Exudate-absorbing material

Abstract

To provide an exudate-absorbing material exhibiting superior exudate absorbability and biocompatibility.SOLUTION: An exudate-absorbing material (α) containing a protein (A) and an organic acid (salt) (B) having a hydroxyl group, wherein the protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y'), a total number of the polypeptide chain (Y) and the polypeptide chain (Y') in the protein (A) is from 1 to 100, the polypeptide chain (Y) is a polypeptide chain in which 2 to 200 of at least one amino acid sequence (X) selected from a VPGVG sequence (1) which is an amino acid sequence shown in SEQ ID NO:1, a GVGVP sequence (4) which is an amino acid sequence shown in SEQ ID NO:4, a GPP sequence, a GAP sequence, and a GAHGPAGPK sequence (3) which is an amino acid sequence shown in SEQ ID NO:3 are continuous, the polypeptide chain (Y') is a polypeptide chain in which 5% or less of amino acids in the polypeptide chain (Y) are substituted with lysine and / or arginine, and a total number of the lysine and the arginine is from 1 to 100.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an exudate-absorbing material. [Background technology]

[0002] Known examples of exudate absorbents include starch-acrylic acid grafts (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 06-200 Summary of the Invention [Problem to be solved by the invention]

[0004] The technology of Patent Document 1 uses a pressure-sensitive adhesive that is temporarily attached, but its biocompatibility is insufficient. Furthermore, its exudate absorption is also insufficient. The object of the present invention is to provide an exudate-absorbing material that is excellent in exudate absorption and biocompatibility. [Means for solving the problem]

[0005] The present inventors have conducted extensive research to solve the above problems and have arrived at the present invention. That is, the present invention provides an exudate-absorbing material (α) containing a protein (A) and an organic acid (salt) (B) having a hydroxyl group, wherein the protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y'), the total number of the polypeptide chains (Y) and the polypeptide chains (Y') in the protein (A) is 1 to 100, the polypeptide chain (Y) is a polypeptide chain consisting of 2 to 200 consecutive amino acids (X) of at least one of the amino acid sequences (X) selected from the amino acid sequence VPGVG (1) shown in SEQ ID NO: 1, the amino acid sequence GVGVP (4) shown in SEQ ID NO: 4, the amino acid sequence GPP, the GAP, and the amino acid sequence GAHGPAGPK (3) shown in SEQ ID NO: 3, and the polypeptide chain (Y') is a polypeptide chain in which 5% or less of the amino acids in the polypeptide chain (Y) are substituted with lysine and / or arginine, and the total number of the lysine and arginine is 1 to 100. [Effects of the Invention]

[0006] The exudate-absorbing material (α) of the present invention has the following effects. (1) Excellent absorption of exudate. (2) Excellent biocompatibility. DETAILED DESCRIPTION OF THE INVENTION

[0007] <Protein (A)> The protein (A) of the present invention has a polypeptide chain (Y) and / or a polypeptide chain (Y'), the total number of the polypeptide chains (Y) and the polypeptide chains (Y') in the protein (A) is 1 to 100, The polypeptide chain (Y) is a polypeptide chain consisting of 2 to 200 consecutive amino acids of at least one amino acid sequence (X) selected from the amino acid sequence VPGVG (1) shown in SEQ ID NO: 1, the amino acid sequence GVGVP (4) shown in SEQ ID NO: 4, the amino acid sequence GPP, the GAP, and the amino acid sequence GAHGPAGPK (3) shown in SEQ ID NO: 3, and the polypeptide chain (Y') is a polypeptide chain in which 5% or less of the amino acids in the polypeptide chain (Y) are substituted with lysine and / or arginine, and the total number of the lysines and arginines is 1 to 100.

[0008] The amino acid sequence (X) constituting the polypeptide chain (Y) may be of one type or of two or more types.

[0009] As the amino acid sequence (X), the VPGVG sequence (1) and the GVGVP sequence (4) are preferred from the viewpoints of exudate absorbency and biocompatibility.

[0010] Specific examples of the polypeptide chain (Y) include (VPGVG) b Array, (GVGVP) c Sequence and (GAHGPAGPK) d In addition, b to d each represent the number of consecutive amino acid sequences (X) and are integers of 2 to 200. When one molecule of protein (A) contains a plurality of polypeptide chains (Y), the polypeptide chains (Y) may be the same or different, and are represented by (VPGVG). b Array, (GVGVP) c Sequence and (GAHGPAGPK) d The sequence may have one or more types selected from the group consisting of sequences. Furthermore, when the protein (A) contains multiple polypeptide chains (Y), the number of consecutive amino acid sequences (X) may be the same or different for each polypeptide chain (Y). That is, the protein (A) may contain multiple polypeptide chains (Y) in which the numbers b to d of consecutive amino acid sequences (X) are the same, or multiple polypeptide chains (Y) in which b to d are different. The polypeptide chain (Y) is selected from the viewpoints of exudate absorption and biocompatibility, and is preferably (VPGVG). b Sequence and (GVGVP) c The sequence is preferred.

[0011] The polypeptide chain (Y) is a polypeptide chain consisting of 2 to 200 consecutive amino acid sequences (X) (the above b to d are 2 to 200), and from the viewpoint of exudate absorbency and biocompatibility, the number of consecutive amino acid sequences (X) is preferably 2 to 100 (the above b to d are 2 to 100), more preferably 2 to 50 (the above b to d are 2 to 50), and particularly preferably 2 to 40 (the above b to d are 2 to 40).

[0012] The polypeptide chain (Y') is a polypeptide chain in which 5% or less of the amino acids in the polypeptide chain (Y) are substituted with lysine and / or arginine, and the total number of substituted lysines and arginines is 1 to 100.

[0013] Whether or not a protein (A) is a polypeptide chain (Y') is determined by whether or not the polypeptide chain (Y) results when all lysines (K) and arginines (R) in the sequence of the protein (A) are replaced with other amino acids [glycine (G), alanine (A), valine (V), proline (P), or histidine (H)].

[0014] In the polypeptide chain (Y'), the proportion of substituted lysine and / or arginine is preferably 0.06% to 5%, more preferably 0.5 to 5%, and particularly preferably 1 to 5%, from the viewpoint of exudate absorbency and biocompatibility.

[0015] The polypeptide chain (Y') may also contain an amino acid sequence (X') in which 60% or less of the amino acids in the amino acid sequence (X) are substituted with lysine and / or arginine. Furthermore, the amino acid sequence (X) and / or the amino acid sequence (X') constituting the polypeptide chain (Y') may each be of one type or two or more types.

[0016] Specific examples of the amino acid sequence (X') include the GKGVP sequence (7) shown in SEQ ID NO: 7, the GKGKP sequence (8) shown in SEQ ID NO: 8, the GKGRP sequence (9) shown in SEQ ID NO: 9, and the GRGRP sequence (10) shown in SEQ ID NO: 10. From the viewpoint of exudate absorbency and biocompatibility, the amino acid sequence (X') is preferably at least one sequence selected from the group consisting of the GKGVP sequence (7), the GKGKP sequence (8) and the GRGRP sequence (10), and more preferably the GKGVP sequence (7) and the GKGKP sequence (8). The total number of polypeptide chains (Y) and polypeptide chains (Y') in one molecule of protein (A) is 1 to 100. The total number of these chains is preferably 1 to 80, and more preferably 1 to 60.

[0017] It is preferable from the viewpoint of exudate absorbency and biocompatibility that the total number of polypeptide chains (Y) and polypeptide chains (Y') in one molecule of protein (A) is within the above range.

[0018] When the protein (A) contains polypeptide chains (Y) with different types and / or different numbers of consecutive amino acid sequences (X), each is counted as one, and the number of polypeptide chains (Y) is the sum of the total. The same applies to polypeptide chains (Y'). The protein (A) in the present invention preferably satisfies the following relational formula (1). 0.50≦[total number of amino acids constituting amino acid sequence (X) contained in protein (A) and amino acid sequence (X') contained in protein (A)] / [total number of amino acids constituting protein (A)]≦0.80 (1)

[0019] The ratio of the number of amino acids can be determined using a protein sequencer. Specifically, it can be determined by the following measurement method. <Measurement method> Protein (A) is decomposed to approximately 30 residues or less using two or more cleavage methods that can cleave at specific amino acid residues. After separation by high-performance liquid chromatography (HPLC), the amino acid sequence is read using a protein sequencer. The obtained amino acid sequence is subjected to peptide mapping to determine the entire sequence of protein (A). Then, "(the total number of amino acids that make up amino acid sequence (X) contained in protein (A) and amino acid sequence (X') contained in protein (A)) / (the total number of amino acids that make up protein (A))" is calculated.

[0020] From the viewpoint of exudate absorbency and biocompatibility, the protein (A) preferably has a polypeptide chain (S) in which 2 to 50 consecutive GAGAGS sequences (2), which are the amino acid sequence shown in SEQ ID NO: 2, are linked together. In the polypeptide chain (S), the number of consecutive GAGAGS sequences (2) is preferably 2 to 40, more preferably 2 to 30, and particularly preferably 2 to 10, from the viewpoints of exudate absorbency and biocompatibility.

[0021] In protein (A), the ratio of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A) [{number of GAGAGS sequences (2) in protein (A) × 6} / {total number of amino acids in protein (A)} × 100] is preferably 5 to 50%, more preferably 10 to 47.5%, and particularly preferably 20 to 45%, from the viewpoints of exudate absorbency and biocompatibility. The ratio of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A) can be determined using a protein sequencer. Specifically, it is determined by the following measurement method.

[0022] <The ratio of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A)> Protein (A) is decomposed to approximately 30 residues or less using two or more cleavage methods that can cleave at specific amino acid residues. After separation by high-performance liquid chromatography (HPLC), the amino acid sequence is read using a protein sequencer. The obtained amino acid sequence is subjected to peptide mapping to determine the complete sequence of protein (A). The ratio of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A) is then calculated using the following formula: The ratio (%) of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A) = [{number of GAGAGS sequences (2) × 6} / {total number of amino acids in protein (A)}] × 100

[0023] When the protein (A) has a total of two or more polypeptide chains of at least one type selected from the group consisting of polypeptide chain (Y), polypeptide chain (Y'), and polypeptide chain (S), an intervening amino acid sequence (Z) may be present between these chains. The intervening amino acid sequence (Z) is a peptide sequence in which one or more amino acids are linked, and is a peptide sequence other than the GAGAGS sequence (2), the amino acid sequence (X), and the amino acid sequence (X'). From the viewpoints of exudate absorbency and biocompatibility, the number of amino acids constituting the intervening amino acid sequence (Z) is preferably 1 to 30, more preferably 1 to 15, and particularly preferably 1 to 10. Specific examples of the intervening amino acid sequence (Z) include the VAAGY sequence (11) shown in SEQ ID NO: 11, the GAAGY sequence (12) shown in SEQ ID NO: 12, and the LGP sequence. The ratio of the number of amino acids in all intervening amino acid sequences (Z) to the total number of amino acids in protein (A) [Σ{(number of amino acids in intervening amino acid sequences (Z)) × (number of intervening amino acid sequences (Z))} / {total number of amino acids in protein (A)} × 100] is preferably 0 to 25%, more preferably 0 to 22.5%, and particularly preferably 0.01 to 15%, from the viewpoint of exudate absorbency and biocompatibility.

[0024] From the viewpoint of in vivo degradability, the protein (A) may have a terminal amino acid sequence (T) at its terminus in addition to the GAGAGS sequence (2), the amino acid sequence (X), the amino acid sequence (X'), and the intervening amino acid sequence (Z). The terminal amino acid sequence (T) may be located at one terminus or at both termini of the protein (A). The terminal amino acid sequence (T) does not include the purification tag described below. The terminal structure of the protein (A) is preferably a structure in which a terminal amino acid sequence (T) is bound to a polypeptide chain (Y). The terminal amino acid sequence (T) is a peptide sequence in which one or more amino acids are bound, and is a peptide sequence other than the GAGAGS sequence (2), the amino acid sequence (X), and the amino acid sequence (X'). From the viewpoint of in vivo degradability, the number of amino acids constituting the terminal amino acid sequence (T) is preferably 1 to 100, more preferably 1 to 50, and particularly preferably 1 to 40. Specific examples of the terminal amino acid sequence (T) include the amino acid sequence MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM (13) shown in SEQ ID NO: 13.

[0025] From the viewpoint of in vivo degradability, the ratio of the number of amino acids in the terminal amino acid sequence (T) to the total number of amino acids in the protein (A) is preferably 0 to 25%, more preferably 0 to 22.5%, and particularly preferably 0.01 to 15%.

[0026] As described below, protein (A) may be produced using bacteria by biotechnology techniques. In such cases, to facilitate the purification or detection of the expressed protein (A), the protein (A) may contain, in addition to the terminal amino acid sequence (T), a protein or peptide having a specific amino acid sequence at the N- or C-terminus (hereinafter referred to as a "purification tag"). Affinity purification tags are used as purification tags. Examples of such purification tags include the 6xHis tag consisting of polyhistidine, V5 tag, Xpress tag, AU1 tag, T7 tag, VSV-G tag, DDDDK tag, S tag, CruzTag09™, CruzTag22™, CruzTag41™, Glu-Glu tag, Ha.11 tag, and KT3 tag. Below are examples of combinations of each purification tag (i) and a ligand (ii) that recognizes and binds to that tag. (i-1) Glutathione-S-transferase (GTS) (ii-1) Glutathione (i-2) Maltose-binding protein (MBP) (ii-2) Amylose (i-3) HQ tag (ii-3) Nickel (i-4) Myc tag (ii-4) Anti-Myc antibody (i-5) HA tag (ii-5) Anti-HA antibody (i-6) FLAG tag (ii-6) Anti-FLAG antibody (i-7) 6xHis tag (ii-7) Nickel or cobalt Methods for introducing the purification tag sequence include inserting a nucleic acid encoding the purification tag at the 5' or 3' end of the nucleic acid encoding protein (A) in an expression vector, or using a commercially available vector for introducing a purification tag.

[0027] In protein (A), the ratio of the total number of amino acids in all intervening amino acid sequences (Z) constituting protein (A), the total number of amino acids in all terminal amino acid sequences (T) constituting protein (A), and the total number of amino acids in the purification tag is preferably 0 to 25%, more preferably 0 to 22.5%, and particularly preferably 0.01 to 15%, based on the total number of amino acids in protein (A), from the viewpoint of in vivo degradability.

[0028] When the protein (A) contains a polypeptide chain (Y) and / or a polypeptide chain (Y') and a polypeptide chain (S), it is preferable that the polypeptide chain (Y) or the polypeptide chain (Y') and the polypeptide chain (S) are chemically bonded alternately from the viewpoint of exudate absorbency and biocompatibility.

[0029] The ratio of the number of GAGAGS sequences (2) to the total number of amino acid sequences (X) and (X') (GAGAGS sequence (2):total of amino acid sequences (X) and (X')) is preferably 1:1.5 to 1:20, more preferably 1:1.5 to 1:6, and particularly preferably 1:2 to 1:5, from the viewpoints of exudate absorbency and biocompatibility. Some preferred examples of the protein (A) are shown below.

[0030] (A1): A protein whose amino acid sequence (X) is the GVGVP sequence (4) (A11): A protein having a polypeptide chain (Y'1) in which one amino acid in a polypeptide chain (Y1) having 2 to 200 consecutive GVGVP sequences (4) is substituted with lysine (K). (A11-1): A protein having a polypeptide chain (Y'1) and a polypeptide chain (S1) consisting of 2 to 200 consecutive GAGAGS sequences (2). (A11-2): A protein having the amino acid sequence (GVGVP)4GKGVP(GVGVP)3 sequence (6)(Y'11) shown in SEQ ID NO: 6 in which one amino acid in the polypeptide chain (Y11) of the (GVGVP)8 sequence (14) is substituted with lysine (K), which is the amino acid sequence shown in SEQ ID NO: 14 in which eight consecutive GVGVP sequences (4) are present, and a polypeptide chain (S1) containing 2 to 200 consecutive GAGAGS sequences (2). (A11-2-1): A protein having a polypeptide chain (S1-1) with the amino acid sequence (GAGAGS)4 sequence (5) shown in SEQ ID NO: 5, in which four consecutive GAGAGS sequences (2) are present, and the amino acid sequence (GVGVP)4GKGVP(GVGVP)3 sequence (6) shown in SEQ ID NO: 6. Specifically, the following proteins are included: (i) A protein (SELP8K) having a molecular mass of approximately 80 kDa and an amino acid sequence (16) shown in SEQ ID NO: 16, which has 12 (GAGAGS)4 sequences (5) and 13 (GVGVP)4GKGVP(GVGVP)3 sequences (6), which are chemically bonded alternately to a (GAGAGS)2 sequence (15) having an amino acid sequence shown in SEQ ID NO: 15. (ii) A protein (SELP8K4) having four (GAGAGS) sequences (5) and four (GVGVP)GKGVP(GVGVP) sequences (6), which are chemically bonded alternately to form an amino acid sequence (27) having a molecular mass of approximately 30 kDa and represented by SEQ ID NO: 27. (A11-2-2): A protein having a polypeptide chain (S1-2) with the (GAGAGS)2 sequence (15), which is the amino acid sequence shown in SEQ ID NO: 15, in which two consecutive GAGAGS sequences (2) are present, and a (GVGVP)4GKGVP(GVGVP)3 sequence (6). (i) A protein (SELP0K) having a molecular mass of approximately 82 kDa and an amino acid sequence (17) represented by SEQ ID NO: 17, which has 17 copies of each of the (GAGAGS)2 sequence (15) and the (GVGVP)4GKGVP(GVGVP)3 sequence (6), which are chemically bonded alternately.

[0031] (A11-3): A protein having the amino acid sequence (GVGVP)6GKGVP(GVGVP)5 sequence (18)(Y'12) shown in SEQ ID NO: 18, in which one amino acid in a polypeptide chain of 12 consecutive GVGVP sequences (4) is substituted with lysine (K), and a polypeptide chain (S1) of 2 to 200 consecutive GAGAGS sequences (2). (A11-3-1): A protein having the (GAGAGS) sequence (19), which is the amino acid sequence shown in SEQ ID NO: 19, in which four consecutive GAGAGS sequences (2) are present, and the (GVGVP) GKGVP (GVGVP) sequence (18). (i) A protein (SELP8K12) having a molecular mass of approximately 105 kDa and an amino acid sequence (20) represented by SEQ ID NO: 20, which has 12 (GAGAGS) sequences (19) and 13 (GVGVP)6GKGVP(GVGVP)5 sequences (18), which are chemically bonded alternately to a (GAGAGS)2 sequence (15).

[0032] (A2): A protein whose amino acid sequence (X) is the VPGVG sequence (1) (A21): A protein having a polypeptide chain (Y2) with 2 to 200 consecutive VPGVG sequences (1) and a GAGAGS sequence (2). (i) A protein (ELP1.1) having 40 units each of the GAGAGS sequence (2), the (VPGVG) sequence (24) which is the amino acid sequence shown in SEQ ID NO: 24, and the (VPGVG) sequence (25) which is the amino acid sequence shown in SEQ ID NO: 25, and having a molecular mass of approximately 200 kDa and a structure formed by chemically bonding 40 blocks each of which is the (VPGVG) sequence (24), the GAGAGS sequence (2), and the (VPGVG) sequence (25) in this order.

[0033] (A3): A protein having a polypeptide chain (Y1) with 2 to 200 consecutive GVGVP sequences (4) and a polypeptide chain (S1) with 2 to 200 consecutive GAGAGS sequences (2). Specifically, the following proteins are included: (i) (GAGAGS)8 sequence (21), which is the amino acid sequence shown in SEQ ID NO: 21, and (GVGVP), which is the amino acid sequence shown in SEQ ID NO: 22 40 A protein (SELP6.1) having five units of each of the sequences (22), which are chemically bonded alternately, and having a molecular mass of approximately 110 kDa and an amino acid sequence (23) shown in SEQ ID NO: 23.

[0034] Among these, the protein of sequence (16) (SELP8K), the protein of sequence (17) (SELP0K), the protein of sequence (20) (SELP8K12), the protein of sequence (23) (SELP6.1), the protein of sequence (26) (ELP1.1), or the protein of sequence (27) (SELP8K4) is preferred. Furthermore, protein (A) may be a protein having an amino acid sequence that is 70% or more homologous to the amino acid sequence of protein (SELP8K) of sequence (16), protein (SELP0K) of sequence (17), protein (SELP8K12) of sequence (20), protein (SELP6.1) of sequence (23), protein (ELP1.1) of sequence (26), or protein (SELP8K4) of sequence (27). Furthermore, this homology is preferably 80% or more, and more preferably 90% or more.

[0035] The molecular mass of protein (A) as determined by SDS-PAGE (SDS polyacrylamide gel electrophoresis) is preferably 15 to 200 kDa, more preferably 30 to 150 kDa, and particularly preferably 70 to 120 kDa, from the viewpoint of in vivo degradability.

[0036] In the present invention, protein (A) can be obtained by extraction from natural products, organic synthesis (enzymatic methods, solid-phase synthesis, liquid-phase synthesis, etc.), genetic recombination, etc. Regarding organic synthesis, methods such as those described in "Lectures on Biochemical Experiments 1, Chemistry of Proteins IV" (published July 1, 1981, edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin Co., Ltd.) and "Continued Lectures on Biochemical Experiments 2, Chemistry of Proteins (Part 2)" (published May 20, 1987, edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin Co., Ltd.) can be applied. Regarding genetic recombination, methods such as those described in Japanese Patent No. 3338441 can be applied. Although protein (A) can be obtained by extraction from natural products, organic synthesis, and genetic recombination, genetic recombination is preferred from the viewpoints of easy modification of the amino acid sequence and inexpensive mass production.

[0037] <Organic acid (salt) having a hydroxyl group (B)> In the present invention, the organic acid (salt) (B) having a hydroxyl group means an organic acid having a hydroxyl group and / or an organic acid salt having a hydroxyl group. Examples of the organic acid (salt) (B) having a hydroxyl group include ascorbic acid (salt) and citric acid (salt). Examples of the salt include sodium salt and potassium salt. Of these, from the viewpoint of exudate absorbency and biocompatibility, ascorbic acid, citric acid, and sodium ascorbate are preferred, and ascorbic acid is more preferred. The organic acid (salt) (B) having a hydroxyl group may be used alone or in combination of two or more kinds.

[0038] <Leachate absorption material (α)> The exudate-absorbing material (α) of the present invention contains a protein (A) and an organic acid (salt) having a hydroxyl group (B). The weight ratio of the organic acid (salt) having a hydroxyl group (B) to the protein (A) [weight of the organic acid (salt) having a hydroxyl group (B) / weight of the protein (A)] is preferably 0.01 to 0.25, and more preferably 0.02 to 0.1, from the viewpoints of exudate absorbency and biocompatibility.

[0039] The exudate absorbent material (α) is preferably in the form of a sponge. In this case, the density is preferably 50 to 600 mg / cm from the viewpoints of handling and exudate absorption. 3 and more preferably 75 to 450 mg / cm 3 , particularly preferably 100 to 350 mg / cm 3 is.

[0040] The exudate-absorbing material (α) can be produced, for example, by the following method. (1) A purified protein (A) and an organic acid (salt) having a hydroxyl group (B) are dissolved in deionized water to obtain an aqueous solution. (2) The aqueous solution is placed in a container, freeze-dried, and molded to obtain an exudate-absorbing material (α).

[0041] The density of the exudate-absorbing material (α) can be appropriately adjusted by adjusting the concentration of the aqueous solution containing the protein (A) and the hydroxyl-containing organic acid (salt) (B) in the freeze-drying step of the molding method, for example, by adjusting the density to within the preferred range described below.

[0042] In the freeze-drying step, the aqueous solution is preferably poured into a mold (such as a metal mold) corresponding to the desired shape, and then freeze-dried. The weight proportion of the protein (A) contained in the aqueous solution is preferably 12.5 to 150 g / L, more preferably 25 to 100 g / L, and particularly preferably 25 to 50 g / L, based on the volume of the aqueous solution.

[0043] In addition, the freeze-drying is preferably carried out in primary and secondary drying steps. The temperature during primary drying is preferably −35 to −5° C. from the viewpoint of moldability. The degree of vacuum during primary drying is preferably 0 to 20 Pa from the viewpoint of moldability. The time for primary drying is preferably 1 to 200 hours from the viewpoint of moldability. The temperature during secondary drying is preferably 5 to 35°C, more preferably 5 to 30°C, from the viewpoint of moldability. The degree of vacuum during secondary drying is preferably 0 to 20 Pa from the viewpoint of moldability. The time for secondary drying is preferably 1 to 200 hours from the viewpoint of moldability. By drying in two stages, primary drying and secondary drying, the exudate absorption and moldability are improved.

[0044] The exudate-absorbing material (α) of the present invention can be used as a dressing material for covering an external wound. For example, it may be used for primary healing and secondary healing, and is more preferably used for secondary healing. More specifically, it can be used for bedsores, ischemic ulcers, diabetic ulcers, open wounds, postoperative dehiscence, infected wounds, burns, and the like. In particular, it can be suitably used for wounds that produce a lot of exudate (for example, ischemic ulcers).

[0045] When using the exudate absorbent material (α) of the present invention, for example, the wound surface may be cleansed, and the exudate absorbent material (α) may be applied to the wound surface. It is preferable to further apply a cover dressing to the wound to fix and protect it. Such a method is also a method of the present invention for treating at least one wound selected from the group consisting of pressure ulcers, ischemic ulcers, diabetic ulcers, open wounds, post-operative dehiscence wounds, infected wounds and burns. In addition, use of the exudate-absorbing material (α) on at least one wound selected from the group consisting of bedsores, ischemic ulcers, diabetic ulcers, open wounds, postoperative dehiscence wounds, infected wounds, and burns is also use of the exudate-absorbing material of the present invention.

[0046] This specification describes the following:

[0047] The present invention (1) relates to an exudate-absorbing material (α) containing a protein (A) and an organic acid (salt) (B) having a hydroxyl group, wherein the protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y'), the total number of the polypeptide chains (Y) and the polypeptide chains (Y') in the protein (A) is 1 to 100, the polypeptide chain (Y) is a polypeptide chain consisting of 2 to 200 consecutive amino acids (X) of at least one of the amino acid sequences (X) selected from the amino acid sequence VPGVG (1) shown in SEQ ID NO: 1, the amino acid sequence GVGVP (4) shown in SEQ ID NO: 4, the amino acid sequence GPP, the amino acid sequence GAP, and the amino acid sequence GAHGPAGPK (3) shown in SEQ ID NO: 3, and the polypeptide chain (Y') is a polypeptide chain in which 5% or less of the amino acids in the polypeptide chain (Y) are substituted with lysine and / or arginine, and the total number of the lysine and arginine is 1 to 100.

[0048] The present invention (2) is an exudate absorbing material according to the present invention (1), wherein the organic acid (salt) (B) having a hydroxyl group is at least one selected from the group consisting of ascorbic acid (salt) and citric acid (salt).

[0049] The present invention (3) is an exudate absorbent material according to the present invention (1) or (2), wherein the weight ratio of the organic acid (salt) (B) having a hydroxyl group to the protein (A) [weight of the organic acid (salt) (B) having a hydroxyl group / weight of the protein (A)] is 0.01 to 0.25.

[0050] The present invention (4) is characterized in that the density of the exudate absorbent material is 50 to 600 mg / cm 3 The exudate-absorbing material according to any one of the present inventions (1) to (3) is as follows:

[0051] The present invention (5) is the exudate-absorbing material according to any one of the present inventions (1) to (4), wherein the protein (A) has a molecular mass of 15 to 200 kDa as determined by SDS-PAGE (SDS polyacrylamide gel electrophoresis).

[0052] The present invention (6) is an exudate absorbing material according to any one of the present inventions (1) to (5), wherein the protein (A) has the amino acid sequence shown in SEQ ID NO: 16, the amino acid sequence shown in SEQ ID NO: 17, the amino acid sequence shown in SEQ ID NO: 20, the amino acid sequence shown in SEQ ID NO: 23, the amino acid sequence shown in SEQ ID NO: 26, the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence having a homology of 70% or more with these amino acid sequences. [Example]

[0053] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0054] <Manufacturing Example 1> [Making SELP8K] ○ Construction of SELP8K-producing strain Plasmid pPTS0345 encoding SELP8K was prepared according to the method described in the Examples of Japanese Patent No. 4088341. The constructed plasmid was transformed into Escherichia coli to obtain a SELP8K-producing strain. Hereinafter, we will describe a method for producing SELP8K (polypeptide (A1)), a polypeptide with a molecular mass of approximately 80 kDa, which has a structure in which 12 (GAGAGS)4 sequences (6) and 13 (GVGVP)4GKGVP(GVGVP)3 sequences (7), which are a type of polypeptide (A), are chemically bonded alternately.

[0055] Cultivation of SELP8K-producing strains An overnight culture of the SELP8K-producing strain grown at 30°C was used to inoculate 50 ml of LB medium in a 250 ml flask. Kanamycin was added to the LB medium to a final concentration of 50 μg / ml to form a culture medium, and the culture medium was incubated at 30°C with stirring (200 rpm). When the turbidity of the culture medium reached OD600 = 0.8 (using a UV1700 spectrophotometer, manufactured by Shimadzu Corporation), 40 ml of the culture medium was transferred to another flask preheated to 42°C and cultured at 42°C for approximately 2 hours. The culture medium was then cooled on ice, the turbidity OD600 of the culture medium was measured, and the E. coli cells were collected by centrifugation.

[0056] Purification of SELP8K Protein was purified from the collected E. coli biomass by the following steps: Step 1: Cell lysis, Step 2: Removal of insoluble debris by centrifugation, Step 3: Ammonium sulfate precipitation, Step 4: Ultrafiltration, Step 5: Cation exchange chromatography, Step 6: Ultrafiltration, and Step 7: Lyophilization. In this manner, purified SELP8K (polypeptide (A1)) with a molecular mass of approximately 85 kDa was obtained.

[0057] Step 1: Cell lysis 200 g of deionized water was added to 100 g of collected E. coli, and the cells were lysed using a high-pressure homogenizer (55 MPa) to obtain a cell lysate containing lysed cells. The cell lysate was then adjusted to pH 4.0 with glacial acetic acid.

[0058] Step 2: Removal of insoluble debris by centrifugation The cell lysate was then centrifuged (6300 rpm, 4°C, 30 minutes) to collect the supernatant.

[0059] Step 3: Ammonium sulfate precipitation A saturated ammonium sulfate solution was added to the supernatant recovered in step 2 so that the ammonium sulfate concentration was 25% by weight. After that, the mixture was left to stand for 8 to 12 hours, and the precipitate was recovered by centrifugation. The recovered precipitate was dissolved in deionized water. Next, a saturated ammonium sulfate solution was added to the dissolved solution so that the ammonium sulfate concentration was 25% by weight. After that, the mixture was left to stand for 8 to 12 hours, and the precipitate was recovered by centrifugation. The recovered precipitate was dissolved in deionized water to obtain a solution.

[0060] Step 4: Ultrafiltration The solution obtained in step 3 was subjected to an ultrafiltration device (holofiber: manufactured by GE Healthcare) with a molecular mass cutoff of 30,000. Ultrafiltration was performed on the solution obtained in step 3 using 20 times the amount of deionized water to obtain a polypeptide solution after ultrafiltration.

[0061] Step 5: Cation exchange chromatography The ultrafiltrated polypeptide solution was added to 10 mM sodium acetate buffer to a polypeptide concentration of 20 g / L, and then subjected to an AKTAPrime (Amersham) equipped with a cation exchange column HiPrepSP XL16 / 10 (GE Healthcare). 500 mM sodium acetate buffer was used as the eluent, and the eluted fraction was collected.

[0062] Step 6: Ultrafiltration The eluted fraction obtained in step 5 was treated in the same manner as in "4: Ultrafiltration" above to obtain a polypeptide solution after ultrafiltration.

[0063] Step 7: Freeze drying The polypeptide solution obtained in step 6 was diluted with deionized water to a polypeptide concentration of 3 g / L, and placed in a stainless steel tray so that the water level was 10 mm or less. The solution was then placed in a freeze dryer (Nihon Techno Service Co., Ltd.) and frozen at -30°C for 24 hours. After freezing, the solution was subjected to primary drying at a vacuum of 5 Pa or less at -30°C for 110 hours, and secondary drying at a vacuum of 5 Pa or less at 30°C for 48 hours to obtain SELP8K (protein (A-1)). Protein (A) was identified using the Western blotting method described below. Furthermore, for protein (A-1), the ratio of "(total number of amino acids constituting amino acid sequence (X) contained in protein (A) and amino acid sequence (X') contained in protein (A)) / (total number of amino acids constituting protein (A))" is 0.54. The ratio of the number of GAGAGS sequences (2) to the total number of amino acid sequences (X) and (X') in one molecule of protein (A-1) (GAGAGS sequences (2) : total of amino acid sequences (X) and (X')) is 1:2.

[0064] Identification of SELP8K(A-1) Analysis was performed by Western blotting using rabbit anti-SELP8K antibody and rabbit anti-6xHis antibody (Roland) against the 6xHis tag in the C-terminal sequence. The Western blotting procedure was as follows. A band showing antibody reactivity with each antibody was observed at the apparent molecular mass of 80 kDa. Table 1 also shows the amino acid composition ratios (measured values) of protein (A) obtained by amino acid composition analysis using an amino acid analysis system (Prominence, Shimadzu Corporation) and the amino acid composition ratios (theoretical values) of SELP8K estimated from the synthetic gene sequence. From these, it was confirmed that protein (A) is a protein (SELP8K) having 13 polypeptide chains (Y'11) of the (GVGVP)4GKGVP(GVGVP)3 sequence (6), in which one of the valines (V) in a polypeptide chain (Y) consisting of eight consecutive GVGVP sequences (4) is replaced with a lysine (K), and 12 polypeptide chains (S1-1) of the (GAGAGS)4 sequence (5), in which four consecutive GAGAGS sequences (2) are chemically bonded alternately to form sequence (16), in which the (GAGAGS)2 sequence (15), which is the amino acid sequence shown in SEQ ID NO: 15, is chemically bonded.

[0065] [Table 1]

[0066] <Western blotting> To 20 μL of the Western blot sample, 10 μL of 3×SDS treatment buffer [containing 150 mM Tris HCl (pH 6.8), 300 mM dithiothreitol, 6% by weight sodium dodecyl sulfate (SDS), 0.3% by weight bromophenol blue, and 30% by weight glycerol] was added, and the mixture was heated at 95°C for 5 minutes to prepare the electrophoresis sample. SDS-PAGE was performed using 15 μL of this electrophoresis sample. The electrophoresis gel was transferred to a polyvinylidene fluoride membrane (hereinafter referred to as "membrane"), which was then immersed in blocking buffer [containing 20 mM Tris (pH 7.6), 137 mM NaCl, 0.1 wt% Tween 20, and 5 wt% skim milk] and shaken at room temperature for 1 hour to block the membrane. After blocking, the membrane was washed with TBS-T [containing 20 mM Tris (pH 7.6), 137 mM NaCl, and 0.1 wt% Tween 20] for 2 minutes. Next, the membrane was immersed in a primary antibody solution (primary antibodies: anti-SELP8K antibody and anti-His-tag antibody (Rockland) diluted 1:500 with TBS-T) and left to stand overnight at 4°C to allow antibody reaction. After the reaction, the membrane was washed four times with TBS-T for 5 minutes each, and then immersed in a solution of a secondary antibody capable of binding to the primary antibody and conjugated with horseradish peroxidase as a labeling enzyme (secondary antibody: ECL anti-rabbit IgG HRP linked F(ab')2 fragment (GE Healthcare) diluted 1:2000 with TBS-T), and left to stand at room temperature for 30 minutes to allow the antibody reaction to occur. After the reaction, the membrane was washed four times with TBS-T for 5 minutes each, and then subjected to an enzymatic reaction using an ECL-Advance Western Blotting Detection Kit (GE Healthcare). The membrane was exposed to light using a Luminometer ForECL (GE Healthcare), and the bands were observed using a chemiluminescence fluorescence imaging device.

[0067] Example 1 12.5 mg of "SELP8K" (protein (A)) and 1.0 mg of ascorbic acid (B-1) were diluted with deionized water to obtain 1 mL of solution (protein (A) concentration: 12.5 mg / mL, weight ratio (B) / (A) = 0.008). 0.5 mL of this solution was poured into a cylindrical mold with a diameter of 9.85 mm and a depth of 17.5 mm. The material was then placed in a freeze dryer (manufactured by Nippon Techno Service Co., Ltd.) and frozen at -40°C for 16 hours. After freezing, the material was subjected to primary drying at a vacuum of 8 Pa or less at -20°C for 90 hours, and secondary drying at a vacuum of 8 Pa or less at 20°C for 24 hours to obtain a cylindrical sponge-like exudate-absorbing material (α-1). The obtained exudate absorbent material was evaluated as described below.

[0068] <Examples 2 to 14 and Comparative Examples 1 to 3> In Example 1, the exudate absorbing materials (α-2) to (α-14) of Examples 2 to 14 and the exudate absorbing materials (rα-1) to (rα-2) of Comparative Examples 1 and 2 were obtained in the same manner as in Example 1, except that the protein (A), the weight of (A), (B), and the weight ratio (B) / (A) were determined according to Table 2. In Comparative Example 3, a commercially available starch-acrylic acid graft was used.

[0069] Example 15 In Production Example 1, except that "plasmid pPT0364 encoding SELP0K" was used instead of "plasmid pPT0345 encoding SELP8K," a protein (SELP0K) having the amino acid sequence (17) shown in SEQ ID NO: 17, which has a molecular mass of approximately 82 kDa and has a structure in which 17 copies of each of the (GAGAGS)2 sequence (15) and the (GVGVP)4GKGVP(GVGVP)3 sequence (6) are chemically bonded alternately, was obtained in the same manner as in Production Example 1. Next, the exudate absorbent material (α-15) of Example 15 was obtained in the same manner as in Example 1, except that the protein (A), the weight of (A), (B), and the weight ratio (B) / (A) in Example 1 were determined according to Table 2.

[0070] Example 16 In Production Example 1, except that "plasmid pPT0345-4 encoding SELP8K4" was used instead of "plasmid pPT0345 encoding SELP8K," the same procedure was followed as in Production Example 1 to obtain a protein (SELP8K4) having the amino acid sequence shown in SEQ ID NO: 27, which has four copies each of the (GAGAGS)4 sequence (5) and the (GVGVP)4GKGVP(GVGVP)3 sequence (6), which are chemically bonded alternately, and has a molecular mass of approximately 30 kDa. Next, the exudate absorbent material (α-16) of Example 16 was obtained in the same manner as in Example 1, except that the protein (A), the weight of (A), (B), and the weight ratio (B) / (A) in Example 1 were determined according to Table 2.

[0071] Example 17 In Production Example 1, except that "plasmid pPT0345-12 encoding SELP8K12" was used instead of "plasmid pPT0345 encoding SELP8K," the same procedure as in Production Example 1 was used to obtain a protein (SELP8K12) having the amino acid sequence (20) shown in SEQ ID NO: 20, which has a molecular mass of approximately 105 kDa and is composed of 12 (GAGAGS)4 sequences (19) and 13 (GVGVP)6GKGVP(GVGVP)5 sequences (18), which are chemically bonded alternately to a (GAGAGS)2 sequence (15). Next, the exudate absorbing material (α-17) of Example 17 was obtained in the same manner as in Example 1, except that the protein (A), the weight of (A), (B), and the weight ratio (B) / (A) in Example 1 were determined according to Table 2.

[0072] Example 18 In Production Example 1, except for using "plasmid pPT0102-1 encoding ELP1.1" instead of "plasmid pPT0345 encoding SELP8K," the same procedure was followed as in Production Example 1 to obtain a protein (ELP1.1) having a molecular mass of approximately 200 kDa and the amino acid sequence (26) shown in SEQ ID NO: 26, which has 40 units each of the GAGAGS sequence (2), the (VPGVG) sequence (24) which is the amino acid sequence shown in SEQ ID NO: 24, and the (VPGVG) sequence (25) which is the amino acid sequence shown in SEQ ID NO: 25, and which has a structure formed by chemically bonding 40 blocks in the order of (VPGVG) sequence (24), GAGAGS sequence (2), and (VPGVG) sequence (25). Next, the exudate absorbing material (α-18) of Example 18 was obtained in the same manner as in Example 1, except that the protein (A), the weight of (A), (B), and the weight ratio (B) / (A) in Example 1 were determined according to Table 2.

[0073] Example 19 The amino acid sequence (GAGAGS)8 shown in SEQ ID NO: 21 and the amino acid sequence (GVGVP) shown in SEQ ID NO: 22 were synthesized in the same manner as in Production Example 1, except that "plasmid pPT0270-1 encoding SELP6.1" was used instead of "plasmid pPT0345 encoding SELP8K." 40 A protein (SELP6.1) having the amino acid sequence (23) shown in SEQ ID NO: 23 and having a molecular mass of approximately 110 kDa, was obtained, which has five units of sequence (22) each, which are chemically bonded alternately. Next, the exudate absorbing material (α-19) of Example 9 was obtained in the same manner as in Example 1, except that the protein (A), the weight of (A), (B), and the weight ratio (B) / (A) in Example 1 were determined according to Table 2.

[0074] (1) Density (unit: mg / cm 3 ) The diameter and height of each sponge-like exudate absorbent material were measured using a dial gauge, and the volume was then measured. The weight was also measured using a balance, and the density (mg / cm) was calculated from the volume and weight. 3 ) was calculated.

[0075] (2) Absorption rate of exudate (unit: seconds) The test was carried out in accordance with JIS L 1907. 10 μL of saline was dropped onto the exudate absorbent material from 10 mm above the material using a micropipette. The time from when the water droplet reached the test piece until the droplet was completely absorbed by the test piece was calculated by analyzing the video. The average of five measurements is shown.

[0076] (3) Biocompatibility Each exudate absorbent material was applied directly to the skin of a mouse and the mouse was kept for one week. After that, the exudate absorbent material was peeled off, and the mouse's skin was observed and evaluated according to the following <evaluation criteria>. <Evaluation criteria> ○○: No inflammation at all ○: Almost no inflammation ×: Inflammation

[0077] [Table 2]

[0078] The results in Table 2 show that the exudate-absorbing material (α) of the present invention is superior to the comparative material in terms of exudate absorption and also in terms of biocompatibility. [Industrial Applicability]

[0079] The exudate-absorbing material (α) of the present invention has excellent exudate absorbency and also has excellent biocompatibility, and therefore can be used in a variety of applications, including daily pharmaceutical applications and medical applications.

Claims

1. An exudate-absorbing material (α) containing a protein (A) and an organic acid (salt) having a hydroxyl group (B), the protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y'), the total number of the polypeptide chains (Y) and the polypeptide chains (Y') in the protein (A) is 1 to 100; the polypeptide chain (Y) is a polypeptide chain having 2 to 200 consecutive amino acids of at least one amino acid sequence (X) selected from the VPGVG sequence (1) shown in SEQ ID NO: 1, the GVGVP sequence (4) shown in SEQ ID NO: 4, the GPP sequence, the GAP sequence, and the GAHGPAGPK sequence (3) shown in SEQ ID NO: 3; The exudate-absorbing material is a polypeptide chain in which 5% or less of the amino acids in the polypeptide chain (Y) are substituted with lysine and / or arginine, and the total number of the lysines and arginines is 1 to 100.

2. 2. The exudate absorbent material according to claim 1, wherein the organic acid (salt) (B) having a hydroxyl group is at least one selected from the group consisting of ascorbic acid (salt) and citric acid (salt).

3. The exudate absorbent material according to claim 1, wherein the weight ratio of the organic acid (salt) (B) having a hydroxyl group to the protein (A) [weight of the organic acid (salt) (B) having a hydroxyl group / weight of the protein (A)] is 0.01 to 0.

25.

4. The density of the exudate absorbent material is 50 to 600 mg / cm ３ 2. The exudate-absorbing material according to claim 1, wherein

5. 2. The exudate absorbent material according to claim 1, wherein the protein (A) has a molecular mass of 15 to 200 kDa as determined by SDS-PAGE (SDS polyacrylamide gel electrophoresis).

6. The exudate absorbing material described in claim 1, wherein the protein (A) has an amino acid sequence shown in SEQ ID NO: 16, an amino acid sequence shown in SEQ ID NO: 17, an amino acid sequence shown in SEQ ID NO: 20, an amino acid sequence shown in SEQ ID NO: 23, an amino acid sequence shown in SEQ ID NO: 26, an amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence having a homology of 70% or more with these amino acid sequences.

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