to any one of [3-1] to [3-11] above, wherein another drug is administered.
[3-13] The method according to any one of [3-1] to [3-12] above, which is a method for preventing esophageal stricture.

[4-1] containing an ion-crosslinkable polysaccharide,
The ion-crosslinkable polysaccharide is crosslinked with a curing agent and used to cover the esophageal lesion.
A composition for covering an esophageal lesion.
[4-2] The ion-crosslinkable polysaccharide is cross-linked with a curing agent, and is used so as to cover the esophageal lesion with a protein or amino-group cross-linked using transglutaminase.
The composition according to the above [4-1].
[4-3] The polysaccharide according to [4-1] or [4-2] above, wherein the ion-crosslinkable polysaccharide is at least one selected from the group consisting of alginic acid, a derivative thereof, and a salt thereof. Composition.
[4-4] The composition according to [4-3] above, wherein the alginic acid derivative is alginic acid modified with a maleimide group, alginic acid modified with a thiol group, or alginic acid modified with an acrylate group.
[4-5] The composition described in [4-4] above, wherein the modification is performed via a spacer.
[4-6] Any one of [4-3] to [4-5] above, wherein the salt of alginic acid or a derivative thereof is sodium alginate, potassium alginate, sodium salt of alginic acid derivative, or potassium salt of alginic acid derivative. The composition according to item.
[4-7] The above [4-1] to [4], wherein the curing agent is at least one metal ion compound selected from the group consisting of Ca ²⁺ , Mg ²⁺ , Ba ²⁺ , Sr ²⁺ , Zn ²⁺ and Fe ^3+. The composition according to any one of -6].
[4-8] The composition according to any one of [4-1] to [4-7] above, wherein the protein or polysaccharide having an amino group is gelatin.
[4-9] The composition according to any one of [4-1] to [4-8] above, wherein the composition is in a liquid form or a powder form having fluidity.
[4-10] The composition according to any one of the above [4-1] to [4-9], wherein the composition is in a liquid form having fluidity and is applied to the surface of the esophageal lesion by spraying. .
[4-11] The composition according to any one of the above [4-1] to [4-10], wherein the composition is in a powder form and is applied to the surface of the esophageal lesion by spraying.
[4-12] The composition according to [4-9] or [4-11] above, wherein the particle diameter of the powder is in the range of 10 μm to 500 μm.
[4-13] The composition according to any one of [4-1] to [4-12] above, which is used in combination with another drug.
[4-14] The composition according to any one of [4-1] to [4-13] above, for hemostasis.
[4-15] The composition according to any one of [4-1] to [4-14] above, which is used for preventing esophageal stricture.

[5-1] containing an ion-crosslinkable polysaccharide,
The ionic crosslinkable polysaccharide is cross-linked with a curing agent and is used to cover the esophageal lesion with the cross-linked ionic crosslinkable polysaccharide.
Kit for covering the damaged part of the esophagus.
[5-2] Further comprising a protein or a polysaccharide having an amino group,
The ion-crosslinkable polysaccharide is crosslinked with a curing agent, and the polysaccharide having a protein or amino group is crosslinked using transglutaminase, the crosslinked ion-crosslinkable polysaccharide and the polysaccharide having a crosslinked protein or amino group. Used to cover esophageal lesions with sugars,
The kit according to [5-1] above.
[5-3] The above-mentioned [5-1] or [5-2], wherein the ion-crosslinkable polysaccharide is at least one selected from the group consisting of alginic acid, a derivative thereof, and a salt thereof. kit.
[5-4] The kit according to [5-3] above, wherein the alginic acid derivative is alginic acid modified with a maleimide group, alginic acid modified with a thiol group, or alginic acid modified with an acrylate group.
[5-5] The kit according to [5-4] above, wherein the modification is performed via a spacer.
[5-6] Any one of the above [5-3] to [5-5], wherein the salt of alginic acid or a derivative thereof is sodium alginate, potassium alginate, sodium salt of alginic acid derivative, or potassium salt of alginic acid derivative. Kit according to item.
[5-7] The above [5-1] to [5], wherein the curing agent is at least one metal ion compound selected from the group consisting of Ca ²⁺ , Mg ²⁺ , Ba ²⁺ , Sr ²⁺ , Zn ²⁺ and Fe ^3+. The kit according to any one of -6].
[5-8] The kit according to any one of [5-1] to [5-7] above, wherein the polysaccharide having protein or amino group is gelatin.
[5-9] The kit according to any one of [5-1] to [5-8] above, wherein the ion-crosslinkable polysaccharide is in a liquid form or a powder form having fluidity.
[5-10] Ionically crosslinkable polysaccharide, and if present, a protein or polysaccharide having an amino group is in a liquid form with fluidity, and ionically crosslinkable polysaccharide, and if present, protein Alternatively, the kit according to any one of [5-1] to [5-9] above, wherein the polysaccharide having an amino group is applied to the surface of the esophageal lesion by spraying.
[5-11] The ionic crosslinkable polysaccharide, and if present, the protein or polysaccharide having an amino group is in powder form, and the ionic crosslinkable polysaccharide, and if present, the protein or amino group The kit according to any one of [5-1] to [5-10] above, wherein the possessed polysaccharide is applied to the surface of the esophageal lesion by spraying.
[5-12] The kit according to [5-9] or [5-11] above, wherein the particle diameter of the powder is in the range of 10 μm to 500 μm.
[5-13] The kit according to any one of [5-1] to [5-12] above, which is used in combination with another drug.
[5-14] The kit according to any one of [5-1] to [5-13] above for hemostasis.
[5-15] The kit according to any one of [5-1] to [5-14] above, which is used for preventing esophageal stricture.

[6-1] Applying ion-crosslinkable polysaccharide to the target esophageal lesion,
The ionically crosslinkable polysaccharide is crosslinked with a curing agent, and the esophageal lesion is covered with the crosslinked ionically crosslinkable polysaccharide.
How to cover esophageal lesions.
[6-2] Further applying a protein or a polysaccharide having an amino group to the target esophageal lesion,
The ion-crosslinkable polysaccharide is crosslinked with a curing agent, and the polysaccharide having a protein or amino group is crosslinked using transglutaminase, the crosslinked ion-crosslinkable polysaccharide and the polysaccharide having a crosslinked protein or amino group. Cover the damaged part of the esophagus with sugars,
The method according to [6-1] above.
[6-3] The above-mentioned [6-1] or [6-2], wherein the ion-crosslinkable polysaccharide is at least one selected from the group consisting of alginic acid, a derivative thereof, and a salt thereof. Method.
[6-4] The method according to [6-3] above, wherein the alginic acid derivative is alginic acid modified with a maleimide group, alginic acid modified with a thiol group, or alginic acid modified with an acrylate group.
[6-5] The method described in [6-4] above, wherein the modification is performed via a spacer.
[6-6] Any one of the above [6-3] to [6-5], wherein the salt of alginic acid or a derivative thereof is sodium alginate, potassium alginate, sodium salt of alginic acid derivative, or potassium salt of alginic acid derivative. The method according to item.
[6-7] The above [6-1] to [6], wherein the curing agent is at least one metal ion compound selected from the group consisting of Ca ²⁺ , Mg ²⁺ , Ba ²⁺ , Sr ²⁺ , Zn ²⁺ and Fe ^3+. The method according to any one of -6].
[6-8] The method according to any one of [6-1] to [6-7] above, wherein the protein or polysaccharide having an amino group is gelatin.
[6-9] The above-mentioned [6-1] to [6], wherein the ion-crosslinkable polysaccharide and the polysaccharide having a protein or amino group, if present, are in a liquid or powder form having fluidity. The method according to any one of [8].
[6-10] Ionically crosslinkable polysaccharide, and if present, a polysaccharide having a protein or amino group, a fluid liquid, ionically crosslinkable polysaccharide, and a protein if present Alternatively, the method according to any one of [6-1] to [6-9] above, wherein the polysaccharide having an amino group is applied to the surface of the esophageal lesion by spraying.
[6-11] The ion-crosslinkable polysaccharide, and if present, the protein or polysaccharide having an amino group is in powder form, and the ion-crosslinkable polysaccharide, and if present, the protein or amino group The method according to any one of [6-1] to [6-10] above, wherein the polysaccharide possessed is applied to the surface of the esophageal lesion by spraying.
[6-12] The method described in [6-9] or [6-11] above, wherein the particle diameter of the powder is in the range of 10 μm to 500 μm.
[6-13] The method according to any one of [6-1] to [6-12] above, wherein another drug is administered.
[6-14] The method according to any one of [6-1] to [6-13] above, which is a method for hemostasis.
[6-15] The method according to any one of [6-1] to [6-14] above, which is a method for preventing esophageal stricture.

According to the present invention, a composition for covering an esophageal lesion is provided.

It is a figure which shows the esophageal damage part to which the composition for esophageal damage part coating | cover is applied, and the esophageal surface after healing. (A) Gel material administration to esophageal injury site, (B) Powder material administration to esophageal injury site, (C) Esophageal surface after healing. It is a figure which shows the result of the tissue adhesiveness evaluation of each material in a normal mucous membrane and a submucosa. (A) Graph showing the adhesion rate (vertical axis) at each bonding time (horizontal axis) of the material containing Alg-Mal and the material containing Alg, (B) Normal on the 10th day when the material containing Alg-Mal was applied Photo of mucous membrane and 13th day submucosa. It is a figure which shows the result of the tissue adhesiveness evaluation of each material in a normal mucous membrane and a submucosa. (A) Graph showing the adhesion rate (vertical axis) at each adhesion time (horizontal axis) of the material containing Alg when CaSO ₄ is added or not added, (B) 5 days when the material containing Alg is applied Photographs of the normal mucosa of the eye and the submucosa of the fourth day. It is a figure which shows the result of the tissue adhesiveness evaluation of the material in the case of Gela particle size 45-90 μm in normal mucosa and submucosa. It is a figure which shows the result of the tissue adhesiveness evaluation of the material in the case of Gela particle size 90-180 μm in normal mucosa and submucosa. It is a figure which shows the result of the stenosis evaluation of in vivo rat esophageal powder material administration (weight change rate). It is a figure which shows the result of the stenosis evaluation of in vivo rat esophageal powder material administration (feeding quantity change). It is a figure which shows the result of stenosis evaluation of in vivo rat esophageal powder material administration (esophageal patency rate). It is a figure which shows the result of stenosis evaluation of In vivo rat esophageal powder material administration (HE dyeing | staining). It is a figure which shows the result of stenosis evaluation of in vivo rat esophageal powder material administration (Sirius red staining). It is a figure which shows the result of the stenosis evaluation of in vivo rat esophageal powder material administration (α-SMA staining). It is a figure which shows the result of the stenosis evaluation of in vivo rat esophageal gel material administration (weight change rate). It is a figure which shows the result of the stenosis evaluation of In vivo rat esophageal gel material administration (feeding amount change). It is a figure which shows the result of stenosis evaluation of in vivo rat esophageal gel material administration (esophageal patency rate). It is a figure which shows the result of stenosis evaluation of In vivo rat esophageal gel material administration (HE dyeing | staining). It is a figure which shows the result of the stenosis evaluation of in vivo rat esophageal gel material administration (Sirius red dyeing | staining). It is a figure which shows the result of the stenosis evaluation of in vivo rat esophageal gel material administration ((alpha) -SMA dyeing | staining). It is a figure which shows the result of the hemostatic effect of In vivo powder material.

Hereinafter, the present invention will be described in detail. The following embodiments are examples for explaining the present invention, and the present invention can be implemented in various forms without departing from the gist thereof.

1. Overview This includes ionic crosslinkable polysaccharides,
The ion-crosslinkable polysaccharide is crosslinked with a curing agent and used to cover the esophageal lesion.
An esophageal lesion coating composition is provided.
The esophageal lesion coating composition of some embodiments comprises an ionically crosslinkable polysaccharide,
The ion-crosslinkable polysaccharide is crosslinked with a curing agent and used to coat the esophageal lesion along with the protein or amino group-crosslinked polysaccharide using transglutaminase.

An outline of the composition for coating an esophageal lesion in this embodiment is described with reference to FIG. 1 so that maleimide-modified alginic acid crosslinked with Ca ²⁺ is coated with gelatin crosslinked with transglutaminase. The case where it is used will be described as an example.

FIG. 1 (A) shows that a solution containing maleimide-modified alginic acid and transglutaminase (“Alg-Mal / TG”) and a solution containing gelatin and Ca ²⁺ (Gela / Ca ²⁺ ) are sprayed together to esophageal lesions. When applied, maleimide-modified alginic acid is cross-linked with Ca ²⁺ and gelatin is cross-linked with transglutaminase at the esophageal lesion.
On the other hand, FIG. 1 (B) shows an esophagus by spraying a powder containing alginic acid and gelatin (“Alg / Gelatin Powder”) and spraying a cross-linking agent solution containing “transglutaminase and Ca ²⁺ ” (“TG / Ca ²⁺ Crosslinking agent”). It is applied to an injured part, and shows a state in which alginic acid is cross-linked with Ca ²⁺ and gelatin is cross-linked with transglutaminase in the esophageal injured part.
The esophageal lesion is covered with a material formed from the composition for covering an esophageal lesion. Here, in this specification, the material formed from the composition for covering an esophageal lesion may be referred to as “esophageal lesion covering material”.

There is epithelial and secreted acidic mucin (sulfomucin) secreted from esophageal gland cells on the normal esophageal mucosal epithelial surface. In addition, this mucin is composed of one central region having many O-linked oligosaccharide chains (serine / threonine-linked sugar chains) and two sub-sites having many cysteines adjacent to both sides of the central region. It consists of a mucosal glycoprotein containing a domain (Cysteine rich region), and the cysteine contained in the primary structure of the mucosal glycoprotein exceeds 10% (Immunol Rev. (2014) 260 (1), p8-20.doi: 10.1111 / imr.12182.). A mucin monomer binds to the Cystein rich region of this mucosal glycoprotein, and a three-dimensional network of mucosal gel layers is constructed by disulfide bonds. Moreover, it is known that mucin contains many glutamine residues and lysine residues.

On the other hand, in the esophageal lesion, collagen (“Collagen”) is abundant in the submucosal layer exposed on the surface of the esophagus.

Gelatin (“Gela”) is a denatured collagen, and when it is cooled in a sol state during heating, it partially becomes a triple helical structure and gels. Because of its high biocompatibility and cell affinity and biodegradability, it is expected to be used as a scaffold material for tissue engineering and a carrier for a drug delivery system (DDS). However, the gelatin solution prepared by cooling easily transitions to the sol state at 37 ° C. which is the in-vivo temperature. Therefore, the gelatin molecule is covalently cross-linked to improve its strength. Is desirable.

Transglutaminase (TG) is an enzyme that binds glutamine and lysine residues contained in proteins or peptides. Since gelatin has unique glutamine and lysine residues, hydrogels can be made. Since the collagen layer is abundant in the submucosal layer exposed on the surface of the esophagus at the esophageal injury site, the collagen is cross-linked by gelatin and TG, whereby the esophageal injury site covering material can adhere to the wound site. Furthermore, not only cell adhesion but also cell migration can be promoted by selecting an appropriate TG dose and hydrogel concentration. Thereby, gelatin cross-linked with transglutaminase (Gela / TG) can promote the healing process of the mucosal epithelial layer after ESD. In addition, since the mucin on the surface of normal esophageal mucosa also contains glutamine residues and lysine residues as described above, the mucin is cross-linked by gelatin and TG, so that the esophageal lesion covering material is wounded. It can also adhere to the normal mucosal layer around the site.

Alginic acid is a polymer composed of two kinds of D-mannuronic acid and L-guluronic acid, and is considered to have high biocompatibility and does not worsen the inflammatory reaction. Further, when Ca ²⁺ is added to a monovalent metal salt of alginic acid (“Alg”), an Egg Box Junction is formed and gelled. In addition, the monovalent metal salt of alginic acid can be expected to have an effect as a hemostatic material in addition to the effect as a wound dressing material that rapidly epithelizes the wound. Furthermore, of the monovalent metal salt of alginic acid, sodium alginate adheres to the mucous membranes of the stomach and esophagus and plays a role in protecting the mucous membranes from attacks such as gastric juice and food. It is also used as a peptic ulcer agent (trade name: Alloid G).

Here, the cysteine residue present in the mucin on the surface of normal esophageal mucosa exceeds 10% as described above. Since the maleimide functional group has a high reactivity to the cysteine residue present on the protein surface via the Michael addition reaction, by using maleimide-modified alginic acid, it binds to cysteine in normal mucosa by thiol-maleimide reaction, The adhesiveness of the esophageal damaged part coating material to the esophageal mucosa can be improved.

From the above, the esophageal damaged part coating material formed from the composition for covering an esophageal damaged part can be used to cover the esophageal damaged part. It was difficult to fix the material to the damaged part of the esophagus due to the peristaltic movement of the esophagus, the passage of food and saliva, the difficulty of adhesion to the mucous membrane, etc. The esophageal lesion covering material formed from the composition can be well adhered to such esophageal lesions.

FIG. 1 (C) is a diagram showing the surface of the esophagus after healing. Furthermore, the esophageal damage part coating material formed from the composition for covering an esophageal damage part suppresses the formation of ulcers in the esophageal damage part, like the esophageal surface after healing shown in FIG. This can prevent stricture of the esophagus.

2. Esophageal lesion "Esophagus" is a tube that connects the pharynx and stomach and serves to send food from the pharynx to the stomach. The wall of the esophagus is divided into four layers from the inside toward the outside: the mucosa, the submucosa, the intrinsic muscle layer, and the outer membrane. The mucosa is divided into three layers from the inside to the outside: the stratified squamous epithelium, the lamina propria, and the mucosal lamina.

“Esophageal damaged part” to which the esophageal damaged part coating material is applied is a part that has suffered esophageal damage.

Esophageal damage includes, for example, esophageal damage associated with resection of esophageal cancer, esophageal damage due to accidental ingestion, esophageal damage due to acid reflux, accidental damage during endoscopy, and acids, alkalis or drugs This includes damage to the esophagus caused by corrosive esophagitis. In some embodiments, the esophageal injury is esophageal injury associated with resection of esophageal cancer. Resection of esophageal cancer is, for example, resection of esophageal cancer by surgery and endoscopic treatment.

Endoscopic treatment is, for example, endoscopic submucosal dissection (ESD) and endoscopic mucosal resection (EMR). In some aspects, the endoscopic treatment is ESD.

Esophageal “damage” is damage to at least one of the mucosa, submucosa, intrinsic muscle layer, and outer membrane of the esophagus, such as (i) damage to the esophageal mucosa, (ii) mucosa and mucosa of the esophagus. Including submucosal damage, (iii) esophageal mucosa, submucosa and proper muscle layer damage, and (iv) esophageal mucosa, submucosa, proper muscle layer, and adventitia damage. In some embodiments, esophageal “damage” is (ii) damage to the mucosa and submucosa of the esophagus, where the submucosa is exposed on the surface of the esophagus.

In some embodiments, the esophageal lesion is a post-ESD esophageal lesion, which is a damaged portion of the esophagus mucosa and submucosa, and the submucosa is exposed on the surface of the esophagus.

3. Esophageal lesion covering composition Here, an esophageal lesion covering composition is provided. The composition for covering an esophageal lesion includes an ion-crosslinkable polysaccharide, and the ion-crosslinkable polysaccharide is crosslinked with a curing agent and used to cover the esophageal lesion. In some embodiments, the composition for covering an esophageal lesion includes an ionic crosslinkable polysaccharide, the ionic crosslinkable polysaccharide is cross-linked with a curing agent and has a protein or amino group cross-linked using transglutaminase. Used to cover esophageal lesions with polysaccharides. The composition for covering an esophageal lesion may contain a monovalent metal salt of alginic acid or a derivative thereof, and the monovalent metal salt of alginic acid or a derivative thereof is crosslinked with a curing agent, and the esophagus together with a protein crosslinked using transglutaminase. It may be used to cover the damaged part.

Also provided is a method for covering an esophageal lesion including applying the composition for covering an esophageal lesion to a subject that needs to cover the esophageal lesion. More specifically, in the method of covering the esophageal lesion, the ion-crosslinkable polysaccharide is applied to the target esophageal lesion, the ion-crosslinkable polysaccharide is crosslinked with a curing agent, and the crosslinked ionic crosslinking is possible. This is a method of covering the damaged part of the esophagus with a polysaccharide. In some embodiments of the esophageal lesion covering method, an ionically crosslinkable polysaccharide and a protein or amino group-containing polysaccharide are applied to a target esophageal lesion, and the ionically crosslinkable polysaccharide is crosslinked with a curing agent. In addition, a protein or a polysaccharide having an amino group is crosslinked using transglutaminase, and the esophageal lesion is covered with a crosslinked ion-crosslinkable polysaccharide and a crosslinked protein or polysaccharide having an amino group. . In the esophageal lesion covering method, a monovalent metal salt of alginic acid or a derivative thereof and a protein are applied to a target esophageal lesion, the monovalent metal salt of alginic acid or a derivative thereof is crosslinked with a curing agent, and the protein is trans-transfected. Covering the esophageal lesion with a monovalent metal salt of cross-linked alginic acid or a derivative thereof and a cross-linked protein, which is cross-linked with glutaminase.

Furthermore, a kit used for a method for covering an esophageal damaged part is provided. More specifically, the kit includes an ionic crosslinkable polysaccharide, the ionic crosslinkable polysaccharide is cross-linked with a curing agent, and is used to cover an esophageal lesion with the cross-linked ionic crosslinkable polysaccharide. The esophageal damaged part coating kit. The kit of some embodiments comprises (a) an ionic crosslinkable polysaccharide, and (b) a protein or polysaccharide with an amino group, the ionic crosslinkable polysaccharide is crosslinked with a curing agent, and the protein or Polysaccharides with amino groups are cross-linked using transglutaminase and used to cover esophageal lesions with cross-linked ionic cross-linkable polysaccharides and cross-linked proteins or polysaccharides with amino groups It is a kit for. The kit may comprise (a) a monovalent metal salt of alginic acid or a derivative thereof, and (b) a protein, wherein the monovalent metal salt of alginic acid or a derivative thereof is crosslinked with a curing agent, and the protein uses transglutaminase. And may be used to coat esophageal lesions with monovalent metal salts of crosslinked alginic acid or derivatives thereof and crosslinked proteins.

In the present specification, the composition for covering an esophageal damaged part, the method for coating an esophageal damaged part, and the kit for covering an esophageal damaged part may be simply referred to as “composition for covering an esophageal damaged part” or “composition”.

That is, some embodiments of the composition for covering an esophageal injury part use a combination of (a) an ion-crosslinkable polysaccharide and (b) a polysaccharide having a protein or an amino group. The composition for covering an esophageal lesion may be used by combining (a) a monovalent metal salt of alginic acid or a derivative thereof and (b) a protein. Materials containing cross-linked ionic cross-linkable polysaccharides (eg monovalent metal salts of cross-linked alginic acid or derivatives thereof) and polysaccharides cross-linked using transglutaminase, ie esophageal damage Cover the esophageal lesions that appeared on the inner surface of the esophagus with the covering material. The esophageal lesion covering material includes a cross-linked ion cross-linkable polysaccharide (eg, cross-linked alginic acid or a monovalent metal salt thereof) and a curing agent. In some embodiments, the esophageal lesion covering material is a crosslinked ionically crosslinkable polysaccharide (eg, a crosslinked monovalent metal salt of alginic acid or a derivative thereof), a curing agent, a crosslinked protein, or a polysaccharide having an amino group. Contains sugars and transglutaminase.

In some embodiments, the composition for covering an esophageal lesion comprises (a) an ion-crosslinkable polysaccharide (for example, a monovalent metal salt of alginic acid or a derivative thereof) and (b) a polysaccharide having a protein or an amino group. Use. “Use in combination” means to use in combination. When the component (a) and the component (b) are used in combination, when the composition is applied to the target, the component (a) and the component (b) are included in the composition, or the target is the composition. It means that (a) component and (b) component should just be contained in the esophageal damage part coating | covering material formed after application. Therefore, the term “used in combination” does not particularly refer to the form in the distribution stage including sales.

For example, when using (a) component and (b) component in combination,
(1) (a) component and (b) component are combined in advance, or (2) two types of compositions obtained by separately preparing (a) component and (b) component are applied. Any of the forms used in combination may be used.
That is, the component (b) may be provided in the form of a composition previously blended with the component (a), and two types of compositions obtained by separately preparing the component (a) and the component (b). You may provide as a kit which combined and packaged.
In some forms, a composition in which the component (a) and the component (b) are preliminarily blended is provided, and separately from this composition, a composition in which transglutaminase and a curing agent are blended in advance is prepared. Preferably, the composition in which the component (a) and the component (b) are blended in advance is in a powder form, and the composition in which transglutaminase and a curing agent are blended in advance is in a liquid form. In this case, the powdered composition is applied to the surface of the esophageal lesion by spraying, while the liquid composition is applied by spraying.
In another some form, the composition which mix | blended (a) component, (b) component, and transglutaminase was provided previously, and a hardening | curing agent is prepared separately from this composition. Preferably, the composition in which the component (a), the component (b), and transglutaminase are blended in advance is in a powder form, and the curing agent is in a liquid form or a powder form. In this case, the powdered composition is applied to the surface of the esophageal lesion by spraying or spraying, while the liquid or powdery curing agent is sprayed.
In still another embodiment, a composition is provided in which (a) component, (b) component, transglutaminase and a curing agent are all blended in advance. Preferably, the composition in which all of component (a), component (b), transglutaminase, and curing agent are blended in advance is in a powder form. In this case, the powdered composition is applied to the surface of the esophageal lesion by spraying.
In some further embodiments, a composition comprising component (a) is provided, and separately from this composition, component (b), transglutaminase, and a curing agent are provided separately. Preferably, the composition containing the component (a), the component (b), the transglutaminase, and the curing agent are each in powder form. In this case, the composition containing the powdery component (a), the powdery component (b), the powdery transglutaminase, and the powdery curing agent are applied to the surface of the esophageal lesion by spraying.
In still another embodiment, a composition in which the component (a) and transglutaminase are preliminarily blended is provided, and in addition to the composition, a composition in which the component (b) is premixed with the curing agent is provided. . Preferably, the composition in which the component (a) and the transglutaminase are blended in advance and the composition in which the component (b) and the curing agent are blended in advance are in a liquid or powder form. In this case, these liquid or powdery compositions are applied to the surface of the esophageal lesion by spraying or spraying.
In still another embodiment, a composition in which the component (a) and transglutaminase are previously blended is provided, and separately from the composition, the component (b) and the curing agent are prepared separately. Preferably, the composition in which the component (a) and the transglutaminase are blended in advance, the component (b), and the curing agent are all in liquid or powder form. In this case, these liquid or powdery compositions are applied to the surface of the esophageal lesion by spraying or spraying.

Each of the component (a) and the component (b) may be provided in a solution state using a solvent, or a solid state such as a powder state (for example, a lyophilized product, particularly a lyophilized powder). It may be provided as a product. When provided as a solid substance, the component (a) and the component (b) may be used in a state where each of them or a mixture has a fluidity such as a solution or a gel using a solvent at the time of administration. Alternatively, it may be used in a solid state.

In some embodiments of the composition, component (a) and component (b) are each in the form of a solid selected from the group consisting of sheet, sponge, or powder; semisolid; Or may be used in a gel form.
Here, the term “powder” can be restated as “powder”, “powder” or “powder”.

The solvent is not particularly limited as long as it is a solvent applicable to a living body. For example, water for injection, purified water, distilled water, ion-exchanged water (or deionized water), milli-Q water, physiological saline, phosphate buffered physiological Examples include saline (PBS). Preferred are water for injection, distilled water, physiological saline and the like that can be used for treatment of humans and animals.

In the case of the composition for covering an esophageal lesion in a preferred embodiment, the content of the ion-crosslinkable polysaccharide (for example, monovalent metal salt of alginic acid or a derivative thereof) in the coating material formed from the composition is 0.00. The content of a protein or a polysaccharide having an amino group (eg, gelatin) is about 1 wt% to 30 wt%. More preferably, the content of the ion-crosslinkable polysaccharide (eg, monovalent metal salt of alginic acid or a derivative thereof) in the coating material formed from the composition is about 1 wt% to 20 wt%, and the protein or amino group is contained. The content of polysaccharide (for example, gelatin) is about 15 wt% to 50 wt%.
Ratio of ionic crosslinkable polysaccharide (eg monovalent metal salt of alginic acid or a derivative thereof) to polysaccharide with protein or amino group (eg gelatin) in the coating material formed from the composition (ionic crosslinkable) The polysaccharide: protein or polysaccharide having an amino group (weight ratio) is preferably 1: 0.1 to 1: 100, more preferably 1: 5 to 1:30.

“Subjects” are human or non-human warm-blooded animals, such as birds, and non-human mammals such as cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, horses. In some embodiments, the “subject” is a human.

The method for applying the composition for covering an esophageal lesion to a subject is not particularly limited. For example, a nebulizer (in the form of a composition, or in the form of a liquid in which the form of (a) component and / or (b) component is fluid) In the case of, for example, centrifugal atomizer (for example, rotating cup atomizer, rotating disk atomizer and wheel atomizer), electrostatic atomizer, ultrasonic atomizer, resonance atomizer, etc. And / or (b) when the form of the component is in the form of powder, for example, on the surface of the esophageal lesion with a duster (eg, Altoshooter (Kaigen Pharma), etc.), syringe, gel pipette, or dedicated filler It may be applied directly. Preferably, it is applied directly to the surface of the esophageal lesion by means of a nebulizer or spreader (eg duster), more preferably by a nebulizer or spreader (eg duster) through an endoscope channel.
When the form of the composition or the form of the component (a) and / or the component (b) is a fluid liquid, the liquid spraying tube (for example, Fine Jet (Top Co., Ltd.), Pentacus An endoscopic cannula (38813000) and a multi-lumen catheter (Fortegro Medical JC02003)) may be connected and used.
In addition, when the form of the composition or the form of the component (a) and / or the component (b) is in the form of powder, it should be used by connecting it to a spraying tube (Fine Jet (Top)). May be.
Apart from the composition, when component (b), curing agent, transglutaminase, and other components are prepared, depending on the form of these components, it may be applied to the subject in the same manner as in the above method. it can.
The composition may be applied to the subject only once, or twice at intervals (eg, 6 hours, 12 hours, 1 day, 2 days, 3 days or 1 week intervals). You may make it apply above (for example, 2 times, 3 times, 4 times, 5 times, or 6 times).

The form of the composition for covering an esophageal lesion, or the form of the component (a) and / or the component (b) is, for example, a fluid liquid (that is, a solution); a sheet, a sponge, or a powder (Especially lyophilized powder) or the like; semisolid; or gel. “Having fluidity” means having the property of changing its form into an indefinite form. In the case of fluid liquid, for example, the composition or (a) component and / or (b) component (hereinafter sometimes referred to as “composition etc.”) is enclosed in a nebulizer or syringe to cause esophageal damage. It is desirable to have fluidity that can be applied by spraying or pouring onto the surface of the part. A gel-like composition or the like can be easily applied to the surface of an esophageal lesion by using a syringe, a gel pipette, a dedicated syringe, or the like. A powdery composition or the like can also be applied to the surface of the esophageal lesion by being sprayed with a spraying device (for example, a duster). In addition, sheet-like, sponge-like, powder-like, gel-like, and fluid liquid compositions are compatible with any surface shape and come into contact with the entire surface of the esophageal lesion to which the composition is applied. You can also The sheet form is a flat plate having a suitable thickness and flexibility, and the sponge form is processed into a porous state. Moreover, what made the composition a solution can also be made into a semi-solid state with a freezing or hardening | curing agent. Further, the gel can be prepared by covalently bonding the composition with a polyvalent cation or a cross-linking reagent as a curing agent (cross-linking agent).

The form of the composition or the like is preferably in the form of a fluid liquid (ie, solution) that can be applied to spraying by a sprayer, or a powder that can be applied to spraying by a sprayer. is there.

The particle diameter of the powder in the case of powder may be controlled using a sieve or the like so as to be within a predetermined range. The particle size can be appropriately selected depending on the type of the spreader. In the present specification, the particle size is measured using, for example, a particle size distribution meter (Parica mini, HORIBA).
In some embodiments, the particle size of the powder such as the composition is, for example, 5 μm to 1000 μm, preferably 10 μm to 500 μm, more preferably 20 μm to 500 μm, and even more preferably 45 μm to 180 μm. It is.
In addition to the above particle sizes, the ratio of the component (a) to the component (b) used in combination (a) component (weight) :( b) component (weight)) is, for example, 1: 0. It is 1 to 1: 100, and preferably 1: 5 to 1:30.

4). Ion-crosslinkable polysaccharides Ion-crosslinkable polysaccharides are polysaccharides that form a gel when mixed with a curing agent described below. The ion-crosslinkable polysaccharide is, for example, at least one selected from the group consisting of alginic acid, carrageenan, pectin, derivatives thereof, or salts thereof. The ionically crosslinkable polysaccharide is preferably at least one selected from the group consisting of alginic acid, derivatives thereof, and salts thereof.

Alginic acid is a biodegradable polymeric polysaccharide and is a polymer in which two types of uronic acids, D-mannuronic acid (M) and L-guluronic acid (G), are polymerized in a straight chain. More specifically, D-mannuronic acid homopolymer fraction (MM fraction), L-guluronic acid homopolymer fraction (GG fraction), and D-mannuronic acid and L-guluronic acid are randomly arranged. This is a block copolymer in which the fractions (MG fraction) are bound arbitrarily. The composition ratio (M / G ratio) of D-mannuronic acid and L-guluronic acid of alginic acid varies depending on the type of organism mainly derived from seaweed, etc. It ranges 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.

Alginic acid derivatives are those obtained by subjecting alginic acid to any modification. Optional modifications made to alginic acid include, for example, maleimide modification, thiol modification, acrylate modification, aldehyde modification, and disulfide modification (eg, pyridyl disulfide). In some embodiments, the alginic acid derivative is maleimide-modified alginic acid, thiol-modified alginic acid, or acrylate-modified alginic acid, preferably maleimide-modified alginic acid.

The maleimide modification of alginic acid is to introduce a maleimide group into the carboxy group part of alginic acid. The cysteine residues present in mucins on the surface of normal esophageal mucosal epithelium are greater than 10%. Since the maleimide functional group has a high reactivity to the cysteine residue present on the protein surface via the Michael addition reaction, by using maleimide-modified alginic acid, it binds to cysteine in normal mucosa by thiol-maleimide reaction, The adhesiveness of the esophageal damaged part coating material to the esophageal mucosa can be improved.

The maleimide group is, for example, the following general formula (1):

Wherein R ¹ and R ² are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, or R ¹ and R ² are a 5-membered ring together with the carbon atom to which they are bonded. Or a 6-membered hydrocarbon group is formed)
It is group represented by these. Such 5-membered or 6-membered hydrocarbon groups are specifically a cyclopentyl group and a cyclohexyl group. In some embodiments, the maleimide group is a group in which R ¹ and R ² are hydrogen atoms in the general formula (1).

The maleimide modification can be performed, for example, according to the method described in Progress in Polymer Science (2012) 37, p106-126; or Biomacromolecules (2014) 15, p445-455, or the like. Specifically, alginic acid is dissolved in 0.3 M NaCl-containing 0.1 M Mes Buffer, and water-soluble carbodiimide (WSCD) and N-hydroxysuccinimide (NHS) are necessary equivalents to the carboxy group of alginic acid (for example, 2 Equivalent, 5 equivalents, or 8 equivalents) Dissolve in Mes Buffer, add dropwise to the alginate solution and stir for an additional 30 minutes. After 30 minutes, 0.5 equivalent of maleimide solution is dropped into Mes Buffer. After adjusting the pH to 6 and stirring for 20 hours, the reaction is stopped by adding sodium bicarbonate until pH 7.5. After dialyzing with pure water containing sodium chloride for 3 days, dialyzing with pure water for 3 days and recovering the desired product through freeze-drying. More specific maleimide modification methods are as described in Examples below.

Other modifications (for example, thiol modification, acrylate modification, aldehyde modification, and disulfide modification) can also be performed by a known method or a method analogous thereto.

The modification rate is a value expressed as a percentage of the number of uronic acid monosaccharide units into which a modifying group (for example, a maleimide group) is introduced among uronic acid monosaccharide units that are repeating units of alginic acids. The modification rate is, for example, 0.5% to 60%, preferably 1% to 30%, more preferably 1% to 10%. The modification rate can be adjusted by a known method or a method analogous thereto. In the case of maleimide modification, the modification rate can be adjusted as follows. That is, for example, the modification rate of maleimide can be increased by increasing the equivalents of WSCD and NHS. The modification rate can be calculated by ¹ H-NMR measurement (D ₂ O).

A spacer may be used when performing maleimide modification. Usable spacers may be those commonly used in the field (for example, spacers described in Greg T. Hermanson, Bioconjugate Technologies, Third Edition (2013)), and more specifically, polyethylene glycol, Polyamides such as peptides, and hydrocarbons are included.

The monovalent metal salt of alginic acid or a derivative thereof is a water-soluble salt formed by ion exchange of the hydrogen atom of the 6-position carboxylic acid of alginic acid or a derivative thereof with a monovalent metal ion such as Na ⁺ or K ^+. is there. Specific examples of the monovalent metal salt of alginic acid or a derivative thereof include sodium alginate, potassium alginate, a sodium salt of an alginate derivative, or a potassium salt of an alginate derivative. In some embodiments, the monovalent metal salt of alginic acid or a derivative thereof is sodium alginate or a sodium salt of an alginic acid derivative. A solution of a monovalent metal salt of alginic acid or a derivative thereof forms a gel when mixed with a curing agent.

The monovalent metal salt of alginic acid or a derivative thereof is a high molecular polysaccharide, and it is difficult to accurately determine the molecular weight, but generally the weight average molecular weight is 10 to 10 million, preferably 10,000 to 10 million. The range is preferably 20,000 to 3 million. For example, a preferable range of the weight average molecular weight measured by gel permeation chromatography (GPC) method is 300,000 to 2,000,000. Further, for example, a preferable range of the weight average molecular weight measured by GPC-MALS method is 1,000 to 300,000, and a more preferable range is 50,000 to 300,000.

Usually, when the molecular weight of the high molecular polysaccharide is calculated by gel filtration chromatography, a measurement error of 10 to 20% or more may occur. For example, the value may vary in the range of about 32 to 480,000 for 400,000, 400,000 to 600,000 for 500,000, and about 800 to 1,200,000 for 1,000,000. Therefore, a suitable weight average molecular weight range for the monovalent metal salt of alginic acid or a derivative thereof is at least 20,000, more preferably 50,000, and even more preferably 100,000. If the molecular weight is too high, it is difficult to produce and causes problems such as excessively high viscosity when used as an aqueous solution, poor solubility, and difficulty in maintaining physical properties during long-term storage. Is preferably 5 million or less, more preferably 3 million or less.

Generally, a polymer substance derived from a natural product does not have a single molecular weight but is an aggregate of molecules having various molecular weights, and thus is measured as a molecular weight distribution having a certain width. A typical measurement technique is gel filtration chromatography. Representative information on the molecular weight distribution obtained by gel filtration chromatography includes weight average molecular weight (Mw), number average molecular weight (Mn), and dispersion ratio (Mw / Mn).

The weight average molecular weight places importance on the contribution to the average molecular weight of a polymer having a large molecular weight and is represented by the following formula.
Mw = Σ (WiMi) / W = Σ (HiMi) / Σ (Hi)
The number average molecular weight is calculated by dividing the total weight of the polymer by the total number of polymers.
Mn = W / ΣNi = Σ (MiNi) / ΣNi = Σ (Hi) / Σ (Hi / Mi)
Here, W is the total weight of the polymer, Wi is the weight of the i-th polymer, Mi is the molecular weight at the i-th elution time, Ni is the number of molecular weights Mi, and Hi is the height at the i-th elution time. .

It is known that the molecular weight of a high molecular weight substance derived from a natural product may vary depending on the measurement method (examples of hyaluronic acid: Chikako YOMOTA et.al., Bull. Natl. Health Sci., Vol. 117, pp 135-139 (1999), Chikako YOMOTA et.al., Bull. Natl. Inst. Health Sci., Vol. 121, pp 30-33 (2003)). As for the molecular weight measurement of alginate, there is a method of calculating from intrinsic viscosity (Intrinsic viscosity), SEC-MALLS (Size Exclusion Chromatography with Multiple Laser Light Scattering, which is calculated by the Ft. (2006), issued by ASTM International). In this document, when measuring the molecular weight by size exclusion chromatography (= gel filtration chromatography), in addition to a calibration curve using pullulan as a standard substance, a multi-angle light scattering detector (MULTIS) (= Measurement by SEC-MALS) is recommended. In addition, there is an example in which the molecular weight by SEC-MALS is used as a standard value on a catalog of alginate (FMC Biopolymer, PRONOVA ^™ sodium alloys catalog).

Here, when the molecular weight of the monovalent metal salt of alginic acid or its derivative is specified, it is a weight average molecular weight calculated by gel filtration chromatography unless otherwise specified.

Typical conditions for gel filtration chromatography include using a calibration curve with pullulan as a standard substance. The molecular weight of pullulan used as the standard substance is preferably at least 1.6 million, 788,000, 404,000, 212,000 and 112,000 as the standard substance. In addition, eluent (200 mM sodium nitrate solution), column conditions, etc. can be specified. As column conditions, it is preferable to use a polymethacrylate resin filler and use at least one to three columns having an exclusion limit molecular weight of 10 million or more. Typical columns are TSKgel GMPW _x1 (diameter 7.8 mm × 300 mm) and G2500PW _XL (diameter 7.8 mm × 300 mm) (manufactured by Tosoh Corporation).

Alginic acid is initially extracted from brown algae and has a high molecular weight and a high viscosity. However, in the process of drying by heat, freeze-drying, and purification, the molecular weight decreases and the viscosity becomes low. Therefore, it is possible to produce a monovalent metal salt of alginic acid or a derivative thereof having a different molecular weight by appropriately controlling the temperature in each step of the production. A monovalent metal salt of alginic acid or a derivative thereof having a high molecular weight can be obtained by controlling the temperature in each step of the production to be lower, and a monovalent metal salt of alginic acid or a derivative thereof having a low molecular weight can be obtained as the temperature increases. . Moreover, the monovalent metal salt of alginic acid or its derivative from which molecular weight differs can also be manufactured by the method of selecting the brown algae used as a raw material suitably, or performing the fractionation by molecular weight in a manufacturing process. Furthermore, about the monovalent metal salt of alginic acid or its derivative produced by each method, after measuring the molecular weight or viscosity, by mixing with the monovalent metal salt of alginic acid or its derivative of another lot having a different molecular weight or viscosity, It is also possible to obtain a monovalent metal salt of alginic acid having a target molecular weight or a derivative thereof.

The alginic acid used here may be naturally derived or synthetic, but is preferably naturally derived. Examples of naturally occurring alginic acid include those extracted from brown algae. Although brown alga containing alginic acid is prosperous in coastal areas around the world, seaweed that can actually be used as a raw material for alginic acid is limited, such as Lessonia in South America, Macrocystis in North America, Laminaria and Ascofilum in Europe, Australia Typical examples are Daviglia. Examples of the brown algae used as a raw material for alginic acid include, for example, the genus Lesonia, the genus Macrocystis, the genus Laminaria (comb), the genus Ascophyllum, the genus Durvillea, Examples include the genus Eisenia and the genus Ecklonia.

5. Curing agent (crosslinking agent)
The curing agent cures by crosslinking a solution of an ion-crosslinked polysaccharide (for example, a monovalent metal salt of alginic acid or a derivative thereof). In the case of a monovalent metal salt of alginic acid or a derivative thereof, the curing agent is, for example, a divalent or higher metal ion compound such as Ca ²⁺ , Mg ²⁺ , Ba ²⁺ , Sr ²⁺ , Zn ²⁺ , Fe ³⁺ , Examples thereof include a crosslinkable reagent having 4 amino groups. More specifically, as a metal ion compound having a valence of 2 or more, CaCl ₂ , MgCl ₂ , CaSO ₄ , ZnCl ₂ , FeCl ₃ , BaCl ₂ , SrCl _2, etc. (preferably, CaCl ₂ , CaSO ₄ , ZnCl ₂ , SrCl ₂ , FeCl ₃ , BaCl _2, etc.) as a crosslinkable reagent having 2 to 4 amino groups in the molecule, a lysyl group (—COCH (NH ₂ ) — (CH ₂ ) ₄ on the nitrogen atom Diaminoalkanes that may have —NH ₂ ), ie, diaminoalkanes and derivatives in which the amino group is substituted with a lysyl group to form a lysylamino group, specifically include diaminoethane, diaminopropane, N— (Lysyl) -diaminoethane and the like can be mentioned.

The amount of the curing agent used depends on the amount and molecular weight of the ion-crosslinkable polysaccharide (eg, monovalent metal salt of alginic acid or its derivative), the G / M ratio of the monovalent metal salt of alginic acid or its derivative, etc. It is desirable to adjust appropriately. The amount of the curing agent used is, for example, 3 mM to 200 mM at a final molar concentration in a solution of an ion-crosslinkable polysaccharide (for example, a monovalent metal salt of alginic acid or a derivative thereof). When the composition is in powder form, the amount of the curing agent used is, for example, 5% to 30% by weight relative to the weight of the ion-crosslinkable polysaccharide (eg, monovalent metal salt of alginic acid or a derivative thereof). Preferably, it is 15 to 25% by weight.

6). Proteins or polysaccharides having amino groups Proteins or polysaccharides having amino groups are proteins or polysaccharides having amino groups capable of cross-linking molecules using transglutaminase. Such proteins include, for example, gelatin, casein, lactoferrin, collagen, keratin, transferrin, albumin, and elastin. Moreover, chitosan can also be used as such a polysaccharide having an amino group. In some embodiments, the protein or polysaccharide with an amino group is a protein, in particular gelatin.

Gelatin is a denatured collagen that is in a sol state when heated and partially gels when cooled and partially forms a triple helical structure. Because of its high biocompatibility and cell affinity and biodegradability, it is expected to be used as a scaffold material for tissue engineering and a carrier for DDS. Gelatin solution prepared by cooling easily transitions to the sol state at 37 ° C, which is the in-vivo temperature, but its strength is improved by covalently cross-linking gelatin molecules with transglutaminase. Can be made.

The gelatin used here is derived from, for example, fish, pig, cow, jellyfish or bird, and preferably derived from fish or pig. Gelatin having a weight average molecular weight of, for example, 30,000 to 200,000 is used. The measurement method of the weight average molecular weight of gelatin can use the same measurement method as the weight average molecular weight of the monovalent metal salt of alginic acid or its derivative. Such gelatin can be produced by using various known methods, or commercially available products can be purchased (for example, from Nitta Gelatin).

The amount of the protein or polysaccharide having amino group used is the kind of polysaccharide having protein or amino group (for example, gelatin or casein), or ion-crosslinkable polysaccharide (for example, monovalent metal salt of alginic acid or its derivative). It is desirable to adjust appropriately according to the usage amount, molecular weight, etc. The amount of gelatin used when the type of protein or polysaccharide having an amino group is gelatin is, for example, 5 wt% with respect to a solution of an ion-crosslinkable polysaccharide (for example, monovalent metal salt of alginic acid or a derivative thereof). % To 40 wt%, preferably 15 wt% to 25 wt%. When the type of polysaccharide having protein or amino group is casein, the amount of casein used is, for example, 5 to 5 with respect to a solution of an ionically crosslinkable polysaccharide (for example, monovalent metal salt of alginic acid or a derivative thereof). 20 wt%.
In some embodiments, an ionically crosslinkable polysaccharide (eg, a monovalent metal salt of alginic acid or a derivative thereof) and a polysaccharide having a protein or amino group (eg, gelatin) in a coating material formed from the composition The ratio of (ionically crosslinkable polysaccharide: protein or polysaccharide having amino groups (weight ratio)) is preferably 1: 0.1 to 1: 100, more preferably 1: 5 to 1: 30.

7). Transglutaminase Transglutaminase (TG) is an enzyme that binds glutamine and lysine residues contained in proteins or peptides. Since proteins have unique glutamine and lysine residues, hydrogels can be generated by binding these residues between proteins and crosslinking the proteins together. Various types of TG can be used, for example, those derived from animals, plants, or microorganisms, or variants thereof, preferably those derived from microorganisms, or variants thereof. More specifically, TG is Activa (trade name) available from Ajinomoto Co., Ltd., Novosirteen (trade name) available from Novo Nordisk Pharma Co., Ltd. Name).

The amount of TG used is preferably adjusted as appropriate according to the type of protein or polysaccharide having an amino group, the amount used, the molecular weight, the amount of lysine and glutamine residues or the amount of amino groups. The amount of TG used is, for example, 0.5% to 40% by weight, preferably 1% to 5% by weight, based on the weight of the protein solution.
The amount of TG used when the composition is in powder form is, for example, 1% to 50% by weight, preferably 10% to 25% by weight, based on the weight of gelatin.

8). Low Endotoxin Treatment In some embodiments, an ion crosslinkable polysaccharide (eg, a monovalent metal salt of alginic acid or a derivative thereof) is a low endotoxin ion crosslinkable polysaccharide (eg, a monovalent of a low endotoxin alginic acid or a derivative thereof). Not a metal salt). In some other embodiments, the ion crosslinkable polysaccharide (eg, monovalent metal salt of alginic acid or a derivative thereof) is a low endotoxin ion crosslinkable polysaccharide (eg, monovalent metal of a low endotoxin alginic acid or derivative thereof). Salt). Proteins or amino group-containing polysaccharides used with these ion-crosslinkable polysaccharides (eg, monovalent metal salts of alginic acid or derivatives thereof) may be low endotoxin proteins or low endotoxin polysaccharides having amino groups. Alternatively, it may not be a low endotoxin protein or a low endotoxin polysaccharide having an amino group. Low endotoxins are those in which endotoxin levels have been reduced to such an extent that they do not substantially cause inflammation or fever. That is, it has been subjected to low endotoxin treatment.

The low endotoxin treatment can be performed by a known method or a method analogous thereto. For example, the method of Takada et al. (See, for example, JP-A-9-32001) for purifying sodium hyaluronate, and the method of Yoshida et al. (Eg, JP-A-8-269102) for purifying β1,3-glucan. Etc.), a method of William et al. (For example, see JP-T-2002-530440, etc.) for purifying biopolymer salts such as alginate, gellan gum, etc., a method of James et al. (For example, international publication) for purifying polysaccharides, etc. 93/13136 pamphlet), the method of Lewis et al. (For example, see US Pat. No. 5,585,591 etc.), the method of Herman Frank et al. (For example, Appl Microbiol Biotechnol (1994) 40: 638) for purifying alginate. -Refer to -643 etc.) or similar It can be carried out by methods. The low endotoxin treatment of the present invention is not limited thereto, but is washed, filtered with a filter (such as an endotoxin removal filter or a charged filter), ultrafiltration, a column (an endotoxin adsorption affinity column, a gel filtration column, a column with an ion exchange resin, etc.) ), Adsorption to hydrophobic substances, resin or activated carbon, organic solvent treatment (extraction with organic solvent, precipitation / precipitation by addition of organic solvent, etc.), surfactant treatment (for example, JP-A-2005-036036) It can be carried out by a known method such as a gazette) or a combination thereof. These processing steps may be appropriately combined with known methods such as centrifugation. It is desirable to select appropriately according to the type of alginic acid or its derivative, the type of protein or polysaccharide having an amino group, and the like.

The endotoxin level can be confirmed by a known method, and can be measured, for example, by a method using Limulus reagent (LAL), a method using Enspercy (registered trademark) ES-24S set (Seikagaku Corporation), or the like. .

The method for treating endotoxin of an ion-crosslinkable polysaccharide (for example, monovalent metal salt of alginic acid or a derivative thereof) is not particularly limited. The endotoxin content of the monovalent metal salt is preferably 500 endotoxin units (EU) / g or less, more preferably 100 EU / g or less, and more preferably 100 EU / g or less, when endotoxin measurement is performed using a Limulus reagent (LAL). Preferably it is 50 EU / g or less, Most preferably, it is 30 EU / g or less. Low endotoxin-treated sodium alginate is commercially available, for example, Sea Matrix (sterilized) (Kimika Co., Ltd. Mochida International), PRONOVA ^™ UP LVG (FMC), and the like.

In addition, the endotoxin content of a polysaccharide having a protein or amino group is not limited, but as a result, the endotoxin content of a polysaccharide having a protein or amino group is measured when endotoxin is measured with a Limulus reagent (LAL). 500 endotoxin units (EU) / g or less, more preferably 100 EU / g or less, still more preferably 50 EU / g or less, particularly preferably 30 EU / g or less. Such a polysaccharide having a protein or amino group can be obtained by subjecting it to the above endotoxin treatment.

9. Viscosity of Esophageal Damage Covering Composition The esophageal damage covering composition of some embodiments is a fluid liquid, ie, a solution. The viscosity of the composition of this embodiment is not particularly limited, but is, for example, 5 mPa · s to 100,000 mPa · s, preferably 10 mPa · s to 20000 mPa · s. Some embodiments of the composition are viscosities that can be applied to the subject, such as with a syringe.

The viscosity of the composition is preferably about 10 mPa · s or more from the viewpoint of adhesion, and preferably about 20000 mPa · s or less from the viewpoint of ease of handling of the composition, more preferably 2000 mPa · s to 10000 mPa · s, 1000 mPa · s to 5000 mPa · s, or 10 mPa · s to 10000 mPa · s.

The viscosity of the composition may be, for example, the concentration of an ionic crosslinkable polysaccharide (eg, alginic acid or derivative thereof), the molecular weight of the ionic crosslinkable polysaccharide (eg, alginic acid or derivative thereof), the M / G of alginic acid or derivative thereof. It can be adjusted by controlling the ratio, the concentration of the polysaccharide having protein or amino group, the molecular weight, temperature, pH, counter ion, etc. of the polysaccharide having protein or amino group.

The viscosity of a solution of an ion-crosslinkable polysaccharide (eg, monovalent metal salt of alginic acid or a derivative thereof) is high when the concentration of the ion-crosslinkable polysaccharide (eg, alginate concentration) in the solution is high. It becomes low when the polysaccharide concentration (for example, alginic acid concentration) which can be ion-crosslinked is low. Moreover, when the molecular weight of polysaccharide (for example, alginic acid) which can be ion-crosslinked is large, it becomes high, and when small, it becomes low. For example, in order to obtain a viscosity of 10 mPa · s to 20000 mPa · s using alginic acid having a molecular weight of about 200 to 500,000 Da, an aqueous alginate solution of about 1% w / v to 3% w / v may be used. When alginic acid having a smaller molecular weight is used, it is necessary to increase the concentration of alginic acid. The viscosity of the aqueous alginate solution can be measured by a known method using, for example, a rotational viscometer (cone plate type) (TVE-20LT, TOKI SANGYO CO., LTD. JAPAN). A known method is, for example, the 16th revision Japanese Pharmacopoeia, General Test Method Viscosity Measurement Method (cone-plate rotational viscometer).

Since the viscosity of a monovalent metal salt solution of alginic acid or a derivative thereof is affected by the M / G ratio, for example, an alginic acid having a preferable M / G ratio can be selected depending on the viscosity of the solution. The M / G ratio of alginic acid used in the present invention is about 0.4 to 4.0, preferably about 0.8 to 3.0, more preferably about 1.0 to 1.6.

As described above, since the M / G ratio is mainly determined by the type of seaweed, the type of brown algae used as a raw material affects the viscosity of a solution of a monovalent metal salt of alginic acid or a derivative thereof. The alginic acid used in the present invention is preferably derived from a brown algae of the genus Lessonia, Macrocystis, Laminaria, Ascofilum, Davilia, more preferably a brown alga of the genus Lessonia, particularly preferably Lessonia It is derived from Niscens (Lessonia nigrescens).

The viscosity of a protein or polysaccharide solution with amino groups is high when the concentration of the protein or polysaccharide with amino groups is high and the concentration of the protein or polysaccharide with amino groups in the solution is low. Lower. Moreover, it is high when the molecular weight of the polysaccharide having protein or amino group is large, and low when it is small. For example, in order to obtain a viscosity of 400 mPa · s to 20000 mPa · s using gelatin having a molecular weight of about 100,000, a gelatin aqueous solution of about 0.5% w / v to 40% w / v can be used. Good. When gelatin having a smaller molecular weight is used, it is necessary to increase the gelatin concentration. The viscosity of the protein aqueous solution can be measured by a known method using, for example, a rotational viscometer (cone plate type) (TVE-20LT, TOKI SANGYO CO., LTD. JAPAN). A known method is, for example, the 16th revision Japanese Pharmacopoeia, General Test Method Viscosity Measurement Method (cone-plate rotational viscometer).

If the viscosity of the composition obtained by mixing an ionically crosslinkable polysaccharide (for example, monovalent metal salt of alginic acid) and a polysaccharide having a protein or amino group is the same as that of each solution before mixing, It becomes almost the same as before mixing. When the viscosity of one solution before mixing is high, the viscosity of the composition obtained can be lowered | hung by mixing the other solution of low viscosity with this. When the solution of one viscosity before mixing is low, the viscosity of the composition obtained can be raised by mixing the other solution of high viscosity with this. In this way, a viscosity of 5 mPa · s to 100,000 mPa · s can be obtained.

10. Mixing method In the composition for esophageal injury, the mixing method of the component (a) and the component (b) is not particularly limited as long as the component (a) and the component (b) can be mixed uniformly. As a mixing method, for example, a method using a static mixer, a method using a double syringe, a method of spraying two liquids simultaneously or continuously, a method of spraying two powdery components simultaneously or continuously, a powder And a method of using a mixer for mixing.

“Static mixer” refers to a static mixing device without a drive unit. More specifically, a static mixer usually consists of a tube and a mixing element without a drive fixed in the tube, thereby splitting the flow and diverting or reversing the flow direction, so that the flow is longitudinal. It is a mixing device that mixes fluids by repeating division, conversion and inversion in the horizontal direction. Some types of static mixers are equipped with a jacket for heat exchange on the outer periphery of the tube, and others are equipped with a tube for heat exchange through which the heat medium passes through the mixing element itself. . For details on the static mixer, see, for example, Thakur et al. (2003) Trans IchemE, 81, Part A: 787-826, which can be referred to.

As the static mixer, a known one can be used. Examples of the known static mixer include, for example, Kenix type (for example, manufactured by Nordson and Noritake); Kenics (Chemineer Inc.); low pressure drop ( Ross Engineering Inc.); SMV (Koch-Glitsch Inc.); SMXL (Koch-Glitsch Inc.); Interfacial Surface Generator. ); Inliner series 50 (Lightnin Inc.); Inli er series 45 (Lightnin Inc.); Custody transfer mixer (Komax systems Inc.); and SMR (. Koch-Glitsch, Inc) and the like.

The double syringe is a device that has a container for separately filling the components to be mixed and is uniformly mixed and discharged when the components are discharged from the double syringe.

As the double syringe, a known one can be used. Examples of known double syringes include those manufactured by Baxter.

Alternatively, the static mixer and the double syringe can be a custom product made to mix the composition.

11. Esophageal stricture prevention method and hemostasis method The composition for covering an esophageal injury site in some embodiments can be used to prevent esophageal stenosis, for example, in an esophageal injury site of a subject in need of preventing esophageal stenosis. Used to apply. “Applying” means that an esophageal lesion covering material formed from the composition is used so as to cover the esophageal lesion on the surface of the esophagus, and preferably covers the normal esophageal surface in and around the esophageal lesion. Means that it is used in a sufficient amount. Specific descriptions such as “esophageal injury part”, “esophageal injury part coating composition”, and “target” are as described above.
“Preventing esophageal stricture” means that esophageal stricture is completely prevented, or that the formation of esophageal stricture is suppressed as compared to the case where the composition for covering an esophageal lesion is not applied, or It means that the formation of stenosis is delayed.

Another embodiment of the composition for covering an esophageal lesion is used for hemostasis of the esophageal lesion. For example, it is used so as to be applied to an esophageal damaged part of a subject that needs to stop hemostasis of the esophageal damaged part. “Applying” means that an esophageal lesion covering material formed from the composition is used so as to cover the esophageal lesion on the surface of the esophagus, and preferably covers the normal esophageal surface in and around the esophageal lesion. Means that it is used in a sufficient amount. Specific descriptions such as “esophageal injury part”, “esophageal injury part coating composition”, and “target” are as described above.
“Stop hemostasis” means that bleeding is completely stopped, or that bleeding is suppressed or hemostasis is accelerated compared to the case where the composition for covering an esophageal injury is not applied.

12 Concomitant drugs The composition for covering an esophageal injury site may be used in combination with other drugs. Specifically, before applying the composition for covering an esophageal lesion to the esophageal lesion, simultaneously with or after, an antibiotic such as streptomycin, penicillin, tobramycin, amikacin, gentamicin, neomycin and amphotericin B, aspirin Non-steroidal antipyretic analgesics (NSAIDs), anti-inflammatory drugs such as acetaminophen, steroidal anti-inflammatory drugs such as triamcinolone acetonide (Kenacort-A), antitumor drugs matomycin C, 5-fluorouracil, etc. A concomitant drug may be administered. These agents may be used by being mixed in the composition for covering an esophageal lesion.

It should be noted that all publications cited in the present specification, for example, prior art documents and publications, patent gazettes and other patent documents, are incorporated herein by reference in their entirety.

The present invention will be described in further detail with reference to the following examples, but the present invention should not be construed as being limited to these examples.

Example 1: Preparation of monovalent metal salt of maleimide-modified alginic acid Maleimide-modified alginic acid was prepared as follows. Sodium alginate (I-3G) (Kimika; Kimika Argin High · G series; viscosity (20 ° C.): 300 to 400 mPa · s (1% aqueous solution)) 500.0 mg, 0.3 M NaCl (200% eggplant type flask) 100 ml of 0.1M MES buffer (Wako Pure Chemicals No. 349-01623) containing Wako Pure Chemicals No. 191-01665) was added and stirred overnight to dissolve. While stirring, 2.3963 g WSCD / HCl (Peptide Laboratories No. 1030) dissolved in 10 ml MES buffer was added dropwise over 10 min. While stirring, 1.4888 g NHS (Wako Pure Chemicals No. 089-04032) dissolved in 10 ml MES buffer was added dropwise over 10 min, and then stirred for 30 min. While stirring, 220.8 mg (0.5 eq) of N- (2-aminoethyl) maleimide hydrochloride (Tokyo Kasei Kogyo No. A2436) dissolved in 10 ml of MES buffer was added dropwise. The pH of the solution was measured, adjusted to pH = 6 with 0.1 M NaOH (Wako Pure Chemicals No. 192-07935), checked for pH at 10, 30, 60 min, and stirred for 20 hours or longer. NaHCO ₃ solid (Wako Pure Chemicals No. 199-05985) was added to bring the pH to 7.50 and the reaction was stopped. The solution was transferred to a dialysis membrane (6-8 kDa) and dialyzed against NaCl for 3 days. Transfered to water and dialyzed for 5 days. Lyophilization was carried out for 5 days to obtain maleimide-modified alginic acid (Alg-Mal) as a white compound (yield 50%). The modification rate of maleimide modification was 20%.

Example 2: Evaluation of tissue adhesion of a material containing a monovalent metal salt of alginic acid and gelatin (test by liquid spray)
The tissue adhesiveness of monovalent metal salt (Alg) of high viscosity alginic acid was evaluated. The tissue adhesion of maleimide-modified Alg (Alg-Mal) was also evaluated in the same manner except that 2 wt% Alg-Mal was used instead of 2 wt% Alg (I-3G).

[material]
A: 2 wt% Alg (I-3G) / 5 wt% TG (effective concentration 0.5 wt%) (+ Blue food dye) 0.5 mL
B: 20 wt% Gela / 0.1 MCa ²⁺ 0.5 mL
Washing water: 0.1% methyl benzoate / 0.9% NaCl

Specifically, the A liquid and the B liquid were prepared as follows.
A: 2 wt% Alg (I-3G) was put into pure water and stirred until dissolved. TG powder (90% maltodextrin content, effective TG concentration 0.5 wt%, Ajinomoto Co., Inc.), which is 5 wt% with respect to the total amount of A solution, was dissolved in the Alg solution and stirred for 30 minutes or more in order to distribute it uniformly. In order to make it easy to visually confirm the material, 0.1 wt% of blue food dye (Kyoritsu Foods Co., Ltd .; food color blue) for food dyeing was added.
B: 20 wt% gelatin (Nitta Gelatin Co., Ltd .; Japanese Pharmacopoeia Gelatin (manufactured exclusively) GLS250) was placed in pure water and stirred at 50 ° C. until completely dissolved. CaCl ₂ (Wako Pure Chemicals No. 036-00485) that was 0.1 M with respect to the total amount of B solution was dissolved in the gelatin solution and stirred for 30 minutes or more in order to distribute it uniformly.

The washing water was prepared as follows. 0.9% NaCl was dissolved in pure water, 0.1% methyl benzoate was added, and the mixture was stirred until completely dissolved. The resulting solution was sterilized by autoclaving.

[procedure]
The porcine esophagus was cut out 2 cm and opened vertically. The mucous membrane was separated from the open esophagus with scissors, the mucosal tissue and the tissue exposing the submucosa were placed on the parafilm, and the weight of each parafilm was measured (W ₀ ). Through a multi-lumen catheter, 0.5 mL each of A liquid and B liquid was sprayed simultaneously on the sample using nitrogen gas (4 L / min). The tissue sample to which the material was administered was placed in a 37 ° C. incubator, and gelation was waited for 10 minutes. The esophageal sample was lifted and the gel on the parafilm was washed away with tap water. After wiping off the moisture, the esophageal sample was reapplied and the weight was measured (W ₁ ). The gel adhering to the mucous membrane and mucosal surface was washed away with 30 mL of water. After wiping off the moisture, the weight was measured as 0 hour weight (W ₂ ). Further, washing with tap water was repeated 3 to 4 times, and after confirming that the material did not peel off, the gel surface of the sample was directed downward and immersed in 60 mL of washing water. It was placed on a shaker and shaken in a 37 ° C. incubator. A sample was taken out after a certain time, and after wiping off moisture, the weight was measured as the weight of each time (W ₂ ).

Calculation of adhesion rate:
W ₁ −W ₀ = Amount of gel on esophageal sample after spray W ₂ −W ₀ = Amount of gel adhered to mucosa / submucosa after washing [(W ₂ −W ₀ ) / (W ₁ −W ₀ )] × 100 % = Adhesion rate to material (%)

[result]
The result is shown in FIG.
In the case of I-3G alginate, peeling occurred on the normal mucosa 2 days and the submucosa 3 days. In comparison, Alg-Mal synthesized from I-3G has a long adhesion time for both the normal mucosa and the submucosa and can be measured up to the 10th day. I was able to observe what I was doing. It seems that the superiority of maleimide modification was observed. From this result, the esophageal stricture prevention effect of each material can be explained as follows. It is considered that I-3G alginate has sufficient adhesion to tissues and stays at the postoperative site where stenosis is likely to occur, thereby preventing stenosis. On the other hand, the adhesion to tissue was further improved by maleimide modification. From this, it can be considered that the adhesion time of AlgAlg-Mal becomes 1 to 2 weeks, stays longer in the postoperative site where stenosis is likely to occur, and prevents stenosis more effectively.

Example 3: tissue adhesive Evaluation Ca ²⁺ upon addition of CaSO ₄ in the material containing a monovalent metal salt and gelatin alginate escapes with time, the possibility of decomposition is accelerated in the cross-linked alginate verified Therefore, CaSO ₄ was added to the material and the tissue adhesion was evaluated.

[material]
A: 2 wt% Alg (I-3G) / 5 wt% TG (effective concentration 0.5 wt%) (+ Blue food dye) 0.5 mL
B-2: 20 wt% Gela / 0.1MCa ²⁺ /0.1MCaSO ₄ 0.5 mL
Washing water: 0.1% methyl benzoate / 0.9% NaCl

The above solution A and washing water were prepared as described in Example 2. Specifically, the solution B-2 was prepared as follows. 20 wt% gelatin was put into pure water and stirred at 50 ° C. until completely dissolved. CaCl ₂ (Wako Pure Chemicals product number 036-00485) which becomes 0.1M with respect to the total amount of the B-2 solution and CaSO ₄ (Wako Pure Chemicals product number 031-00935) which becomes 0.1M with respect to the total amount of the B-2 solution. ) Was dissolved in the gelatin solution and stirred for 30 minutes or more in order to distribute it uniformly.

[procedure]
Tissue adhesion evaluation was performed in the same procedure as in Example 2, except that the B-2 solution was used instead of the B solution.

[result]
The result is shown in FIG.
By adding CaSO _4, the swelling of the gel was suppressed, the adhesion time to the normal mucosa was prolonged, and no swelling was seen even when it was peeled off on the fifth day. Adhesion time to the submucosa was almost unchanged regardless of the addition of CaSO ₄ when evaluated by weight, but was present until the fifth day according to visual evaluation. If Ca ²⁺ escapes with time, the decomposition of the crosslinked alginic acid may be accelerated. Actually, by adding CaSO ₄ having low solubility, Ca ²⁺ was gradually released, and decomposition of the alginate gel was suppressed.

Example 4: Evaluation of tissue adhesion of a material containing a monovalent metal salt of alginic acid and casein [Material]
A-3: 2 wt% Alg (I-3G) / 1 wt% TG (effective concentration 0.1 wt%) (+ Blue food dye) 0.5 mL
B-3: 10 wt% casein / 0.1 MCa ²⁺ 0.5 mL
Washing water: 0.1% methyl benzoate / 0.9% NaCl

[procedure]
The washing water was prepared as described in Example 2, specifically, the A-3 and B-3 liquids as follows.
First, the A-3 solution was prepared as follows. 2 wt% Alg (I-3G) was put into pure water and stirred until dissolved. TG powder (containing 90% maltodextrin, effective TG concentration 0.1 wt%) that is 1 wt% with respect to the total amount of liquid A was dissolved in the Alg solution and stirred for 30 minutes or more in order to distribute it uniformly. In order to make it easy to confirm the material visually, 0.1 wt% of blue food dye for food staining was added.
Next, the B-3 solution was prepared as follows. Add 10wt% casein (Wako Pure Chemicals product number 03-323271) in pure water, adjust to pH 7.0 with 0.1M NaOH (Wako Pure Chemicals product number 192-07935) while stirring, and continue stirring overnight. The micelle was sufficiently swollen with water to obtain a casein solution. CaCl ₂ (Wako Pure Chemicals No. 036-00485), which is 0.1 M based on the total amount of B solution, was dissolved in the casein solution and stirred for 30 minutes or more in order to distribute it uniformly.

[procedure]
Tissue adhesion evaluation was performed in the same procedure as in Example 2 except that the A-3 and B-3 solutions were used instead of the A and B solutions.

[result]
After the material administration, Alg was cross-linked by Ca ²⁺ and casein was cross-linked by TG to form a two-component mixed gel. On the other hand, as a result of washing with washing water, the material peeled off at an early stage as compared with other materials. Thus, it was found that the low concentration of casein is slightly inferior in adhesiveness compared to gelatin. However, since the material using gazein has adhesiveness to the tissue, it can be considered that the material stays at a postoperative site where stenosis is likely to occur and prevents stenosis.

Example 5: Evaluation of tissue adhesion of a material containing a monovalent metal salt of alginic acid and gelatin (test by powder spraying)
The tissue adhesiveness of monovalent metal salt (Alg) of high viscosity alginic acid was evaluated.

[material]
A-5: Alg (I-3G) / Gela powder (Alg: Gela = 1: 10 (weight ratio)) 2 g
B-5: 5 wt% TG (effective concentration 0.5 wt%) / 0.1 MCa ²⁺ 2.5 mL
Washing water: 0.1% methyl benzoate / 0.9% NaCl

Specifically, the above A-5 powder and B-5 liquid were prepared as follows.
A-5 powder: Alg (I-3G) and gelatin (Sigma G1890) were stirred at a weight ratio of 1:10 using a mixer. Here, the size of the powder was adjusted as follows. The gelatin for spraying was passed through a sieve having a mesh size of 500 μm, 355 μm, 180 μm, 90 μm and 45 μm (manufactured by Tokyo Screen Co., Ltd., JIS Z 8801) to control the size of the particles. The particle size is measured using a particle size distribution meter (Parica mini, HORIBA).
Solution B-5: CaCl ₂ (Wako Pure Chemicals No. 036-00485) that was 0.1 M based on the total amount of the solution was placed in pure water and stirred until it was completely dissolved. TG powder (containing 90% maltodextrin, effective TG concentration 0.5 wt%) that is 5 wt% with respect to the total amount of the solution was put into the solution and stirred until it was completely dissolved.

[procedure]
The porcine esophagus was cut out 2 cm and opened vertically. The mucous membrane was separated from the opened esophagus with scissors, and each esophageal sample exposing the mucosal tissue and submucosa was placed on the parafilm, and the weight of each parafilm was measured. This measured value was regarded as “tissue inherent weight”. The vertical and horizontal lengths of the esophageal sample were measured, and the area was calculated. This calculated value was regarded as “tissue area”. Connect Altoshooter (Kagen Pharma) with A-5 powder in a medicine bottle to a spray tube (Fine Jet (length 1.6 m, measured outer diameter approximately 2 mm, measured inner diameter approximately 1 mm) (top)) This was used to spray A-5 powder on the esophageal sample. After waiting for a water absorption time of 1 minute, the weight of the esophageal sample to which the A-5 powder adhered was measured. This measured value was regarded as “the weight of the tissue after powder application”. Moreover, the powder which did not adhere | attach was collect | recovered and the weight was also measured. Through a fine jet (Top Co., Ltd.) liquid spray tube, 0.5 mL of the B-5 solution was sprayed onto the sample. The tissue sample to which the material was administered was placed in a 37 ° C. incubator, and gelation was waited for 10 minutes. The esophageal sample was lifted and the gel on the parafilm was washed away with tap water. After wiping off moisture, the esophageal sample was reapplied and the weight was measured. This measured value was regarded as “weight of tissue after spraying powder and liquid crosslinking agent”. The gel adhering to the mucous membrane and mucosal surface was washed away with 30 mL of water. After wiping off the moisture, the weight was measured as the weight of 0 hours. Further, washing with tap water was repeated 3 to 4 times, and after confirming that the material did not peel off, the gel surface of the sample was directed downward and immersed in 60 mL of washing water. It was placed on a shaker and shaken in a 37 ° C. incubator. A sample was taken out after a certain time, and after wiping off moisture, the weight was measured as the weight of each time.

Calculation of powder adhesion amount, gel amount after administration of cross-linking agent and material weight:
Powder adhesion amount (mg / cm ² ) = (weight of tissue after powder dispersion (mg) −original weight of tissue (mg)) / tissue area (cm ² )
Gel amount after administration of cross-linking agent (mg / cm ² ) = (weight of tissue after spraying powder and liquid cross-linking agent (mg) −original weight of tissue (mg)) / tissue area (cm ² )
Material Weight (g) = tissue weight at each time point (g) −tissue original weight (g)

[result]
The results are shown in Table 1, FIG. 4 and FIG.

Table 1: Adhesion amount change by Gela particle size

In the case of Gela particle size of 45 to 90 μm, 48 hours after immersion in washing water, the material peeled off the mucous membrane while maintaining the gel shape (FIG. 4). The material adhering to the submucosal layer was deformed and peeled off after 96 hours (FIG. 4).
It was found that when the gela particle size was 90 to 180 μm, the amount of powder adhesion was larger (Table 1) and the material adhesion time was longer than when the gela particle size was 45 to 90 μm (FIG. 5).
The Gela particle size of 45 to 90 μm is close to the Alg (I-3G) particle size (50 μm). The dose of Alg (I-3G) is 1/10 that of gelatin. On the other hand, Alg (I-3G) absorbs and swells quickly, so it may swell considerably after absorption and become larger than Gela particles. Moreover, Alg (I-3G) is rapidly cross-linked by Ca ²⁺ , and the outside of the Gela particles is covered with Alg (I-3G), and the contact area between the tissue, solution, or Gela is reduced, and Gela and mucin by TG are reduced. The cross-linking reaction may be slightly reduced. Consistent with this hypothesis is that the adhesion is improved when the Gela particles are larger.
Therefore, Alg stays longer in the postoperative site where stenosis is likely to occur, effectively preventing stenosis, and Alg stays longer in the postoperative site where stenosis is likely to occur by controlling the particle size of Gela. It is possible.

Example 6: Evaluation of stenosis after administration of In vivo rat esophageal powder material Evaluation of stenosis after administration of In vivo rat esophageal powder material was performed.

[Equipment]
Suture: Non-absorbing thread 3-0, 6-0, Absorbing thread 4-0
16G Surfflow Round Diamond File

[material]
Alg group (Alg single administration group):
Powder material (A-6-1): Alg (I-3G) 10 mg / animal Liquid cross-linking agent (B-6-1): 0.1 MCa ²⁺ 0.2 mL / animal

Gela group (Gela alone administration group):
Powder material (A-6-2): Gelatin 10 mg / animal Liquid cross-linking agent (B-6-2): 5 wt% TG (effective concentration 0.5 wt%) 0.2 mL / animal

Alg / Gela group:
Powder material (A-6-3): Alg (I-3G) / Gela powder (Alg: Gela = 1: 10 (weight ratio)) 10 mg / animal Liquid cross-linking agent (B-6-3): 5 wt% TG (Effective concentration 0.5wt%) / 0.1MCaCl ₂ 0.2mL / animal

Specifically, the powder materials (A-6-1) to (A-6-3) were respectively prepared as follows.
Powder material (A-6-1): Alg (I-3G) was weighed at 10 mg / animal.
Powder material (A-6-2): Gelatin was weighed at 10 mg / animal.
Powder material (A-6-3): Alg (I-3G) and Gelatin were stirred at a weight ratio of 1:10 using a mixer.

Further, the liquid crosslinking agents (B-6-1) to (B-6-3) were specifically prepared as follows.
Liquid cross-linking agent (B-6-1): CaCl ₂ (Wako Pure Chemicals No. 036-00485) of 0.1 M with respect to the total amount of the solution was placed in pure water and stirred until it was completely dissolved.
Liquid cross-linking agent (B-6-2): TG powder (90% maltodextrin contained, effective TG concentration 0.5%) (Ajinomoto Co., Inc.) (Ajinomoto Co., Inc.), which is 5 wt% based on the total amount of the solution, is completely dissolved. Stir until
Liquid cross-linking agent (B-6-3): CaCl ₂ (Wako Pure Chemicals No. 036-00485) of 0.1 M with respect to the total amount of the solution was placed in pure water and stirred until it was completely dissolved. TG powder (containing 90% maltodextrin, effective TG concentration 0.5 wt%) that is 5 wt% with respect to the total amount of the solution was put into the solution and stirred until it was completely dissolved.

[reagent]
0.05 w / v% trypsin-0.53 mmol / L EDTA · 4Na solution (Wako Pure Chemical Industries, Ltd.)
Saline (Otsuka Pharmaceutical Co., Ltd.)

anesthesia

[animal]
SD rat Male 9 weeks old 16 rats 1 week evaluation N = 4 / group 2 weeks evaluation N = 4 / group

[procedure]
As three kinds of mixed anesthetics, 1.875 mL of medetomidine hydrochloride, 2 mL of midazolam, and 2.5 mL of butorphano tartrate were mixed with 18.625 mL of physiological saline, and 0.5 mL was administered to 100 g of rat body weight.

Thereafter, the rat abdomen was incised 3 cm in length. The stomach was pulled out from the abdominal incision and placed on the gauze. A cut of about 3 mm was made in the upper part of the stomach with scissors. A file was inserted from the upper cut of the stomach and scrubbed in about 6-7 times in the top, bottom, left and right directions. The file was extracted, and about 15 mm from the stomach was ligated with a 3-0 thread, and the knot was pinched with a pair of pears to fix the surflow. 0.05 w / v% trypsin EDTA was injected and maintained for 1 minute. I released the ligature and pulled out the surf. The file was scrubbed about 6-7 times in each of the vertical and horizontal directions to extract the file. Then, the following treatment was performed in each of the Control group and the material group.

Control group: The incision of the stomach was sutured with 6-0 non-absorbing thread and closed with 4-0 absorbing thread and 3-0 non-absorbing thread.
Material group: After administration of powder material with a spraying device, liquid cross-linking agent was injected, the stomach cut was closed, and the abdomen was closed.

After laparotomy, 0.75 mL of atipamezole hydrochloride was mixed with 9.25 mL of physiological saline, and 0.5 mL was administered to 100 g of rat body weight as a binding agent for three types of mixed anesthesia, and awakened from anesthesia.

Thereafter, changes in body weight and changes in food intake were measured as follows. Rats were weighed daily at a fixed time. The weight change rate was calculated by the following formula:
Weight change rate (%) = [weight at each time point (g) / preoperative weight (g)] × 100

In addition, 100 g / animal was fed at a fixed time every day, and the remaining amount of food was measured the next day. Food intake was calculated using the following formula:
Intake amount (g) = [100 g−remaining amount of food (g)]

In addition, the esophageal opening rate was measured as follows. Esophageal specimens were removed from rats euthanized by carbon dioxide, and the appearance was evaluated by incision in the longitudinal direction. The width of the narrowed ulcer was taken as the width of the narrowed esophagus and the length was measured. The width of the normal esophagus 2 cm away from it was also measured in the same manner. The esophageal opening rate was calculated with the following formula:
Esophageal opening rate (%) = [stenotic esophageal width / normal esophageal width] × 100

[result]
The results of measuring the weight change rate and the change in food intake are shown in FIGS. 6 and 7, respectively. The Alg group and the Alg / Gela group had a gradual decrease in body weight and decreased food consumption compared to the control group and the Gela group.

The body weight decreased until the 5th, and the amount of food consumption increased after that, but the body weight remained almost unchanged. The Gela group gained weight the day after returning to food, but continued to decrease until the day of recovery.

Figure 8 shows the esophageal opening rate. In all groups, the esophageal opening rate on the 14th day after surgery was slightly lower than that on the 7th day. There was no significant difference between the Gela group and the Control group regarding the esophageal opening rate on the 7th and 14th days, but the Alg group and the Alg / Gela group were significantly higher than the esophageal opening rate of the Control group. It is considered that the severity of the esophagus was reduced in the Alg group and the Alg / Gela group.

Here, the significant difference test was performed as follows. All results were evaluated as mean values and standard deviations. Tukey's HSD test (Tukey honestificant difference test) was performed on the difference in the average values between the two groups in the data of each group.

Example 7: Histological evaluation of stenosis after administration of In vivo rat esophageal powder material A histological evaluation of stenosis after administration of In vivo rat esophageal powder material was performed.

(1) Cryosection [reagent]
10 v / v% formalin buffer saline 30 w / v% sucrose solution C. T compound liquid nitrogen

[procedure]
The esophageal sample was immersed and fixed in a 10 v / v% formalin buffer for 4 hours or more. Washed with saline. It was immersed overnight in a 30 w / v% sucrose solution. O. C. Encapsulated in a T compound. It was allowed to stand in liquid nitrogen and frozen. The frozen tissue sample was frozen in a deep freezer at −80 degrees (° C.) for 3 hours or more. Thin slices were made using a frozen microtome. Affixed to a slide glass.

Here, the esophageal sample was prepared as follows. Esophageal specimens were excised from rats euthanized by carbon dioxide and cut in the longitudinal direction with the esophageal epithelium side up.

(2) HE staining [reagent]
Caratach hematoxylin 0.5 w / v% eosin alcohol ethanol Remosol

[procedure]
O. C. The T compound was lightly rinsed with tap water. It was immersed in a caratach hematoxylin solution for 10 minutes. Colored and washed in running water for 10 minutes. It was immersed in eosin solution for 3 minutes. Lightly washed with water for about 3 seconds. It was immersed in a 70 v / v% ethanol solution for 1 minute. The container was changed and immersed in a 100 v / v% ethanol solution for 3 minutes (× 2 times). It was immersed in Remosol for 3 minutes (× 2 times). After drying in a draft, the mount quick was placed on the tissue sample and covered with a cover glass. After the mount quick solidified, it was observed using a microscope.

[result]
The length of the regenerated epithelium was measured with ImageJ (NIH: ImageJ ver. 1.51) based on the photograph of HE staining, and the reepithelialization rate was calculated. Specifically, the method for calculating the re-epithelialization rate is as follows. The length of the part from which the mucosal muscle was removed was defined as the length of the submucosal exfoliation wound. The length of the portion not covered by the epithelium was defined as the length that was not re-epithelialized. The reepithelialization rate was calculated with the following formula:
Epithelial regeneration distance = [length of exfoliated wound-length not re-epithelialized]
Re-epithelialization rate (%) = [epithelial regeneration distance / exfoliated wound length]

The result is shown in FIG. From the results of HE staining, in all of the Alg group, Gela group, and Alg / Gela group, epithelial development was observed on the 14th day (POD14) rather than the 7th day (POD7). In addition, the epithelialization rate was faster in the Alg group and the Alg / Gela group than in the Gela group, and the Alg / Gela group was the fastest.

(3) Sirius red staining [reagent]
1% Sirius red solution 0.5% Acetic acid Ethanol Remosol

[procedure]
O. C. The T compound was lightly rinsed with tap water. It was immersed in 1 wt% sirius red liquid for 10 minutes. Washed twice with 0.5 v / v% acetic acid solution. The container was changed and immersed in a 100 v / v% ethanol solution for 3 minutes (× 2 times). It was immersed in Remosol for 3 minutes to make it clear. After drying in a draft, the mount quick was placed on the tissue sample and covered with a cover glass. After the mount quick solidified, it was observed using a microscope.

[result]
From the results of Sirius red staining, on the 14th day, collagen was observed not only in the submucosa but also in the muscle layer in all of the Alg group, Gela group, and Alg / Gela group. The amount of collagen produced based on the photograph of Sirius red staining was measured with ImageJ, and the amount of accumulated collagen was calculated. Specifically, the method for calculating the amount of accumulated collagen is as follows. Since collagen is stained red by Sirius red staining, the area stained red by image analysis using ImageJ was measured, and the portion selected in the tissue was defined as the area of collagen. Collagen accumulation was calculated using the following formula:
Collagen accumulation (%) = [collagen fiber area / total tissue area] × 100%

The result is shown in FIG. From the results of Sirius red staining, the amount of accumulated collagen increased on the 14th day (POD14) rather than the 7th day (POD7) in all of the Alg group, Gela group and Alg / Gela group.

(4) α-SMA staining [reagent]
Primary antibody: Monoclonal Anti-Actin, α-smooth muscle antibody produced in mouse (Sigma Aldrich A2547)
Secondary antibody: Alexa Fluor 488 goat anti-mouse IG (H + L) (Molecular probe) (abcam ab150113)
・ Triton X-100 (Sigma Aldrich X100-100ML)
・ PBS (-) (Wako 166-23555)
・ Bovine serum albumin (Sigma Aldrich A-7906)
-Vectashield mounting agent (Mounting Medium with DAPI) (Vec H-1200)

[procedure]
O. C. The T compound was washed away. A liquid blocker super pop pen drawn a water-repellent circle around the sample to prevent the solution from flowing out. The slide was washed with PBST (0.1 v / v% Triton X-containing PBS) for 5 min × 3 times. Blocker solution (3 wt% BSA + 1 v / v% sheep serum in PBST) was placed on the sample and blocked for 1 h at room temperature. Washed twice with PBST for 2 min × 2. The primary antibody was diluted 500 times with PBST, placed on the sample and allowed to react overnight at 4 ° C. Washed with PBST for 5 min × 3 times. The secondary antibody diluted 500 times was placed on the sample and reacted for 1 h. Washed with PBST for 10 min × 3 times. It dehydrated with 100 v / v% ethanol. Cleared with remozole. A drop of Vectashield mounting agent was added to the slide, and the cover glass was gently pushed to remove excess moisture around it. Coverslip with clear nail police and dry.

[result]
The expression level of α-SMA was calculated based on the photograph of immunostaining. Specifically, the method for calculating the expression level of α-SMA is as follows. Cell nuclei were fluorescently labeled blue with a Vectashield mounting agent (with DAPI). The number of cells labeled in blue was calculated by image analysis using ImageJ, and this was used as the number of cells in the entire tissue. Furthermore, since α-SMA is fluorescently labeled in green, the number of cells labeled in green was calculated in the same manner, and this was used as the number of cells expressing α-SMA. The expression level of α-SMA was calculated by the following formula:
α-SMA expression level (%) = [number of α-SMA expressing cells / number of cells in whole tissue]

The result is shown in FIG. From the results of immunostaining, the α-SMA expression level on the 14th day increased in all of the Alg group, Gela group, and Alg / Gela group compared to the 7th day. In particular, from the stained photographs of the Gela group, α-SMA expression of vascular wall cells (pericytes or vascular smooth muscle cells) was observed, and it was confirmed that there were many blood vessels. It is thought that gelatin promoted angiogenesis. Angiogenesis has become one of the phenomena observed in the formation of granulation formed during inflammation and wounding. As a result of increased blood flow and cellular activity, many collagens were produced. From the results of immunostaining, the expression level of α-SMA, which is a myofibroblast marker, was significantly lower in the Alg group and the Alg / Gela group than in the other groups. For this reason, it was considered that the contraction of cells and tissues was suppressed, and it was found that esophageal stricture could be prevented.

Example 8: Evaluation of stenosis after administration of an in vivo rat esophageal gel material An evaluation of stenosis after administration of an in vivo rat esophageal gel material was performed.

[Equipment]
Suture: Non-absorbing thread 3-0, 6-0, Absorbing thread 4-0
16G Surfflow Round Diamond File

[material]
Alg group (Alg single administration group):
Liquid material (A-8-1): 2 wt% Alg (I-3G) 0.2 mL / animal Liquid cross-linking agent (B-8-1): 12 wt% carboxymethylcellulose (CMC) +0.1 MCa ²⁺ 0.2 mL / animal

Gela group (Gela alone administration group):
Liquid material (A-8-2): 20 wt% Gelatin 0.2 mL / animal Liquid cross-linking agent (B-8-2): 12 wt% CMC + 5 wt% TG (effective concentration 0.5 wt%) 0.2 mL / animal

Alg / Gela group:
Liquid material (A-8-3): 2 wt% Alg (I-3G) / 5 wt% TG (effective concentration 0.5 wt%) 0.2 mL / animal Liquid cross-linking agent (B-8-3): 20 wt% Gela / 0.1 M CaCl ₂ 0.2 mL / animal

Alg-Mal / Gela group:
Liquid material (A-8-4): 2 wt% Alg-Mal / 5 wt% TG (effective concentration 0.5 wt%) 0.2 mL / animal Liquid cross-linking agent (B-8-4): 20 wt% Gela / 0.1 MCaCl ₂ 0.2mL / animal

Alg-Mal / Gela_TA (triamcinolone acetonide) group:
Liquid material (A-8-5): 2 wt% Alg-Mal / 5 wt% TG (effective concentration 0.5 wt%) 0.2 mL / animal Liquid cross-linking agent (B-8-5): 20 wt% Gela / 0.1 MCaCl ₂ / TA400μg 0.2mL / animal

The liquid materials (A-8-1) to (A-8-5) were specifically prepared as follows.
Liquid material (A-8-1): 2 wt% Alg (I-3G) was added to pure water and stirred until dissolved.
Liquid material (A-8-2): 20 wt% gelatin was added to pure water and stirred at 50 ° C. until completely dissolved.
Liquid material (A-8-3): 2 wt% Alg (I-3G) was added to pure water and stirred until dissolved. TG (effective concentration 0.5 wt%, Ajinomoto Co., Inc.), which is 5 wt% based on the total amount of A-8-3 solution, was dissolved in the Alg solution and stirred for 30 minutes or more.
Liquid material (A-8-4): 2 wt% Alg-Mal was added to pure water and stirred until dissolved. TG (effective concentration 0.5 wt%, Ajinomoto Co., Inc.), which is 5 wt% based on the total amount of the A-8-4 solution, was dissolved in the Alg-Mal solution and stirred for 30 minutes or more in order to distribute it uniformly.
Liquid material (A-8-5): 2 wt% Alg-Mal was added to pure water and stirred until dissolved. TG (effective concentration 0.5 wt%, Ajinomoto Co., Inc.), which is 5 wt% based on the total amount of A-8-5 solution, was dissolved in the Alg-Mal solution and stirred for 30 minutes or more.

In addition, the liquid crosslinking agents (B-8-1) to (B-8-5) were specifically prepared as follows.
Liquid cross-linking agent (B-8-1): 12 wt% CMC was added to pure water and stirred until dissolved. CaCl ₂ (Wako Pure Chemicals No. 036-00485) that was 0.1 M with respect to the total amount of B-8-1 solution was dissolved and stirred for 30 minutes or more.
Liquid crosslinking agent (B-8-2): 12 wt% CMC was added to pure water and stirred until dissolved. TG (effective concentration 0.5 wt%) that was 5 wt% based on the total amount of the B-8-2 solution was dissolved and stirred for 30 minutes or more.
Liquid cross-linking agent (B-8-3): 20 wt% gelatin was added to pure water and stirred at 50 ° C. until completely dissolved. CaCl ₂ (Wako Pure Chemicals No. 036-00485), which is 0.1 M based on the total amount of the B-8-3 solution, was dissolved in the gelatin solution and stirred for 30 minutes or more.
Liquid cross-linking agent (B-8-4): 20 wt% gelatin was added to pure water and stirred at 50 ° C. until completely dissolved. CaCl2 (Wako Pure Chemicals product number 036-00485), which becomes 0.1 M with respect to the total amount of the B-8-4 solution, was dissolved in the gelatin solution and stirred for 30 minutes or more.
Liquid cross-linking agent (B-8-5): 20 wt% gelatin was added to pure water and stirred at 50 ° C. until completely dissolved. CaCl2 (Wako Pure Chemicals product number 036-00485), which is 0.1 M based on the total amount of the B-8-5 solution, was dissolved in the gelatin solution and stirred for 30 minutes or more. 400 μg of TA (Wako 205-10963) was mixed with gelatin solution for 0.2 mL of B-8-5 solution, and stirred for 30 minutes or more.

anesthesia

The rat abdomen was incised 3 cm in length. The stomach was pulled out from the abdominal incision and placed on the gauze. A cut of about 3 mm was made in the upper part of the stomach with scissors. A file was inserted from the upper cut of the stomach and scrubbed in about 6-7 times in the top, bottom, left and right directions. The file was extracted, and about 15 mm from the stomach was ligated with a 3-0 thread, and the knot was pinched with a pair of pears to fix the surflow. 0.05 w / v% trypsin EDTA was injected and maintained for 1 minute. I released the ligature and pulled out the surf. The file was scrubbed about 6-7 times in each of the vertical and horizontal directions to extract the file. Then, the following treatment was performed in each of the Control group and the material group.

Control group: The incision of the stomach was sutured with 6-0 non-absorbing thread and closed with 4-0 absorbing thread and 3-0 non-absorbing thread.
Material group: Liquid material and liquid cross-linking agent were placed in a 1 mL syringe, injected by surflow, the stomach incision was closed, and the abdomen was closed.

Thereafter, the rate of change in body weight and change in food intake were measured as follows. Rats were weighed daily at a fixed time. The weight change rate was calculated by the following formula:
Weight change rate (%) = [weight at each time point (g) / preoperative weight (g)] × 100

100 g / animal was fed at a fixed time every day, and the remaining amount of food was measured the next day. Food intake was calculated using the following formula:
Food intake (g) = [100 g-remaining food (g)]

In addition, the esophageal opening rate was measured as follows. Esophageal specimens were removed from rats euthanized with carbon dioxide, and the appearance was evaluated by incision in the longitudinal direction. The width of the narrowed ulcer was taken as the width of the narrowed esophagus and the length was measured. The width of a normal esophagus 2 cm away from the same was also measured. The esophageal opening rate was calculated by the following formula:
Esophageal opening rate (%) = [stenotic esophageal width / normal esophageal width] × 100

[result]
The results of measuring the weight change rate and the change in food intake are shown in FIGS. 12 and 13, respectively. The Alg group, the Alg / Gela group, the Alg-Mal / Gela group and the Alg-Mal / Gela_TA group had a gradual decrease in body weight and decrease in food consumption compared to the control group and the Gela group.

Figure 14 shows the esophageal opening rate. In all groups, the esophageal opening rate on the 14th day after surgery was slightly lower than that on the 7th day. There was no significant difference between the Gela group and the Control group regarding the esophageal opening rate on the 7th and 14th days, but the Alg group, the Alg / Gela group, the Alg-Mal / Gela group and the Alg-Mal / Gela_TA group It was significantly higher than the esophageal opening rate of the Control group. In the Alg group, Alg / Gela group, Alg-Mal / Gela group and Alg-Mal / Gela_TA group, it is considered that the severity of esophageal stricture was reduced.

Example 9: Histological evaluation of stenosis in administration of gel material to in vivo rat esophagus A histological evaluation of stenosis in administration of gel material to in vivo rat esophagus was performed.

(1) Cryosection [reagent]
10 v / v% formalin buffer saline 30 w / v% sucrose solution C. T compound liquid nitrogen

[result]
The length of the regenerated epithelium was measured with ImageJ based on the HE stained photograph, and the re-epithelialization rate was calculated. Specifically, the method for calculating the re-epithelialization rate is as follows. The length of the part from which the mucosal muscle was removed was defined as the length of the submucosal exfoliation wound. The length of the portion not covered by the epithelium was defined as the length that was not re-epithelialized. The reepithelialization rate was calculated with the following formula:
Epithelial regeneration distance = [length of exfoliated wound-length not re-epithelialized]
Re-epithelialization rate (%) = [epithelial regeneration distance / exfoliated wound length] × 100

The result is shown in FIG. From the results of HE staining, the Alg group, Gela group, Alg / Gela group, Alg-Mal / Gela group and Alg-Mal / Gela_TA group were all on the 14th day (DAY14) rather than the 7th day (DAY7). Epithelial development was seen. In addition, the epithelialization rate was faster in the Alg group, Alg / Gela group, Alg-Mal / Gela group and Alg-Mal / Gela_TA group than in the Gela group, and the Alg-Mal / Gela group was the fastest.

[result]
From the results of Sirius red staining, on the 14th day, collagen not only in the submucosa but also in the muscle layer in all of the Alg group, Gela group, Alg / Gela group, Alg-Mal / Gela group and Alg-Mal / Gela_TA group. Was observed. The amount of collagen produced based on the photograph of Sirius red staining was measured with ImageJ, and the amount of accumulated collagen was calculated. Specifically, the method for calculating the amount of accumulated collagen is as follows. Since collagen is stained red by Sirius red staining, the area stained red by image analysis using ImageJ was measured, and the portion selected in the tissue was defined as the area of collagen. The amount of accumulated collagen was calculated using the following formula:
Collagen accumulation (%) = [collagen fiber area / total tissue area]

The result is shown in FIG. From the results of Sirius red staining, the Alg group, Gela group, Alg / Gela group, Alg-Mal / Gela group and Alg-Mal / Gela_TA group were all in the 14th day (DAY14) rather than the 7th day (DAY7). ) Increased the amount of collagen accumulated.

[result]
The expression level of α-SMA was calculated based on the photograph of immunostaining. Specifically, the method for calculating the expression level of α-SMA is as follows. Cell nuclei were fluorescently labeled blue with a Vectashield mounting agent (with DAPI). The number of cells labeled in blue was calculated by image analysis using ImageJ, and this was used as the number of cells in the entire tissue. Furthermore, since α-SMA is fluorescently labeled in green, the number of cells labeled in green was calculated in the same manner, and this was used as the number of cells expressing α-SMA. The expression level of α-SMA was calculated by the following formula:
α-SMA expression level (%) = [number of α-SMA expressing cells / number of cells in whole tissue] × 100

The result is shown in FIG. From the results of immunostaining, α-SMA expression in the Alg group, Gela group, Alg / Gela group, Alg-Mal / Gela group and Alg-Mal / Gela_TA group on day 14 compared to day 7 The amount increased. In particular, from the stained photographs of the Gela group, α-SMA expression of vascular wall cells (pericytes or vascular smooth muscle cells) was observed, and it was confirmed that there were many blood vessels. It is thought that gelatin promoted angiogenesis. Angiogenesis has become one of the phenomena observed in the formation of granulation formed during inflammation and wounding. As a result of increased blood flow and cellular activity, many collagens were produced. In the case of esophageal stricture, it can be inferred that it has become a vicious circle. From the results of immunostaining, the expression level of α-SMA, which is a marker of myofibroblasts, was significantly lower in the Alg group, Alg / Gela group, Alg-Mal / Gela group and Alg-Mal / Gela_TA group than in the other groups. It was. For this reason, it was considered that the contraction of cells and tissues was suppressed, and it was found that esophageal stricture could be prevented.

Example 10: Examination of the hemostatic effect of the powder material In order to verify the hemostatic effect of the powder material, an experiment was conducted using a mouse bleeding model.

[animal]
ICR mouse male 8 weeks old, weight 33-37g
[reagent]
Saline forceps 3.24 wt% sodium citrate aqueous solution

[Equipment]
1mL syringe for insulin injection Filter paper (φ90mm, 5C, ADVANTEC)
50mL centrifuge tube (Bio Lite)

[material]
No liquid crosslinking agent: N = 10
Powder material (A-10-1): Alg (I-3G) / Gelatin = 1: 10 (weight ratio) 0.1 g / unit

With liquid crosslinking agent: N = 9
Powder material (A-10-2): Alg (I-3G) / Gelatin = 1: 10 (weight ratio) 0.1 g / unit Liquid cross-linking agent (B-10-2): 5% TG (effective concentration 0) 0.5 wt%) / 0.1MCa ²⁺ 0.5 mL / animal

Specifically, the powder materials (A-10-1) and (A-10-2) were prepared as follows.
Powder material (A-10-1): Alg (I-3G) and Gelatin were stirred at a weight ratio of 1:10 using a mixer.
Powder material (A-10-2): Alg (I-3G) and Gelatin were stirred at a weight ratio of 1:10 using a mixer.

Further, the liquid crosslinking agent (B-10-2) was specifically prepared as follows.
Liquid cross-linking agent (B-10-2): CaCl ₂ (Wako Pure Chemicals No. 036-00485) of 0.1 M with respect to the total amount of the solution was put in pure water and stirred until it was completely dissolved. TG (effective concentration 0.5 wt%, Ajinomoto Co., Inc.), which is 5 wt% based on the total amount of the solution, was added to the solution and stirred until it was completely dissolved.

[procedure]
Add 0.3 mL of 8-fold diluted somnopentyl to ICR mice i. p. Administration and anesthesia were performed. The mouse was attached to the cork board, and the abdomen was opened in a U shape. Parafilm and pre-weighed filter paper were placed on an organ other than the liver in a horizontal state. The liver LL leaf pulled out in a horizontal state was placed on a filter paper. While taking a video at an angle of 45 °, a 5 mm wide incision was made with a scalpel at a location approximately 1 cm below the LL leaf of the liver. Following the incision, the material was administered and allowed to bleed for 3 minutes (control left untreated). Here, the following treatment was performed in each of the Control group and the material group.
Control group: After incision with a scalpel, no material was administered and bleeding was performed for 3 minutes.
Powder group: After incision with a scalpel, powder material (A-10-1) was administered to the incision site and allowed to bleed for 3 minutes.
Crosslinked group: After incision with a scalpel, powder material (A-10-2) was administered to the incision site, and then liquid cross-linking agent (B-10-2) was immediately administered and allowed to bleed for 3 minutes.
After 3 minutes, the filter paper was collected, placed in a 50 mL centrifuge tube, and 50 mL pure water was added to hemolyze the blood adhering to the filter paper. In addition, 0.1 mL blood was collected from the heart of the mouse after the test using a 1 mL syringe containing 0.1 mL of a 3.24 wt% sodium citrate aqueous solution, and used for preparing a calibration curve when the amount of bleeding was converted. 500 μL of hemolyzed blood was placed in a microcell, the amount of hemoglobin was measured at an absorbance of 540 nm, and the amount of bleeding was calculated. Specifically, the amount of bleeding was calculated as follows: blood collected from the heart of a mouse was mixed with 0.01 mL, 0.1 mL, 0.5 mL, and 1 mL in 50 mL of pure water, and the wavelength was 540 nm. Absorbance was measured with an ultraviolet-visible spectrophotometer (JASCO V-630). A calibration curve was drawn based on the measured absorbance and blood loss. The absorbance of each sample was measured and converted into a bleeding amount using a calibration curve.

[result]
Using a mouse bleeding model, even when the powder material was administered alone, it remained around the incision without flowing into the bloodstream, and a hemostatic effect was observed. On the other hand, when the liquid cross-linking agent was sprayed, the powder became transparent instantly and the color of blood exuding from the incision was seen.

FIG. 18 shows the results of evaluating the amount of bleeding using the absorbance derived from hemoglobin in order to examine the hemostatic effect. It was clarified that the amount of bleeding in the material administration group was significantly reduced compared to the Control group, and the hemostatic effect of Alg / Gelatin was shown. On the other hand, there was no significant difference between the cross-linking agent administration and the non-administration group.

Claims

Containing ionic crosslinkable polysaccharides,
The ion-crosslinkable polysaccharide is crosslinked with a curing agent and used to cover the esophageal lesion.
A composition for covering an esophageal lesion.
The ion-crosslinkable polysaccharide is cross-linked with a curing agent and used to coat the esophageal lesion with a protein or amino group cross-linked using transglutaminase.
The composition of claim 1.
The composition according to claim 1 or 2, wherein the ion-crosslinkable polysaccharide is at least one selected from the group consisting of alginic acid, derivatives thereof, and salts thereof.
The composition according to claim 3, wherein the alginic acid derivative is alginic acid modified with a maleimide group, alginic acid modified with a thiol group, or alginic acid modified with an acrylate group.
The composition according to claim 4, wherein the modification is performed through a spacer.
The composition according to any one of claims 3 to 5, wherein the salt of alginic acid or a derivative thereof is sodium alginate, potassium alginate, a sodium salt of an alginate derivative, or a potassium salt of an alginate derivative.
The composition according to any one of claims 1 to 6, wherein the curing agent is at least one metal ion compound selected from the group consisting of Ca 2+ , Mg 2+ , Ba 2+ , Sr 2+ , Zn 2+ and Fe 3+. object.
The composition according to any one of claims 1 to 7, wherein the polysaccharide having protein or amino group is gelatin.
The composition according to any one of claims 1 to 8, wherein the composition is in a liquid form having fluidity or a powder form.
The composition according to any one of claims 1 to 9, wherein the composition is in a liquid form having fluidity and is applied to the surface of an esophageal lesion by spraying.
The composition according to any one of claims 1 to 10, wherein the composition is in a powder form and is applied to the surface of an esophageal lesion by spraying.
The composition according to claim 9 or 11, wherein the particle diameter of the powder is in the range of 10 µm to 500 µm.
The composition according to any one of claims 1 to 12, which is used in combination with another drug.
The composition according to any one of claims 1 to 13, for hemostasis.
The composition according to any one of claims 1 to 14, for use in preventing esophageal stricture.
Containing ionic crosslinkable polysaccharides,
The ionic crosslinkable polysaccharide is cross-linked with a curing agent and is used to cover the esophageal lesion with the cross-linked ionic crosslinkable polysaccharide.
Kit for covering the damaged part of the esophagus.
Further comprising a polysaccharide having a protein or amino group,
The ion-crosslinkable polysaccharide is crosslinked with a curing agent, and the polysaccharide having a protein or amino group is crosslinked using transglutaminase, the crosslinked ion-crosslinkable polysaccharide and the polysaccharide having a crosslinked protein or amino group. Used to cover esophageal lesions with sugars,
The kit according to claim 16.
Apply ion-crosslinkable polysaccharide to the target esophageal lesion,
The ionically crosslinkable polysaccharide is crosslinked with a curing agent, and the esophageal lesion is covered with the crosslinked ionically crosslinkable polysaccharide.
How to cover esophageal lesions.
Apply more protein or amino acid-containing polysaccharide to the target esophageal lesion,
The ion-crosslinkable polysaccharide is crosslinked with a curing agent, and the polysaccharide having a protein or amino group is crosslinked using transglutaminase, the crosslinked ion-crosslinkable polysaccharide and the polysaccharide having a crosslinked protein or amino group. Cover the damaged part of the esophagus with sugars,
The method of claim 18.