Materials that prevent leakage of digestive juices and materials that protect organs from digestion caused by digestive juices

Abstract

The present invention provides a material or method that can replace conventional materials and methods for preventing digestive fluid leakage. The digestive fluid leakage preventing material of the present invention comprises a self-assembling peptide gel, has an adhesion retention rate of at least 40% on a collagen sheet, and a decomposition rate of 35% or less based on pancreatic juice treatment at 37°C for 7 days.

Description

Technical Field

[0001] The present invention relates to a material for preventing leakage of digestive fluid and a material for protecting organs from digestion caused by digestive fluid. Background Art

[0002] During surgery on digestive organs, digestive fluids can leak from the organs after surgery. For example, pancreatic juice, which produces enzymes that break down proteins, fats, and carbohydrates, can leak from the pancreas after surgery. This condition, known as pancreatic fistula, can dissolve the pancreas itself and surrounding tissues in a digestive manner, inducing inflammation and is therefore a significant postoperative complication.

[0003] Representative pancreatic surgeries include pancreaticoduodenectomy for tumors near the pancreatic head and pancreatic tail resection for tumors in the pancreatic body or tail. In pancreaticoduodenectomy, the pancreatic head, bile duct, duodenum, and part of the stomach are removed, and the pancreas, bile duct, and stomach are sutured to the small intestine, respectively. In pancreatic tail resection, the pancreatic body or tail is resected and automatically sutured using a linear stapler, or the stumps are closed by suturing. At this time, in order to prevent the leakage of pancreatic juice caused by incomplete suturing of the anastomosis and / or incomplete closure of the stump, fibrin glue is sometimes applied to the anastomosis and / or stump. However, fibrin glue is digested by activated pancreatic juice, and therefore is not satisfactory from the perspective of preventing pancreatic juice leakage.

[0004] In all of the above surgical procedures, a drainage tube is placed near the anastomosis or pancreatic stump. The presence of pancreatic fistula is determined based on the amylase level in the drainage fluid and the duration of the leakage. If pancreatic fistula is confirmed, treatment is performed, such as medication administration and external drainage of pancreatic fluid using the drainage tube. For example, Patent Document 1 proposes a method for closing pancreatic fistulas using self-assembling peptides. However, this method intends to close pancreatic fistulas by using the self-assembling peptide to plug the hole left by the drainage tube.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application No. 2019-508175 Summary of the Invention

[0008] The present invention has been made to solve the above-mentioned conventional problems, and its main object is to provide a digestive juice leakage preventing material that can exhibit a good effect from the viewpoint of preventing pancreatic juice leakage.

[0009] According to one aspect of the present invention, a material for preventing leakage of digestive fluid can be provided, which is composed of a self-assembling peptide gel containing self-assembling peptides and water, has an attachment residual rate to collagen sheets of at least 40%, and a decomposition rate of less than 35% based on pancreatic fluid treatment at 37°C for 7 days.

[0010] In one embodiment, the self-assembling peptide comprises a self-assembling peptide having a positive charge at physiological pH.

[0011] In one embodiment, the self-assembling peptide comprises a self-assembling peptide having a charge of +1 to +4 at physiological pH.

[0012] In one embodiment, the viscosity measured using a rotational viscometer at a temperature of 25° C. and a rotation speed of 0.5 rpm is 3.0 Pa·s or more.

[0013] In one embodiment, the self-assembling peptide comprises a self-assembling peptide comprising the following amino acid sequence (A).

[0014] Amino acid sequence (A): a1b1c1b2a2b3db4a3b5c2b6a4

[0015] (In this amino acid sequence, a1 to a4 are basic amino acid residues; b1 to b6 are uncharged polar amino acid residues and / or hydrophobic amino acid residues, wherein at least five of b1 to b6 are hydrophobic amino acid residues; c1 and c2 are acidic amino acid residues; and d is a hydrophobic amino acid residue or an uncharged polar amino acid residue)

[0016] In one embodiment, the self-assembling peptide comprises at least one selected from the group consisting of the self-assembling peptides represented by SEQ ID NOs: 1 to 13 and 16 to 18.

[0017] In one embodiment, the material for preventing digestive juice leakage is used to prevent pancreatic juice from leaking from the pancreas.

[0018] According to another aspect of the present invention, a method for preventing digestive fluid from leaking from the digestive tract is provided, comprising applying the above-mentioned material for preventing digestive fluid from leaking to a digestive organ that needs to be prevented from leaking digestive fluid.

[0019] According to another aspect of the present invention, there is provided an organ protection material that is protected from digestion caused by digestive juices. The material is composed of a self-assembling peptide gel containing self-assembling peptides and water, and has an attachment residual rate to collagen sheets of at least 40%, and a decomposition rate of less than 35% based on pancreatic juice treatment at 37°C for 7 days.

[0020] In one embodiment, the self-assembling peptide comprises a self-assembling peptide having a positive charge at physiological pH.

[0021] In one embodiment, the self-assembling peptide comprises a self-assembling peptide having a charge of +1 to +4 at physiological pH.

[0022] In one embodiment, the viscosity measured using a rotational viscometer at a temperature of 25° C. and a rotation speed of 0.5 rpm is 3.0 Pa·s or more.

[0023] In one embodiment, the self-assembling peptide comprises a self-assembling peptide comprising the following amino acid sequence (A).

[0024] Amino acid sequence (A): a1b1c1b2a2b3db4a3b5c2b6a4

[0025] (In this amino acid sequence, a1 to a4 are basic amino acid residues; b1 to b6 are uncharged polar amino acid residues and / or hydrophobic amino acid residues, wherein at least five of b1 to b6 are hydrophobic amino acid residues; c1 and c2 are acidic amino acid residues; and d is a hydrophobic amino acid residue or an uncharged polar amino acid residue)

[0026] In one embodiment, the self-assembling peptide comprises at least one selected from the group consisting of the self-assembling peptides represented by SEQ ID NOs: 1 to 13 and 16 to 18.

[0027] In one embodiment, the organ protection material is used to protect an organ from digestion caused by pancreatic juice.

[0028] According to another aspect of the present invention, there is provided a method for protecting an organ from digestion caused by digestive fluid, comprising applying the above-mentioned organ protection material to the surface of the organ of the object to be protected.

[0029] According to the present invention, by using a self-assembling peptide gel having an adhesion residual rate to a collagen sheet of a predetermined value or higher and a decomposition rate by pancreatic juice treatment of a predetermined value or lower, a material can be provided that is effective in preventing pancreatic juice from leaking from the pancreas. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a graph showing the attachment residual rate of the self-assembling peptide gel.

[0031] Figure 2 It is a graph showing the amylase level in rat ascites.

[0032] Figure 3 This is a microscopic photograph of the pancreatic resection site of a rat 3 days after surgery. DETAILED DESCRIPTION

[0033] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to these embodiments.

[0034] A. Definition of Terms

[0035] (1) As used herein, the term "self-assembling peptide" refers to a peptide that can spontaneously assemble in an aqueous solution to form a nanometer-sized fibrous aggregate (hereinafter referred to as "nanofiber") through the interaction of peptide molecules. The interactions between peptide molecules are not particularly limited and include, for example, hydrogen bonds, interionic interactions, electrostatic interactions such as van der Waals forces, and non-covalent interactions such as hydrophobic interactions. The formation of nanofibers can be confirmed, for example, by electron microscopic observation.

[0036] (2) In this specification, "gel" refers to a viscoelastic substance having both viscous and elastic properties. In one embodiment, a substance having a viscosity of 3 Pa·s or greater as measured at 25°C and 0.5 rpm using a rotational viscometer can be referred to as a gel.

[0037] (3) In this specification, "self-assembling peptide gel" refers to a substance in which nanofibers formed by the spontaneous assembly of self-assembling peptides form a three-dimensional network structure and hold water molecules therein to form a gel.

[0038] (4) In this specification, the term "hydrophilic amino acid" includes basic amino acids such as arginine (Arg / R), lysine (Lys / K), and histidine (His / H); acidic amino acids such as aspartic acid (Asp / D) and glutamic acid (Glu / E); and uncharged polar amino acids such as tyrosine (Tyr / Y), serine (Ser / S), threonine (Thr / T), asparagine (Asn / N), glutamine (Gln / Q), and cysteine ​​(Cys / C). The letters in parentheses represent the three-letter and single-letter amino acids, respectively.

[0039] (5) As used herein, the term "hydrophobic amino acids" includes non-polar amino acids such as alanine (Ala / A), leucine (Leu / L), isoleucine (Ile / I), valine (Val / V), methionine (Met / M), phenylalanine (Phe / F), tryptophan (Trp / W), glycine (Gly / G), and proline (Pro / P). The letters in parentheses represent the three-letter and single-letter amino acids, respectively.

[0040] B. Materials to prevent digestive fluid from leaking

[0041] The material for preventing leakage of digestive juice according to an embodiment of the present invention is composed of a self-assembling peptide gel containing self-assembling peptides and water, and has an attachment residual rate to the collagen sheet of more than 40%, and a decomposition rate based on pancreatic juice treatment at 37°C for 7 days of less than 35%. According to the material for preventing leakage of digestive juice, it is possible to stably attach in a manner covering the suture and / or closed portion of the digestive organ without being substantially digested (decomposed) by the digestive juice, and thus it is possible to appropriately prevent leakage of digestive juice from the digestive organ. In addition, by promoting cell proliferation at the attachment site, the healing of the leakage portion (suture and / or closed portion) can be promoted. It should be noted that in this specification, preventing leakage of digestive juice is a concept that includes preventing leakage of digestive juice.

[0042] Examples of the digestive organs include the stomach, duodenum, small intestine, liver, gallbladder, pancreas, spleen, etc. In one embodiment, leakage of pancreatic juice from the pancreas and / or leakage of bile from the gallbladder (bile duct) can be prevented.

[0043] The above-mentioned material for preventing leakage of digestive fluid has a typical adhesion residual rate of 40% or more, preferably 45% or more, and more preferably 50% or more on the collagen sheet. The upper limit of the adhesion residual rate is not particularly limited, and may be, for example, 80%, or 70%. If the adhesion residual rate is within the above range, it can be stably and continuously adhered in a manner that covers the suture and / or closure of the digestive organ. The adhesion residual rate can be measured by the method described in the examples. In addition, the adhesion residual rate can be increased, for example, by using a self-assembling peptide having a positive charge at a physiological pH value (pH = 7.4), increasing the peptide concentration, increasing the viscosity, and the like.

[0044] The decomposition rate of the above-mentioned material for preventing leakage of digestive juice based on pancreatic juice treatment (exposure to pancreatic juice) at 37°C for 7 days is typically 35% or less, preferably 30% or less, more preferably 15% or less, and further preferably 0% to 10%. If the decomposition rate based on pancreatic juice treatment is within the above range, it can be stably and continuously attached to the surface of the digestive organ without being significantly decomposed by the digestive juice that should be prevented from leaking. Here, the decomposition rate based on pancreatic juice treatment can be measured using simulated pancreatic juice (for example, a 1 mg / mL aqueous solution of a digestive enzyme containing 1 g of pancreatin according to the Japanese Pharmacopoeia) as described in the examples. Peptides are usually decomposed by pancreatic juice containing peptidases, but self-organized peptide aggregates (specifically, self-assembled peptide gels) are not easily decomposed by pancreatic juice and therefore can appropriately play a qualitative role as a material for preventing leakage of digestive juice.

[0045] The viscosity of the digestive fluid leakage preventing material can be, for example, 3.0 Pa·s or greater, preferably 9.0 Pa·s or greater, more preferably 15.0 Pa·s or greater, even more preferably 25.0 Pa·s or greater, and even more preferably 35.0 Pa·s or greater. Alternatively, the viscosity of the digestive fluid leakage preventing material can be, for example, 1000 Pa·s or less, or, for example, 100 Pa·s or less. This concentration is measured using a rotational viscometer at 25°C and 0.5 rpm.

[0046] The pH of the material preventing digestive fluid leakage is preferably such that the self-assembling peptides constituting the self-assembling peptide gel can maintain their charge at physiological pH. The pH of the material preventing digestive fluid leakage can be, for example, 5.0 to 9.0, preferably 5.5 to 8.0, and more preferably 5.5 to 7.5.

[0047] As described above, the material for preventing leakage of digestive fluid is composed of a self-assembling peptide gel containing self-assembling peptides and water. The self-assembling peptide gel may further contain pharmaceutically acceptable additives depending on the intended purpose.

[0048] B-1. Self-assembling peptides

[0049] As the above-mentioned self-assembling peptide, a self-assembling peptide having a positive charge at a physiological pH value (pH=7.4) can be preferably used. Specifically, a self-assembling peptide having a charge of, for example, +1 to +4, preferably +1 to +3, more preferably +2 or +3, and further preferably +2 per molecule at a physiological pH value can be used. Such self-assembling peptides can appropriately form nanofibers and gels. In addition, the self-assembling peptide gel formed by such a self-assembling peptide can exert the following reversible self-assembly ability: the fluidity increases by physical stimulation such as stirring, vibration, and shear force, and the fluidity decreases by stopping the physical stimulation. Thus, there is the following advantage: the load when administering using a slender instrument such as a syringe or catheter is reduced. Furthermore, the self-assembling peptide gel formed using a positively charged self-assembling peptide can make the surface of organs such as digestive organs and mucous membranes excellent in retention. As a reason for achieving such an effect, the present invention is not limited, but it is speculated that the self-assembling peptide gel formed using a positively charged self-assembling peptide is positively charged as a whole, and electrostatic interaction is generated between the negatively charged cell surface, resulting in an increase in the adhesion between the two.

[0050] On the other hand, since the self-assembling peptide gel formed by self-assembling peptides that have no charge or negative charge at physiological pH (as a result, the self-assembling peptide gel is uncharged or negatively charged as a whole) cannot obtain electrostatic attraction between itself and the negatively charged cell surface, the retention on the surface of organs such as digestive organs and mucous membranes may become insufficient.

[0051] In addition, self-assembling peptides that are uncharged at physiological pH typically have irreversible self-assembly capabilities, and nanofiber formation and gelation begin through changes in the pH of the peptide solution or through reaction with inorganic salts. Therefore, since such self-assembling peptides gel after being administered to the sutures and / or closures of the digestive organs in a solution state, they diffuse along the surface of the administration site until gelation occurs, resulting in difficulty in rapidly forming a gel of sufficient thickness.

[0052] The charge of the self-assembling peptide refers to the total charge of the amino acid residues contained in the peptide molecule. The charge at physiological pH can be calculated using a program available on the PROTEIN CALCULATOR v3.4 website (http: / / protcalc.sourceforge.net / ), for example.

[0053] The number of amino acid residues constituting the self-assembling peptide is, for example, 9 or more, preferably 10 to 40, more preferably 10 to 32, and even more preferably 12 to 32. The N-terminal amino group and / or C-terminal carboxyl group of the peptide may be appropriately protected with a protecting group such as an acetyl group or an amide group.

[0054] As the above-mentioned self-assembling peptide, for example, the following peptide can be used: the amino acid residues at odd positions (or even positions) are acidic amino acid residues and basic amino acid residues arranged alternately, and any 1 to 4, preferably 2 to 3 amino acid residues of the acidic amino acid residues are substituted with uncharged polar amino acid residues or hydrophobic amino acid residues, and the amino acid residues at even positions (or odd positions) are all hydrophobic amino acid residues. Such a peptide forms a β-fold consisting of a hydrophobic face configured only with hydrophobic amino acid residues and a hydrophilic face containing hydrophilic amino acid residues in an aqueous solution. The two β-folds extend in a state of overlapping with the hydrophobic face as the inner side, and as a result, nanofibers can be formed. The nanofibers are further assembled by the interaction between the hydrophilic faces to form a network structure, which can be gelled. It should be noted that the confirmation of the formation of the β-fold structure can be carried out, for example, by measuring the molar ellipticity using circular dichroism and determining the case where the molar ellipticity at 216nm becomes a negative value. Alternatively, the following method can be used to confirm the presence of a chromatogram at 1620 cm －1 The peak based on β-sheet appears near 1690 cm －1 A peak based on antiparallel β-sheets appears nearby.

[0055] As the above-mentioned self-assembling peptide, a peptide comprising the following amino acid sequence (A) can be exemplified. The self-assembling peptide can be a peptide consisting of the following amino acid sequence (A). As long as the effects of the present invention can be obtained, a peptide having several, for example, 1 to 5, arbitrary amino acid residues attached to its N-terminus and / or C-terminus can also be used. In addition, the N-terminal amino group of the peptide can be acetylated, and the C-terminal carboxyl group can be amidated as needed.

[0056] Amino acid sequence (A): a1b1c1b2a2b3db4a3b5c2b6a4

[0057] (In this amino acid sequence, a1 to a4 are basic amino acid residues; b1 to b6 are uncharged polar amino acid residues and / or hydrophobic amino acid residues, wherein at least five of b1 to b6 are hydrophobic amino acid residues; c1 and c2 are acidic amino acid residues; and d is a hydrophobic amino acid residue or an uncharged polar amino acid residue)

[0058] In one embodiment, in the above amino acid sequence, b1 to b6 are all hydrophobic amino acid residues. b1 to b6 can each independently be an alanine residue, a valine residue, a leucine residue, or an isoleucine residue, preferably an alanine residue or a leucine residue, more preferably all b1 to b6 are leucine residues, or five are leucine residues and one is an alanine residue.

[0059] In one embodiment, in the above amino acid sequence, d is an alanine residue, a leucine residue, an asparagine residue, a serine residue or a glutamine residue.

[0060] In one embodiment, in the above amino acid sequence, a1 to a4 are all arginine or lysine, preferably arginine.

[0061] In one embodiment, in the above amino acid sequence, c1 and c2 are both aspartic acid or glutamic acid, preferably aspartic acid.

[0062] The self-assembling peptides that can be used in the present invention are not limited to the peptides comprising the above-mentioned amino acid sequence (A). For example, the peptides described in WO2007 / 000979 can be used.

[0063] Specific examples of the self-assembling peptides include peptides of SEQ ID NOs. 1 to 13 and 16 to 18. Among them, peptides of SEQ ID NOs. 1 to 4 are more preferred. As long as the effects of the present invention are achieved, the self-assembling peptides may be used alone or in combination of two or more.

[0064] [Table 1]

[0065] Amino acid sequence Charge at pH 7.4 Serial number n-RLDLRLALRLDLR-c +2 1 n-RLDLRLSLRLDLR-c +2 2 n-RLALRLDLRLDLR-c +2 3 n-KRLDLNLRLDLRK-c +3 4 n-RLDLRLLLRLDLR-c +2 5 n-RADLRLALRLDLR-c +2 6 n-RLDLRLALRLDAR-c +2 7 n-RADLRLLLRLDLR-c +2 8 n-RADLRLLLRLDAR-c +2 9 n-RLDLRALLRLDLR-c +2 10 n-RLDLRLLARLDLR-c +2 11 n-RLNLRLDLRLNL-c +2 12 n-RAQARAQARAQARAQA-c +4 13 n-RASARASARASARASA-c +4 16 n-RASARASARASARADA-c +3 17 n-RASARADARADARADA-c +1 18

[0066] The proportion of the self-assembling peptide in the material for preventing leakage of digestive fluid (self-assembling peptide gel) can be appropriately set according to the desired adhesion residual rate, decomposition rate after pancreatic fluid treatment, etc. The proportion can be, for example, 0.3 w / w% to 6.0 w / w%, preferably 0.5 w / w% to 5.0 w / w%, more preferably 0.6 w / w% to 4.0 w / w%, further preferably 0.8 w / w% to 3.0 w / w%, and even more preferably 1.0 w / w% to 2.0 w / w%.

[0067] The self-assembling peptide can be prepared by any appropriate method such as liquid phase synthesis, solid phase synthesis, molecular biology, etc. The self-assembling peptide may be in the form of a salt depending on the preparation method, and materials for preventing digestive fluid leakage using the self-assembling peptide in the form of a salt are also included in the embodiments of the present invention.

[0068] B-2. Water

[0069] Examples of water include distilled water, deionized water, and pure water. Water, together with the additives described below, can constitute an aqueous medium such as physiological saline, buffered saline (PBS, etc.), phosphate buffer, and isotonic aqueous buffer.

[0070] The proportion of water in the digestive fluid leakage preventing material (self-assembling peptide gel) is, for example, 80% by weight or more, preferably 85% by weight or more, and more preferably 90% by weight or more.

[0071] B-3. ​​Added ingredients

[0072] As pharmaceutically acceptable additives, any appropriate additive can be selected by those skilled in the art according to the purpose, etc. Specific examples include pH adjusters, isotonic agents, pharmaceutical agents, preservatives, excipients, stabilizers, fillers, solubilizers, etc. The additives may be used alone or in combination of two or more.

[0073] Examples of the pH adjuster include citric acid, trisodium citrate, succinic acid, monosodium succinate, disodium succinate, sodium acetate, sodium bicarbonate, sodium carbonate, phosphoric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, histidine, and lysine.

[0074] Examples of the isotonicity agent include sodium chloride, polyethylene glycol, dextran, mannitol, sorbitol, inositol, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose, and raffinose.

[0075] Any appropriate drug can be used as long as the effects of the present invention can be achieved. Specific examples of the drug include anti-inflammatory agents, protease inhibitors (e.g., gabexate mesylate), steroids, and antibiotics.

[0076] B-4. Method for manufacturing a material that prevents digestive fluid leakage

[0077] The above-mentioned material that prevents the digestive fluid from leaking can be manufactured by any appropriate manufacturing method. For example, the material that prevents the digestive fluid from leaking can be obtained by mixing the self-assembling peptide, water and any additive. Each component or the mixture before mixing can be sterilized as needed. As a mixing method and a sterilizing method, any appropriate method applicable in the technical field can be used respectively. The self-assembling peptide gel that constitutes the material that prevents the digestive fluid from leaking is also stable under high temperature conditions, and therefore can also be sterilized by high pressure steam.

[0078] C. Methods to prevent digestive juice leakage

[0079] According to another aspect of the present invention, a method for preventing digestive fluid leakage is provided. The method for preventing digestive fluid leakage according to an embodiment of the present invention comprises applying the material for preventing digestive fluid leakage described in item B to a digestive organ that needs to be prevented from digestive fluid leakage.

[0080] As a coating method, for example, the material for preventing leakage of digestive fluid can be coated on the surface of the digestive organ in a manner that covers or plugs the position where digestive fluid will leak (incomplete suture position, perforation, cut surface, etc.). In one embodiment, the material for preventing leakage of digestive fluid is discharged to the target site by using tubular instruments such as syringes, needles, pipettes, catheters, tubes, and spread using spatulas, cotton swabs, etc. as needed. In addition, for example, a sprayer can also be used for spray-based coating. As described above, the material for preventing leakage of digestive fluid can increase its fluidity by imparting physical stimulation such as shear force, and return to its original fluidity by stopping the physical stimulation. Therefore, when using the above-mentioned tubular instrument, by increasing the fluidity of the material for preventing leakage of digestive fluid, it can be easily discharged with a smaller pressing force. On the other hand, by quickly returning to its original fluidity after discharge, the material for preventing leakage of digestive fluid can be stably attached to the surface of the digestive organ.

[0081] The coating amount may be any appropriate amount as long as the effects of the present invention are achieved. For example, the coating amount may be such that the coating thickness is 0.01 mm or more and less than 3 cm, preferably 0.05 mm or more and less than 1 cm.

[0082] Examples of digestive organs that require prevention of digestive fluid leakage include the stomach, duodenum, small intestine, liver, gallbladder, pancreas, and spleen. The materials and methods for preventing digestive fluid leakage can be preferably applied to the duodenum, gallbladder, and pancreas, and are effective in preventing pancreatic fistulas and / or bile fistulas. It should be noted that the digestive organs are preferably those of humans or non-human animals (e.g., non-human primates, dogs, cats, rats, cattle, pigs, sheep, etc.).

[0083] D. Organ protection materials

[0084] According to another aspect of the present invention, an organ protection material is provided. This embodiment of the organ protection material comprises a self-assembling peptide gel containing self-assembling peptides and water, exhibiting an adhesion retention rate of 40% or greater to a collagen sheet and a decomposition rate of 35% or less based on pancreatic juice treatment at 37°C for 7 days. This organ protection material can prevent the surrounding organs from being significantly digested (decomposed) by digestive fluid, even in the event of digestive fluid leakage.

[0085] Organs to be protected are organs (viscera) present in the abdominal cavity, and examples thereof include internal organs such as the stomach, duodenum, small intestine, large intestine, liver, gallbladder, pancreas, spleen, and kidneys, and circulatory organs such as blood vessels.

[0086] The attachment residual rate of the above-mentioned organ protection material to the collagen sheet is typically 40% or more, preferably 45% or more, and more preferably 50% or more. The upper limit of the attachment residual rate is not particularly limited, and may be, for example, 80%, or, for example, 70%. If the attachment residual rate is within the above range, it can be stably and continuously attached to the surface of the organ to be protected. The attachment residual rate can be measured by the method described in the examples. In addition, the attachment residual rate can be increased, for example, by using a peptide having a positive charge at a physiological pH value (pH = 7.4), increasing the peptide concentration, increasing the viscosity, etc.

[0087] The decomposition rate of the above-mentioned organ protection material based on pancreatic juice treatment at 37°C for 7 days is typically 35% or less, preferably 30% or less, more preferably 15% or less, and further preferably 0% to 10%. If the decomposition rate based on pancreatic juice treatment is within the above range, it is possible to prevent the organ of the protected object from coming into contact with the digestive juice without being significantly decomposed by the digestive juice. Here, the decomposition rate based on pancreatic juice treatment can be measured using simulated pancreatic juice (for example, a 1 mg / mL aqueous solution of a digestive enzyme containing 1 g of Japanese Pharmacopoeia pancreatin in 1 g) as described in the examples. Peptides are usually decomposed by pancreatic juice containing peptidases, but since the self-organized peptide aggregates (specifically, self-assembled peptide gels) are not easily decomposed by pancreatic juice, they can appropriately function as organ protection materials.

[0088] The viscosity of the organ-protecting material can be, for example, 3.0 Pa·s or greater, preferably 9.0 Pa·s or greater, more preferably 15.0 Pa·s or greater, even more preferably 25.0 Pa·s or greater, and even more preferably 35.0 Pa·s or greater. Alternatively, the viscosity of the organ-protecting material can be, for example, 1000 Pa·s or less, or, for example, 100 Pa·s or less. This concentration is measured using a rotational viscometer at 25°C and 0.5 rpm.

[0089] The pH of the organ-protecting material is preferably such that the self-assembling peptides constituting the self-assembling peptide gel can maintain their charge at physiological pH. The pH of the organ-protecting material may be, for example, 5.0 to 9.0, preferably 5.5 to 8.0, and more preferably 5.5 to 7.5.

[0090] As described above, the organ protection material is composed of a self-assembling peptide gel containing self-assembling peptides and water. The self-assembling peptide gel may further contain pharmaceutically acceptable additives depending on the intended purpose. As the self-assembling peptide gel, the same self-assembling peptide gel as that constituting the material for preventing digestive fluid leakage described in item B can be used.

[0091] E. Organ protection methods

[0092] According to another aspect of the present invention, a method for protecting an organ from digestion by digestive fluid is provided. The organ protection method according to an embodiment of the present invention comprises applying the organ protection material described in item D to the surface of the organ to be protected.

[0093] As a coating method, for example, the organ protective material can be coated on the organ surface in a manner that covers at least a portion of the organ surface of the protected object. In one embodiment, the organ protective material is discharged to the target site by using a tubular device such as a syringe, a needle, a pipette, a catheter, a tube, and spread using a spatula, a cotton swab, etc. as needed. In addition, for example, a spray-based coating can also be performed using a sprayer. As described above, the fluidity of the organ protective material can be increased by imparting physical stimulation such as shear force, and can be restored to its original fluidity by stopping the physical stimulation. Therefore, when using the above-mentioned tubular device, by increasing the fluidity of the organ protective material, it can be easily discharged with a smaller pressing force. On the other hand, by quickly returning to its original fluidity after discharge, the organ protective material can be stably attached to the organ surface.

[0094] The coating amount may be any appropriate amount as long as the effects of the present invention are achieved. For example, the coating amount may be such that the coating thickness is 0.01 mm or more and less than 3 cm, preferably 0.05 mm or more and less than 1 cm.

[0095] Examples of organs that require protection from digestion by digestive juices include internal organs such as the stomach, duodenum, small intestine, large intestine, liver, gallbladder, pancreas, spleen, and kidneys, and circulatory organs such as blood vessels. It should be noted that these organs are preferably organs of humans or non-human animals (e.g., non-human primates, dogs, cats, rats, cattle, pigs, sheep, etc.).

[0096] Example

[0097] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

[0098] Viscosity

[0099] The viscosity (Pa·s) of the sample was measured using a rotational viscometer (manufactured by Toki Kogyo Co., Ltd., product name "TVE-20LT") as a viscosity measuring device. Specifically, as follows. First, weigh about 0.2 g of the sample in a sample cup, attach it to the viscometer body, and circulate water adjusted to 25±0.2°C for more than 15 minutes. Then, rotate the conical rotor (manufactured by Toki Kogyo Co., Ltd., 3°×R9.7) at 0.5 rpm within the measurement range M, and take the average value of the viscosity displayed on the screen about 15, 20, and 25 minutes after the start of rotation as the viscosity.

[0100] Decomposition rate based on pancreatic juice treatment

[0101] On a 0.1 g sample (gel) placed in a 1.5 mL tube, simulated pancreatic juice (made by Maruishi Pharmaceutical Co., Ltd., product name: 1 mg / mL aqueous solution of pancreatin powder "Maruishi" (a digestive enzyme agent containing 1 g of pancreatin according to the Japanese Pharmacopoeia in 1 g)) or 200 μL of PBS was allowed to stand at 37°C for 7 days. 1 mL of trifluoroacetic acid (made by Wako Pure Chemical Industries, Ltd., for peptide synthesis) was added to the tube after standing and thoroughly mixed. 14 mL of trifluoroacetic acid was further added to the mixture, and the solution, which was fixed to 25 mL with distilled water, was used as a sample for HPLC determination. The sample for HPLC determination was subjected to HPLC determination under the following conditions, and the decomposition rate after pancreatic juice treatment was obtained based on the following formula. It should be noted that the distilled water used in the preparation was subjected to HPLC determination under the same conditions as the sample solution, and was subtracted from each chromatogram to perform baseline correction.

[0102] Decomposition rate by pancreatic juice treatment (%) = (peak area of ​​self-assembling peptide in PBS-immersion sample - peak area of ​​self-assembling peptide in simulated pancreatic juice-immersion sample) ÷ peak area of ​​self-assembling peptide in PBS-immersion sample × 100

[0103] <HPLC measurement conditions>

[0104] Apparatus 1 (for analysis of self-assembled peptide gels 1 to 4): Waters M515 Pump, In-Line Degasser AF, 717 Autosampler, 996 Photodiode Array Detector, 2410 Refractive Index Detector, column heater

[0105] Apparatus 2 (for analysis of self-assembled peptide gels 5 to 7): Waters e2695 Separations Module, Waters 2998 Photodiode Array Detector, Waters SMH Column Heater, Empower 3 Feature Release 3 Hotfix 1, column heater: JASCO 860-CO

[0106] Column: YMC-Triart C18 3mm×250mm,

[0107] Column heater set temperature: 67°C

[0108] Detection wavelength: 205nm

[0109] Analysis time: 60 minutes (Self-assembling peptide gels 3 and 5-7) or 110 minutes (Self-assembling peptide gels 1, 2, and 4)

[0110] Mobile phase: Liquid A: distilled water 600 mL, acetonitrile 200 mL, trifluoroacetic acid 0.8 mL

[0111] Solution B: 250 mL of distilled water, 250 mL of acetonitrile, 0.5 mL of trifluoroacetic acid

[0112] [Table 2]

[0113] Composition of mobile phase

[0114] Time [min] Liquid A[%] Liquid B [%] 0.0 95 5 15.00 95 5 30.00 75 25 40.00 75 25 50.00 0 100 60.00 0 100 61.00 95 5

[0115] [Table 3]

[0116] Composition of mobile phase

[0117] Time [min] Liquid A[%] Liquid B [%] 0.0 100 0 75.00 60 40 105.00 0 100 110.00 0 100 110.01 100 0

[0118] Flow rate: 0.4 mL / min

[0119] Injection volume: 60 μL

[0120] Adhesion Residual Rate

[0121] Utilize ring saw (8mm) to dig out collagen sheet (made by Nichihide Collagen Industry Co., Ltd., product number "40322223"), immerse in physiological saline for more than 30 minutes. Take out from physiological saline, apply 10 μL of sample (self-assembling peptide gel) on the collagen sheet after gently removing water, measure the weight (A) of the collagen sheet after coating. Then, the surface opposite to the surface coated with the sample of the collagen sheet is gently moistened with physiological saline containing the product name "Pancreatin Maruishi" 0.1%, and is attached to 6-well plates (made by Corning, product name "cell culture 6-well multiwall plate (cell culture 6well multiwall plate)") in such a way that the sample coating surface becomes the upper surface. The plate is centrifuged at 800rpm, 20 seconds using a plate centrifuge (made by Kubota Trading Co., Ltd., product name "PlateSpin"). After centrifugation, the collagen sheet was peeled off the plate, its weight (B) was measured, and the adhesion residual rate was calculated based on the following formula. It should be noted that for each sample, the above measurement and adhesion residual rate calculation were performed with N=8, and the average value was used as the adhesion residual rate for each sample.

[0122] Adhesion residual rate (%) = (B) / (A) × 100

[0123] [Preparation Example 1 Self-assembling Peptide Gel 1]

[0124] A high-pressure steam sterilized self-assembling peptide gel (pH 5.9) containing 1.5 w / w% of a self-assembling peptide (SEQ ID NO: 1: Ac-RLDLRLALRLDLR-CONH2), an isotonicity agent, a pH adjuster, and water was used as self-assembling peptide gel 1. The decomposition rate of self-assembling peptide gel 1 after treatment with pancreatic juice was 8.8%, and the viscosity was 35 Pa·s or more (above the measurement range). In addition, Figure 1 As shown, the adhesion residual rate is 54.4%.

[0125] [Preparation Example 2 Self-assembling Peptide Gel 2]

[0126] 1.5 w / w% of the self-assembling peptide (SEQ ID NO: 14: Ac-SAEASAEASAEAKAEA-CONH2), an isotonic agent, a pH adjuster, and water were mixed to obtain a self-assembling peptide gel 2 (pH 5.8). The decomposition rate of the self-assembling peptide gel 2 after treatment with pancreatic juice was less than 10%. Figure 1 As shown, the adhesion residual rate is 33.4%.

[0127] [Preparation Example 3 Self-assembling Peptide Gel 3]

[0128] A self-assembling peptide gel (pH 2.2) containing 1.5 w / w% of a self-assembling peptide (SEQ ID NO: 15: Ac-RADARADARADARADA-CONH2) and water was used as self-assembling peptide gel 3. The decomposition rate of self-assembling peptide gel 3 after pancreatic juice treatment was less than 10%, and the viscosity was 11.0 Pa·s. Figure 1 As shown, the adhesion residual rate is 36.5%.

[0129] [Preparation Example 4 Self-assembling Peptide Gel 4]

[0130] 0.8 w / w% of the self-assembling peptide (SEQ ID NO: Ac-RLDLRLALRLDLR-CONH2), an isotonic agent, a pH adjuster, and water were mixed to obtain a self-assembling peptide gel 4 (pH 6.0). The decomposition rate of the self-assembling peptide gel 4 after treatment with pancreatic juice was 28%, and the viscosity was 11.2 Pa·s. Figure 1 As shown, the adhesion residual rate is 45.9%.

[0131] [Preparation Example 5 Self-Assembling Peptide Gel 5]

[0132] 1.5 w / w% of the self-assembling peptide (SEQ ID NO: 16: Ac-RASARASARASARASA-CONH2), an isotonic agent, a pH adjuster, and water were mixed to obtain a self-assembling peptide gel 5 (pH 6.8). The decomposition rate of the self-assembling peptide gel 5 after treatment with pancreatic juice was 30.4%. Figure 1 As shown, the adhesion residual rate is 57.7%.

[0133] [Preparation Example 6 Self-Assembling Peptide Gel 6]

[0134] 1.5 w / w% of the self-assembling peptide (SEQ ID NO: 17: Ac-RASARASARASARADA-CONH2), an isotonic agent, a pH adjuster, and water were mixed to obtain a self-assembling peptide gel 6 (pH 6.2). The decomposition rate of the self-assembling peptide gel 6 after treatment with pancreatic juice was 5.7%. Figure 1 As shown, the adhesion residual rate is 70.6%.

[0135] [Preparation Example 7 Self-Assembling Peptide Gel 7]

[0136] 1.5 w / w% of the self-assembling peptide (SEQ ID NO: 18: Ac-RASARADARADARADA-CONH2), an isotonic agent, a pH adjuster, and water were mixed to obtain a self-assembling peptide gel 7 (pH 7.7). The decomposition rate of the self-assembling peptide gel 7 after treatment with pancreatic juice was 2.1%. Figure 1 As shown, the adhesion residual rate was 59.0%.

[0137] [Test Example 1]

[0138] SD rats (8 weeks old) were opened in the abdomen under deep anesthesia and a portion of the pancreas was removed. Self-assembling peptide gel 1 or self-assembling peptide gel 3 was applied to a thickness of 0.1 mm or more in a manner that covered the resection surface, and the abdominal cavity was closed. In addition, rats whose abdominal cavities were closed without applying any substance to the resection surface were used as controls. Three days after the operation, the abdomen was opened again to recover the ascites, and euthanasia was performed (N=9). Then, the amylase value (Amy) in the ascites was measured. The results are shown in Figure 2 (Mean ± SD). In addition, microscopic observation photos of the pancreatic resection site 3 days after surgery are shown in Figure 3 .

[0139] according to Figure 2 It was found that the self-assembling peptide gel 1, which had an adhesion residual rate to the collagen sheet of a predetermined value or higher and a decomposition rate by pancreatic juice treatment of a predetermined value or lower, could more appropriately prevent pancreatic juice leakage than the self-assembling peptide gel 3.

[0140] In addition, according to Figure 3 It can be seen that the amount of residual self-assembling peptide gel on the pancreatic surface coated with self-assembling peptide gel 3 is significantly less than that on the pancreatic surface coated with self-assembling peptide gel 1. This shows that the difference in the adhesion and residual rate of the self-assembling peptide gel on the digestive organs leads to a difference in the effect of preventing digestive fluid leakage. In addition, if we consider Figure 1 , suggesting that the charge of the self-assembling peptides leads to differences in the adhesion force with the organ surface.

[0141] Industrial applicability

[0142] The digestive fluid leakage preventing material and organ protection material of the present invention can be suitably used in the medical field. Sequence Listing <110> Melicon Co., Ltd. National University Corporation Osaka University <120> Materials that prevent leakage of digestive juices and materials that protect organs from digestion caused by digestive juices <130> MNC17198 <150> JP2020-032544 <151> 2020-02-28 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 1 Arg Leu Asp Leu Arg Leu Ala Leu Arg Leu Asp Leu Arg 1 5 10 <210> 2 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 2 Arg Leu Asp Leu Arg Leu Ser Leu Arg Leu Asp Leu Arg 1 5 10 <210> 3 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 3 Arg Leu Ala Leu Arg Leu Asp Leu Arg Leu Asp Leu Arg 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 4 Lys Arg Leu Asp Leu Asn Leu Arg Leu Asp Leu Arg Lys 1 5 10 <210> 5 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 5 Arg Leu Asp Leu Arg Leu Leu Leu Arg Leu Asp Leu Arg 1 5 10 <210> 6 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 6 Arg Ala Asp Leu Arg Leu Ala Leu Arg Leu Asp Leu Arg 1 5 10 <210> 7 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 7 Arg Leu Asp Leu Arg Leu Ala Leu Arg Leu Asp Ala Arg 1 5 10 <210> 8 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 8 Arg Ala Asp Leu Arg Leu Leu Leu Arg Leu Asp Leu Arg 1 5 10 <210> 9 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 9 Arg Ala Asp Leu Arg Leu Leu Leu Arg Leu Asp Ala Arg 1 5 10 <210> 10 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 10 Arg Leu Asp Leu Arg Ala Leu Leu Arg Leu Asp Leu Arg 1 5 10 <210> 11 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 11 Arg Leu Asp Leu Arg Leu Leu Ala Arg Leu Asp Leu Arg 1 5 10 <210> 12 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 12 Arg Leu Asn Leu Arg Leu Asp Leu Arg Leu Asn Leu 1 5 10 <210> 13 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 13 Arg Ala Gln Ala Arg Ala Gln Ala Arg Ala Gln Ala Arg Ala Gln Ala 1 5 10 15 <210> 14 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 14 Ser Ala Glu Ala Ser Ala Glu Ala Ser Ala Glu Ala Lys Ala Glu Ala 1 5 10 15 <210> 15 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 15 Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala 1 5 10 15 <210> 16 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 16 Arg Ala Ser Ala Arg Ala Ser Ala Arg Ala Ser Ala Arg Ala Ser Ala 1 5 10 15 <210> 17 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 17 Arg Ala Ser Ala Arg Ala Ser Ala Arg Ala Ser Ala Arg Ala Asp Ala 1 5 10 15 <210> 18 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Self-assembling peptides <400> 18 Arg Ala Ser Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala 1 5 10 15

Claims

1. A material for preventing leakage of digestive fluid, comprising a self-assembling peptide gel containing a self-assembling peptide and water, The residual rate of collagen sheet adhesion is at least 40%. The decomposition rate based on pancreatic juice treatment at 37°C for 7 days is less than 35%. The self-assembling peptide has a charge of +1 to +4 at physiological pH. The self-assembling peptide comprises at least one selected from the group consisting of a self-assembling peptide comprising the amino acid sequence (A) and the self-assembling peptides represented by SEQ ID NOs. 3, 4, 12, 13, and 16 to 18, Amino acid sequence (A): a1b1c1b2a2b3db4a3b5c2b6a4 In the amino acid sequence, a1 to a4 are basic amino acid residues; b1 to b6 are uncharged polar amino acid residues and / or hydrophobic amino acid residues, wherein, At least five of b1 to b6 are hydrophobic amino acid residues; c1 and c2 are acidic amino acid residues; and d is a hydrophobic amino acid residue or an uncharged polar amino acid residue.

2. The material for preventing digestive fluid leakage according to claim 1, wherein: The viscosity measured using a rotational viscometer at a temperature of 25° C. and a rotation speed of 0.5 rpm is 3.0 Pa·s or more.

3. The material for preventing digestive fluid leakage according to claim 1, wherein: The material for preventing digestive juice from leaking out is used to prevent pancreatic juice from leaking out of the pancreas.

4. An organ protection material for protection against digestion caused by digestive juice, comprising a self-assembling peptide gel containing a self-assembling peptide and water, The residual rate of collagen sheet adhesion is at least 40%. The decomposition rate based on pancreatic juice treatment at 37°C for 7 days is less than 35%. The self-assembling peptide has a charge of +1 to +4 at physiological pH. The self-assembling peptide comprises at least one selected from the group consisting of a self-assembling peptide comprising the amino acid sequence (A) and the self-assembling peptides represented by SEQ ID NOs. 3, 4, 12, 13, and 16 to 18, Amino acid sequence (A): a1b1c1b2a2b3db4a3b5c2b6a4 In the amino acid sequence, a1 to a4 are basic amino acid residues; b1 to b6 are uncharged polar amino acid residues and / or hydrophobic amino acid residues, wherein, At least five of b1 to b6 are hydrophobic amino acid residues; c1 and c2 are acidic amino acid residues; and d is a hydrophobic amino acid residue or an uncharged polar amino acid residue.

5. The organ protection material according to claim 4, wherein The viscosity measured using a rotational viscometer at a temperature of 25° C. and a rotation speed of 0.5 rpm is 3.0 Pa·s or more.

6. The organ protection material according to claim 4, wherein The organ protection material is used to protect the organ from digestion caused by pancreatic juice.

Images