Bronchial embolization material

Abstract

A problem addressed by the present invention is to provide a bronchial embolization material capable of tissue repair and replacement. This bronchial embolization material contains a protein (A), 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 1-100; in the polypeptide chain (Y) are a contiguous 2-200 of at least one amino acid sequence (X) among a VPGVG sequence (1), which is an amino acid sequence represented by SEQ ID NO: 1, a GVGVP sequence (4), which is an amino acid sequence represented by SEQ ID NO: 4, a GPP sequence, a GAP sequence, and a GAHGPAGPK sequence (3), which is an amino acid sequence represented by SEQ ID NO: 3; in the polypeptide chain (Y'), no more than 5% of the amino acids in the polypeptide chain (Y) have been substituted by lysine and / or arginine; the total number of lysines and the arginines is 1-100; the protein (A) satisfies the relational expression (1) below; the density of the bronchial embolization material is 90-450 mg / cm3; and the bronchial embolization material satisfies relational expression (2) below.　(1) 0.50 ≦ (total number of amino acids constituting the amino acid sequence (X) contained in the protein (A) and amino acids constituting an amino acid sequence (X') contained in the protein (A)) / (total number of amino acids constituting the protein (A)) ≦ 0.80 (where the amino acid sequence (X') is an amino acid sequence in which no more than 60% of the amino acids in the amino acid sequence (X) have been substituted by lysine and / or arginine)　(2) 0.01≦Q / P (where: P is the weight of a dry product (DP) obtained by drying the bronchial embolization material for three hours at 100°C at 1 atm, then drying said material for 15 hours at 40°C at 1 atm; and Q is the weight of the product (DQ) obtained by soaking the dry product (DP) for 72 hours at 25°C at 1 atm in water having 1,000-fold the weight of P, removing the soaked product, drying said product for three hours at 100°C at 1 atm, and then drying said product for 15 hours at 40°C at 1 atm.)

Description

[Technical field]

[0001] The present invention relates to a bronchial embolization material. [Background technology]

[0002] Conventionally, a method for blocking the bronchi using a bronchial embolization material to prevent air leak from the bronchi or the stump of the lung due to lung resection has been known (Patent Document 1). However, the material described in Patent Document 1 is made of silicone, so tissue repair and replacement are not achieved and the silicone remains in the affected area, causing various problems (infection, delayed healing, need for removal, etc.). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2004-024864 A Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a bronchial embolization material capable of repairing and replacing tissue. [Means for solving the problem]

[0005] The present inventors conducted extensive research to solve the above problems and arrived at the present invention. That is, the present invention provides a bronchial embolization material containing a protein (A), the protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y'), the total number of the polypeptide chains (Y) and (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 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; 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, The protein (A) satisfies the following relational formula (1): The density of the bronchial embolization material is 90 to 450 mg / cm 3 and The above-mentioned bronchial embolization material satisfies the following relational formula (2). 0.50≦[total number of amino acids constituting the amino acid sequence (X) contained in the protein (A) and amino acids constituting the amino acid sequence (X′) contained in the protein (A)] / [total number of amino acids constituting the protein (A)]≦0.80 (1) [In the relational formula (1), the amino acid sequence (X') is an amino acid sequence in which 60% or less of the amino acids in the amino acid sequence (X) are substituted with lysine and / or arginine.] 0.01≦Q / P (2) [In the relational formula (2), P is the weight of the dried product (DP) obtained by drying the bronchial embolization material at 100°C under 1 atmosphere for 3 hours, and then drying at 40°C under 1 atmosphere for 15 hours; Q is the weight of the dried product (DP) obtained by immersing the dried product (DP) in water having a weight 1,000 times that of P at 25°C under 1 atmosphere for 72 hours, removing the dried product, drying it at 100°C under 1 atmosphere for 3 hours, and then drying it at 40°C under 1 atmosphere for 15 hours (DQ).] Effect of the Invention

[0006] The bronchial embolization material of the present invention can be used as an embolization material against air leaks from the stumps of the bronchi or lungs caused by lung resection, and has the effect of promoting tissue repair and replacement. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] The bronchiectomy material of the present invention is a bronchiectomy material containing protein (A). The protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y'), the total number of the polypeptide chains (Y) and (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 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; 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, The protein (A) satisfies the following relational formula (1). 0.50≦[total number of amino acids constituting the amino acid sequence (X) contained in the protein (A) and amino acids constituting the amino acid sequence (X′) contained in the protein (A)] / [total number of amino acids constituting the protein (A)]≦0.80 (1) The amino acid sequence (X') in the relational formula (1) is an amino acid sequence in which 60% or less of the amino acids in the amino acid sequence (X) are substituted with lysine and / or arginine.

[0008] The bronchial embolization material of the present invention contains a protein (A). The protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y'). Therefore, the bronchial embolization material of the present invention has excellent biocompatibility and appropriate elasticity capable of responding to the expansion and contraction of the lungs.

[0009] The type of amino acid sequence (X) constituting the polypeptide chain (Y) may be one type, or two or more types.

[0010] From the viewpoints of biocompatibility, elasticity, and regenerative ability of bronchial and pulmonary tissues of the bronchial embolization material, the amino acid sequence (X) is preferably the VPGVG sequence (1) or the GVGVP sequence (4).

[0011] The polypeptide chain (Y) is specifically (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 from 2 to 200. When a single 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 the formula (VPGVG). b Array, (GVGVP) c Sequence and (GAHGPAGPK) d The sequence may include one or more of the sequences selected from the group consisting of sequences. Furthermore, when the protein (A) has 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 have multiple polypeptide chains (Y) with the same number of consecutive amino acid sequences (X), b to d, or may have multiple polypeptide chains (Y) with different numbers of consecutive amino acid sequences (X), b to d. From the viewpoint of biocompatibility and elasticity of the bronchial embolization material, the polypeptide chain (Y) is (VPGVG). b Sequence and (GVGVP) c The sequence is preferred.

[0012] The polypeptide chain (Y) is a polypeptide chain having 2 to 200 consecutive amino acid sequences (X) (the above b to d are 2 to 200). From the viewpoint of the biocompatibility and elasticity of the bronchial embolism material, 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).

[0013] 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.

[0014] Whether or not a protein is a polypeptide chain (Y') is determined by whether or not the polypeptide chain (Y) is formed 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)].

[0015] 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 the biocompatibility of the bronchial embolism material.

[0016] 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 type of amino acid sequence (X) and / or amino acid sequence (X') constituting the polypeptide chain (Y') may be one type or two or more types.

[0017] 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 the biocompatibility of the bronchial embolism material, 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).

[0018] 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 is preferably 1 to 80, and more preferably 1 to 60. It is preferable from the viewpoints of biocompatibility and elasticity of the bronchiectomy material that the total number of polypeptide chains (Y) and polypeptide chains (Y') in one molecule of protein (A) is within the above range.

[0019] In addition, when the protein (A) has 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 them. The same applies to the polypeptide chains (Y').

[0020] As described above, the protein (A) in the present invention must satisfy the relational formula (1). 0.50 ≦ [total number of amino acids constituting amino acid sequence (X) contained in protein (A) and amino acid constituting amino acid sequence (X') contained in protein (A)] / [total number of amino acids constituting protein (A)] ≦ 0.80 (1) If the ratio of the above amino acid numbers is less than 0.50, the regenerative ability of bronchial and pulmonary tissues is impaired, and if it exceeds 0.80, water solubility is impaired. From the viewpoint of improving the regenerative capacity of bronchial and pulmonary tissues, the ratio of the above amino acid numbers is preferably 0.50 to 0.70, more preferably 0.525 to 0.675, and particularly preferably 0.525 to 0.65.

[0021] The ratio of the above amino acid numbers can be determined by 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 that, it is separated by high performance liquid chromatography (HPLC) and 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 the amino acids that make up amino acid sequence (X') contained in protein (A)] / [the total number of amino acids that make up protein (A)]" is calculated.

[0022] From the viewpoint of the biocompatibility of the bronchial embolization material and the regenerative ability of bronchial and lung tissues, it is preferable that the protein (A) has a polypeptide chain (S) in which 2 to 50 consecutive GAGAGS amino acid residues (2), which are the amino acid sequence shown in SEQ ID NO: 2, are linked together. In terms of the biocompatibility of the bronchoembolization material, the number of consecutive GAGAGS sequences (2) in the polypeptide chain (S) is preferably 2 to 40, more preferably 2 to 30, and particularly preferably 2 to 10.

[0023] 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 viewpoint of improving the regenerative ability of bronchial and lung tissues. The ratio of the number of amino acids in all of the GAGAGS sequences (2) to the total number of amino acids in the protein (A) can be determined using a protein sequencer. Specifically, it is determined by the following measurement method.

[0024] <Proportion 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 that, it is separated by high performance liquid chromatography (HPLC) and the amino acid sequence is read using a protein sequencer. From the obtained amino acid sequence, peptide mapping is performed to determine the entire sequence of protein (A). Then, the ratio of the number of amino acids in all GAGAGS sequences (2) to the total number of amino acids in protein (A) is calculated using the measurement formula described below. 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

[0025] When the protein (A) has at least one polypeptide chain selected from the group consisting of the polypeptide chain (Y), the polypeptide chain (Y') and the polypeptide chain (S), the protein (A) may have an intervening amino acid sequence (Z) between them. The intervening amino acid sequence (Z) is a peptide sequence in which one or more amino acids are bound, and is not the GAGAGS sequence (2), the amino acid sequence (X) or the amino acid sequence (X'). From the viewpoint of the biocompatibility and elasticity of the bronchial embolism material, 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) which is the amino acid sequence shown in SEQ ID NO: 11, the GAAGY sequence (12) which is the amino acid sequence 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 viewpoints of the bioaffinity and elasticity of the bronchial embolism material.

[0026] 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 present 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 not the GAGAGS sequence (2), the amino acid sequence (X), or 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 sequence (13) shown in SEQ ID NO: 13.

[0027] 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%.

[0028] As described below, the protein (A) may be produced using bacteria by biotechnological techniques. In such cases, in order to facilitate purification or detection of the expressed protein (A), the protein (A) may have a protein or peptide having a special amino acid sequence at the N- or C-terminus (hereinafter, these are referred to as "purification tags") in addition to the terminal amino acid sequence (T). As the purification tag, a tag for affinity purification is used. Examples of such purification tags include a 6xHis tag consisting of polyhistidine, a V5 tag, an Xpress tag, an AU1 tag, a T7 tag, a VSV-G tag, a DDDDK tag, an S tag, a CruzTag09TM, a CruzTag22TM, a CruzTag41TM, a Glu-Glu tag, a Ha.11 tag, and a KT3 tag. Below, examples of combinations of each purification tag (i) and a ligand (ii) that recognizes and binds to that tag are shown. (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) 6×His tag (ii-7) Nickel or cobalt Methods for introducing the purification tag sequence include a method for inserting a nucleic acid encoding a purification tag into the 5' or 3' end of a nucleic acid encoding protein (A) in an expression vector, and a method for using a commercially available vector for introducing a purification tag.

[0029] 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, from the viewpoint of in vivo degradability, 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).

[0030] 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 alternately chemically bonded to each other, from the viewpoint of the biocompatibility and elasticity of the bronchial embolism material.

[0031] The ratio of the number of GAGAGS sequences (2) to the total number of amino acid sequences (X) and amino acid sequences (X') (GAGAGS sequence (2):total of amino acid sequences (X) and amino acid sequences (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 viewpoint of improving the regenerative ability of bronchial and pulmonary tissues.

[0032] Some preferred examples of the protein (A) are given below. (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 replaced with lysine (K). (A11-1): A protein having a polypeptide chain (Y'1) and a polypeptide chain (S1) having 2 to 200 consecutive GAGAGS sequences (2). (A11-2): A protein having an amino acid sequence (GVGVP)4GKGVP(GVGVP)3 sequence (6) (Y'11) shown in SEQ ID NO: 6 in which one amino acid in a polypeptide chain (Y11) of the (GVGVP)8 sequence (14) which is an amino acid sequence shown in SEQ ID NO: 14 in which 8 consecutive GVGVP sequences (4) are present is replaced with lysine (K), and a polypeptide chain (S1) having 2 to 200 consecutive GAGAGS sequences (2). (A11-2-1): A protein having a polypeptide chain (S1-1) having a (GAGAGS)4 sequence (5) which is an amino acid sequence shown in SEQ ID NO: 5 in which the GAGAGS sequence (2) is four consecutive amino acids, and a (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 about 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 each of the (GAGAGS)4 sequence (5) and the (GVGVP)4GKGVP(GVGVP)3 sequence (6), which are chemically bonded alternately to form an amino acid sequence (27) shown in SEQ ID NO: 27, with a molecular mass of approximately 30 kDa. (A11-2-2): A protein having a polypeptide chain (S1-2) that is 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 about 82 kDa and an amino acid sequence (17) shown in SEQ ID NO: 17, which has 17 units each of the (GAGAGS)2 sequence (15) and the (GVGVP)4GKGVP(GVGVP)3 sequence (6), which are chemically bonded alternately.

[0033] (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 replaced with lysine (K), and a polypeptide chain (S1) of 2 to 200 consecutive GAGAGS sequences (2). (A11-3-1): A protein having the (GAGAGS)4 sequence (19), which is the amino acid sequence shown in SEQ ID NO: 19, in which the GAGAGS sequence (2) is four consecutive amino acids, and the (GVGVP)6GKGVP(GVGVP)5 sequence (18). (i) A protein (SELP8K12) having a molecular mass of about 105 kDa and an amino acid sequence (20) shown in SEQ ID NO: 20, which has 12 (GAGAGS)4 sequences (19) and 13 (GVGVP)6GKGVP(GVGVP)5 sequences (18), which are chemically bonded alternately to a (GAGAGS)2 sequence (15).

[0034] (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 each of the GAGAGS sequence (2), the (VPGVG)4 sequence (24) which is the amino acid sequence shown in SEQ ID NO: 24, and the (VPGVG)8 sequence (25) which is the amino acid sequence shown in SEQ ID NO: 25, and having a structure in which 40 blocks each of the (VPGVG)4 sequence (24), the GAGAGS sequence (2), and the (VPGVG)8 sequence (25) are chemically bonded in this order, and having a molecular mass of about 200 kDa and an amino acid sequence shown in SEQ ID NO: 26.

[0035] (A3): A protein having a polypeptide chain (Y1) containing 2 to 200 consecutive GVGVP sequences (4) and a polypeptide chain (S1) containing 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 an amino acid sequence (23) shown in SEQ ID NO: 23, which has a molecular mass of about 110 kDa and has a structure in which five units of the sequence (22) are chemically bonded alternately.

[0036] 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) are preferred. Protein (A) may also be a protein having an amino acid sequence that is 70% or more identical 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). Moreover, this homology is preferably 80% or more, and more preferably 90% or more.

[0037] From the viewpoint of degradability in vivo, 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.

[0038] In the present invention, the hydrophobicity of the protein (A) is preferably 0.2 to 1.2, more preferably 0.4 to 1.0, and particularly preferably 0.42 to 0.80, from the viewpoint of improving the regenerative ability of bronchial and pulmonary tissues. The hydrophobicity of protein (A) indicates the degree of hydrophobicity of the protein (A) molecule, and can be calculated by applying the number of each amino acid residue constituting the protein (A) molecule (Mα), the hydrophobicity of each amino acid (Nα), and the total number of amino acid residues in one protein (A) molecule (MT) to the following formula: Note that the hydrophobicity of each amino acid is calculated using the following values ​​described in a non-patent document (Albert L. Lehninger, David L. Nelson, Lehninger's New Biochemistry, Vol. 1, Hirokawa Shoten, September 2010, pp. 346-347). Hydrophobicity = Σ(Mα × Nα) / (MT) Mα: The number of each amino acid residue in one molecule of the artificial protein (A) Nα: Hydrophobicity of each amino acid MT: Total number of amino acid residues in one molecule of artificial protein (A) A (Alanine): 1.8 R(arginine):-4.5 N (asparagine): -3.5 D(Aspartic acid):-3.5 C (cysteine): 2.5 Q(Glutamine):-3.5 E (glutamic acid): -3.5 G (Glycine): -0.4 H(histidine):-3.2 I (Isoleucine): 4.5 L (Leucine): 3.8 K(lysine):-3.9 M (Methionine): 1.9 F (phenylalanine): 2.8 P(proline):-1.6 S(Serine):-0.8 T (Threonine): -0.7 W (tryptophan): -0.9 Y (tyrosine): -1.3 V(Valine): 4.2 For example, when the artificial protein (A) has the sequence (GVGVP)4GKGVP(GVGVP)3 (6), the hydrophobicity of the artificial protein (A) = {16 (number of G) x (-0.4) + 15 (number of V) x 4.2 + 8 (number of P) x (-1.6) + 1 (number of K) x (-3.9)} / 40 (total number of amino acid residues) = 1.0.

[0039] Since the bronchi embolization material of the present invention contains the protein (A) having the above-mentioned amino acid sequence, it is degraded by enzymes in the living body and therefore has excellent biodegradability.

[0040] In the present invention, the protein (A) can be obtained by extraction from natural products, organic synthesis (enzymatic method, solid-phase synthesis, liquid-phase synthesis, etc.), and gene recombination. Regarding the organic synthesis, the method described in "Biochemical Experiment Lecture 1, Chemistry of Proteins IV (July 1, 1981, edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin Co., Ltd.)" and the method described in "Continued Biochemical Experiment Lecture 2, Chemistry of Proteins (Part 2) (May 20, 1987, edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin Co., Ltd.)" can be applied. Regarding the gene recombination, the method described in Japanese Patent No. 3338441 can be applied. Although the protein (A) can be obtained by both extraction from natural products, organic synthesis, and gene recombination, the gene recombination method is preferred from the viewpoints of easily changing the amino acid sequence and inexpensive mass production.

[0041] The density of the bronchial embolization material of the present invention is 90 to 450 mg / cm 3 The bronchial embolization material of the present invention is preferably in the form of a sponge. The density of the bronchial embolization material is 90mg / cm 3 If the dose is less than 450 mg / cm, the bronchial embolization effect will worsen. 3 Above this level, bronchial and pulmonary tissue repair is impaired. From the viewpoint of the bronchi embolization effect and tissue repair of the bronchi and lungs, the density of the bronchi embolization material is 90 to 300 mg / cm 3 is preferably 95 to 250 mg / cm 3More preferably, the density is 100 to 225 mg / cm. 3 It is particularly preferred that: In order to adjust the density to the above range, a method may be used in which the concentration of an aqueous solution containing protein (A) is adjusted to a preferred range described later in the freeze-drying step of the molding method for the bronchial embolization material described later in detail.

[0042] The bronchial embolization material of the present invention must satisfy the following relational formula (2). 0.01≦Q / P (2) In the relational formula (2), P is the weight of the dried product (DP) obtained by drying the bronchial embolization material at 100° C. under 1 atmospheric pressure for 3 hours and then at 40° C. under 1 atmospheric pressure for 15 hours. In addition, in the relational formula (2), Q is the weight of the dried product (DQ) obtained by immersing the dried product (DP) in water having a weight 1,000 times that of P at 25°C at 1 atmosphere for 72 hours, removing it, drying it at 100°C at 1 atmosphere for 3 hours, and then drying it at 40°C at 1 atmosphere for 15 hours.

[0043] If Q / P is less than 0.01, the tissue repair effect in the bronchi and lungs is deteriorated. Moreover, from the viewpoint of the bronchoembolization effect and tissue repair of the bronchi and lungs, Q / P is preferably 0.01 to 0.90. In order to set the Q / P ratio within the above range, it is preferable to carry out an organic solvent treatment step in the molding method for the bronchial embolization material, which will be described in detail later. Furthermore, by adjusting the hydrophobicity of the protein (A) to the preferred value, Q / P can be adjusted to fall within the above range.

[0044] It is preferable that the bronchial embolization material of the present invention further satisfies the following relational formula (3). 0.3≦R / P (3) In the relational formula (3), P is the weight of the dried product (DP) obtained by drying the bronchial embolization material at 100° C. under 1 atmospheric pressure for 3 hours and then at 40° C. under 1 atmospheric pressure for 15 hours. In addition, in the relational formula (3), R is the weight of the dried product (DR) obtained by immersing the dried product (DP) in water having a weight 1,000 times that of P at 25°C at 1 atmosphere for 2 hours, removing it, drying it at 100°C at 1 atmosphere for 3 hours, and then drying it at 40°C at 1 atmosphere for 15 hours.

[0045] When R / P is within the above range, the tissue repair effect on the bronchi and lungs is further improved. Furthermore, from the viewpoint of the bronchoembolization effect and the repair of bronchial and pulmonary tissues, R / P is more preferably 0.3 to 1.00, particularly preferably 0.95 to 1.00, particularly preferably 0.96 to 1.00, and most preferably 1.00. In order to set the R / P ratio within the above range, it is preferable to carry out an organic solvent treatment step in the molding method for the bronchial embolization material, which will be described later in detail.

[0046] The bronchiectomy material of the present invention is preferably produced by molding the protein (A) by the following method. A preferred molding method includes a method in which an aqueous solution containing the protein (A) is freeze-dried, followed by treatment with an organic solvent (sometimes abbreviated as "organic solvent treatment"). The freeze-drying step and the organic solvent treatment step will be described below.

[0047] In the freeze-drying step, an aqueous solution containing the protein (A) is preferably poured into a mold (such as a metal mold) corresponding to the desired shape, and then freeze-dried. From the viewpoint of adjusting the density of the bronchoembolization material within the above range, the weight proportion of the polypeptide (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.

[0048] In addition, the freeze-drying is preferably carried out in a primary drying and secondary drying manner. The temperature during the primary drying is preferably −35 to −5° C. from the viewpoint of moldability. The degree of vacuum during the primary drying is preferably 0 to 20 Pa from the viewpoint of moldability. From the viewpoint of moldability, the time for the primary drying is preferably 10 to 100 hours. The temperature during secondary drying is preferably from 5 to 35°C, and more preferably from 5 to 30°C, from the viewpoint of moldability. The degree of vacuum during the secondary drying is preferably 0 to 20 Pa from the viewpoint of moldability. From the viewpoint of moldability, the secondary drying time is preferably 10 to 100 hours.

[0049] In the organic solvent treatment step, the dried product obtained in the freeze-drying step is preferably immersed in an organic solvent (preferably at 1 to 40°C for 1 to 168 hours) and then the organic solvent is removed by heating (preferably under conditions of 5 to 30°C and humidity of 10 to 80 RH%). The immersion time of 1 hour or more is preferable from the viewpoint of adjusting the "Q / P" value to the above range, and the immersion time of 168 hours or less is preferable from the viewpoint of the storage stability of the protein (A). In the organic solvent treatment step, the amount of the organic solvent is preferably an amount that completely immerses the dried product obtained in the freeze-drying step.

[0050] The organic solvent is not particularly limited, and may be a hydrophobic organic solvent such as hexane, or a hydrophilic organic solvent. The SP value and / or HLB value of the hydrophilic organic solvent is preferably within the more preferred range described below. Specific examples of the hydrophilic organic solvent include alcohols having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), ketones having 3 to 6 carbon atoms (acetone, ethyl methyl ketone, methyl isobutyl ketone, etc.), glycols having 2 to 6 carbon atoms (ethylene glycol, propylene glycol, diethylene glycol, etc.) and their monoalkyl (1 to 2 carbon atom) ethers, dimethylformamide, and alkyl acetates having 3 to 5 carbon atoms (methyl acetate, ethyl acetate, etc.). Among the above organic solvents, from the viewpoints of moldability of the bronchoembolization material, the bronchoembolization effect, and the repair of bronchial and pulmonary tissues, preferred are alcohols having 1 to 4 carbon atoms, and more preferred are methanol and ethanol.

[0051] The SP value of the organic solvent is set to 5 to 20 (cal / cm) from the viewpoints of moldability of the bronchial embolization material, bronchial embolization effect, and tissue repair of the bronchus and lungs. 3 ) 1 / 2 It is preferable that the density is 12 to 20 (cal / cm 3 ) 1 / 2 More preferably, it is 13.5 to 15.0 (cal / cm 3 ) 1 / 2 It is particularly preferred that: The SP value in the present invention is calculated by the method described in Polymer Engineering and Science, Vol. 14, pp. 151-154, by Robert F. Fadors et al. The HLB value of the organic solvent is preferably 0 to 75, more preferably 20 to 60, and particularly preferably 30 to 60, from the viewpoints of moldability of the bronchoembolization material, bronchoembolization effect, and tissue repair of the bronchi and lungs. The HLB value in the present invention is an index showing the balance between hydrophilicity and lipophilicity, and can be calculated from the ratio of the organic value and inorganic value of an organic compound, for example, by the Oda method described on page 212 of "Introduction to Surfactants" [published by Sanyo Chemical Industries, Ltd. in 2007, written by Takehiko Fujimoto]. HLB=10×Inorganic / Organic The organic and inorganic values ​​for deriving the HLB can be calculated using the values ​​in the table on page 213 of the aforementioned "Introduction to Surfactants."

[0052] The bronchial embolization material of the present invention is used to occlude bronchi in the treatment of bronchial stump fistula, intractable pneumothorax, empyema with a fistula, and the like that occurs after lung cancer surgery. Specifically, it is used in bronchial fistula embolization and omentum and muscle flap filling procedures. In bronchoscopic embolization, a transairway approach is made using a bronchoscope, and the bronchoembolization material of the present invention is inserted and fixed in the causative bronchus of the fistula. In meshwork and muscle flap filling surgery, a thoracic approach such as RATS, VATS, or thoracotomy can be used to directly suture and insert and fix the bronchial embolization material of the present invention into the fistula instead of a meshwork / muscle flap. These methods are also methods of treating bronchial stump fistula, refractory pneumothorax, and empyema with a fistula according to the present invention.

[0053] The bronchial embolization material of the present invention has the effect of repairing and replacing tissues, and therefore is useful as a therapeutic material since it reduces the rate of infection and recurrent pneumothorax after embolization compared to conventional silicone-based materials.

[0054] In addition, use of the above-mentioned bronchial embolization material for obstructing a bronchus in at least one disease selected from the group consisting of bronchial stump fistula, refractory pneumothorax, and empyema with a fistula is also use of the bronchial embolization material of the present invention. EXAMPLES

[0055] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the following, "parts" refers to "parts by weight."

[0056] <Example 1: Production of bronchial embolization material> [Creating SELP8K] ○ Preparation 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, a method for producing SELP8K (polypeptide (A1)), a polypeptide having a molecular mass of about 80 kDa and a structure in which 12 (GAGAGS)4 sequences (6), which are a type of polypeptide (A), and 13 (GVGVP)4GKGVP(GVGVP)3 sequences (7) are chemically bonded alternately, using this SELP8K-producing strain will be described.

[0057] 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 prepare 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 absorptiometer UV1700: manufactured by Shimadzu Corporation), 40 ml of the culture medium was transferred to another flask warmed to 42°C and cultured at 42°C for about 2 hours. Thereafter, the culture medium was cooled on ice, the turbidity OD600 of the culture medium was measured, and the E. coli was collected by centrifugation.

[0058] Purification of SELP8K Using the collected E. coli, the protein was purified from the 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: freeze-drying. In this manner, purified SELP8K (polypeptide (A1)) with a molecular mass of approximately 85 kDa was obtained. Then, step 8: molding (freeze-drying) and step 9: molding (organic solvent treatment) were performed to obtain a bronchial embolization material.

[0059] 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.

[0060] Step 2: Removal of insoluble debris by centrifugation The cell lysate was then centrifuged (6300 rpm, 4° C., 30 minutes) to collect the supernatant.

[0061] Step 3: Ammonium sulfate precipitation A saturated ammonium sulfate solution was added to the supernatant collected 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 collected by centrifugation. The collected precipitate was dissolved in deionized water. Next, a saturated ammonium sulfate solution was added to the dissolved liquid 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 collected by centrifugation. The collected precipitate was dissolved in deionized water to obtain a solution.

[0062] Step 4: Ultrafiltration The solution obtained in step 3 was subjected to an ultrafiltration device (hollow fiber: manufactured by GE Healthcare) with a molecular mass cutoff of 30,000. Ultrafiltration was carried out on the solution obtained in step 3 using 20 times the amount of deionized water to obtain a polypeptide solution after ultrafiltration.

[0063] Step 5: Cation exchange chromatography The polypeptide solution after ultrafiltration was added to 10 mM sodium acetate buffer so that the polypeptide concentration was 20 g / L, and then the solution was subjected to AKTAPrime (Amersham) equipped with a cation exchange column HiPrepSP XL16 / 10 (GE Healthcare). The eluted fraction was collected using 500 mM sodium acetate buffer as the eluent.

[0064] 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.

[0065] Step 7: Freeze drying The polypeptide solution obtained in step 6 was diluted with deionized water so that the polypeptide concentration was 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 (manufactured by Nippon Techno Service Co., Ltd.) and frozen at -30°C for 24 hours. After freezing, the solution was primarily dried at a vacuum of 5 Pa or less and -30°C for 110 hours, and secondary dried at a vacuum of 5 Pa or less and 30°C for 48 hours to obtain SELP8K (protein (A-1)). Protein (A) was identified using the Western blotting method described below. Regarding protein (A-1), the ratio of "[the total number of amino acids constituting amino acid sequence (X) contained in protein (A) and the amino acids constituting amino acid sequence (X') contained in protein (A)] / [the total number of amino acids constituting protein (A)]" is 0.54. In one molecule of protein (A-1), 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 1:2.

[0066] Process 8: Molding (freeze-drying process) The protein (A-1) obtained in step 7 was diluted with deionized water to obtain a SELP8K solution [protein (A-1) concentration: 12.5 g / L], which was then placed in a container with a base area of ​​1.9 mm 2 1 ml of the mixture was placed in a cylindrical mold with a depth of 1.7 mm, and freeze-dried under the same conditions as in step 7 to form a sponge shape.

[0067] Process 9: Molding (organic solvent treatment) The SELP8K molded in step 8 was immersed in 10 mL of methanol and allowed to stand for 45 hours at 4° C. Thereafter, the methanol was completely removed under conditions of 25° C. and 30% RH humidity to obtain a bronchial embolization material (Z-1).

[0068] 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 of the C-terminal sequence. The Western blotting procedure was as follows. A band showing antibody reactivity to each antibody was observed at the apparent molecular mass of 80 kDa. In addition, Table 1 shows the amino acid composition ratios (actual 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 predicted 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) consisting of four consecutive GAGAGS sequences (2), which are chemically bonded alternately to form a sequence (16) in which the (GAGAGS)2 sequence (15), which is the amino acid sequence shown in SEQ ID NO: 15, is chemically bonded.

[0069] [Table 1]

[0070] <Western blotting> 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 to 20 μL of the sample for Western blotting, and the mixture was heated at 95° C. for 5 minutes to prepare a sample for electrophoresis. SDS-PAGE was performed using 15 μL of this sample for electrophoresis. The gel after electrophoresis was transferred to a polyvinylidene fluoride membrane (hereinafter abbreviated as “membrane”), which was then immersed in a blocking buffer [containing 20 mM Tris (pH 7.6), 137 mM NaCl, 0.1% by weight Tween 20, and 5% by weight skim milk] and shaken at room temperature for 1 hour to perform blocking treatment of the membrane. After blocking, the membrane was washed with TBS-T [containing 20 mM Tris (pH 7.6), 137 mM NaCl, and 0.1% by weight Tween 20] for 2 minutes. Next, the membrane was immersed in a primary antibody solution (primary antibody: a solution in which anti-SELP8K antibody and anti-His-tag antibody (manufactured by Rockland) were diluted 1:500 with TBS-T) and left to stand overnight at 4 ° C. to carry out an antibody reaction. After the reaction, the membrane was washed 4 times with TBS-T for 5 minutes, and then immersed in a solution of a secondary antibody that can bind to the primary antibody and is bound to horseradish peroxidase as a labeling enzyme (secondary antibody: a solution in which ECL anti-rabbit IgG HRP linked F(ab')2 fragment (manufactured by GE Healthcare) was diluted 1:2000 with TBS-T) and left to stand at room temperature for 30 minutes to carry out an antibody reaction. After the reaction, the membrane was washed four times with TBS-T for 5 minutes each, and then the enzyme reaction was carried out using the ECL-Advance Western Blotting Detection Kit (GE Healthcare). The bands were visualized by exposing the membrane to a high-sensitivity instant black-and-white film (FUJIFILM Corporation) using a Luminometer For ECL (GE Healthcare).

[0071] <Examples 2 to 6: Production of bronchial embolization material> The same procedure as in Example 1 was carried out, except that the concentration of protein (A-1) in the SELP8K solution in step 8 was changed from 12.5 g / L to 25, 40, 50, 100, or 150 g / L, to obtain bronchial embolization materials (Z-2) to (Z-6).

[0072] <Examples 7 to 9: Production of bronchial embolization material> Bronchial embolization materials (Z-7) to (Z-9) were obtained in the same manner as in Example 4, except that the methanol used in step 9 in Example 4 was changed to hexane, ethanol or ethylene glycol.

[0073] <Example 10: Production of bronchial embolization material> In Example 4, except for using "plasmid pPT0364 encoding SELP0K" instead of "plasmid pPT0345 encoding SELP8K", the same procedure as in Example 4 was used to obtain a bronchial embolization material (Z-10) containing a protein (SELP0K) having an 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.

[0074] <Example 11: Production of bronchial embolization material> In Example 4, except for using "plasmid pPT0345-4 encoding SELP8K4" instead of "plasmid pPT0345 encoding SELP8K", the same procedure as in Example 4 was used to obtain a bronchial embolization material (Z-11) containing a protein (SELP8K4) having an amino acid sequence (27) 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.

[0075] <Example 12: Production of bronchial embolization material> In Example 4, except for using "plasmid pPT0345-12 encoding SELP8K12" instead of "plasmid pPT0345 encoding SELP8K," the same procedure as in Example 4 was followed to obtain a bronchial embolization material (Z-12) containing a protein (SELP8K12) having a molecular mass of approximately 105 kDa and an amino acid sequence shown in SEQ ID NO: 20, which has 12 (GAGAGS)4 sequences (19) and 13 (GVGVP)6GKGVP(GVGVP)5 sequences (18), which are chemically bonded alternately to a (GAGAGS)2 sequence (15).

[0076] <Example 13: Production of bronchial embolization material> In Example 4, except for using "plasmid pPT0102-1 encoding ELP1.1" instead of "plasmid pPT0345 encoding SELP8K," the same procedure as in Example 4 was used to obtain a bronchial embolization material (Z-13) containing a protein (ELP1.1) having a molecular mass of approximately 200 kDa and a structure formed by chemically bonding 40 blocks each of the GAGAGS sequence (2), the (VPGVG)4 sequence (24) which is the amino acid sequence shown in SEQ ID NO: 24, and the (VPGVG)8 sequence (25) which is the amino acid sequence shown in SEQ ID NO: 25, in which the (VPGVG)4 sequence (24), the GAGAGS sequence (2), and the (VPGVG)8 sequence (25) are bonded in this order.

[0077] <Example 14: Preparation of bronchial embolization material> In Example 4, except that "plasmid pPT0270-1 encoding SELP6.1" was used instead of "plasmid pPT0345 encoding SELP8K", the same procedure as in Example 4 was used to synthesize (GAGAGS)8 sequence (21) having the amino acid sequence shown in SEQ ID NO: 21 and (GVGVP) having the amino acid sequence shown in SEQ ID NO: 22. 40A bronchial embolization material (Z-14) was obtained that contains a protein (SELP6.1) having an amino acid sequence (23) shown in SEQ ID NO: 23, which has five units of sequence (22) that are chemically bonded alternately, and has a molecular mass of approximately 110 kDa.

[0078] <Comparative Examples 1-2: Production of Comparative Bronchial Embolic Materials> In Example 1, the concentration of protein (A-1) in the SELP8K solution in step 8 was changed from 12.5 g / L to 10 or 200 g / L, and the procedure was repeated to obtain comparative bronchial embolization materials (Z'-1) to (Z'-2).

[0079] <Comparative Example 3: Production of a comparative bronchial embolization material> The same procedure as in Example 4 was carried out except that "Step 9: Molding (organic solvent treatment)" was not carried out, and the molded product obtained in step 8 was used as a comparative bronchial embolization material (Z'-3).

[0080] The density, Q / P, and R / P were evaluated by the following method using the bronchial embolization materials obtained in Examples 1 to 14 and the comparative bronchial embolization materials obtained in Comparative Examples 1 to 3. The results are shown in Table 2.

[0081] <density> The density of the bronchial embolization material was calculated from the volume (the base area and height of the obtained cylindrical bronchial embolization material were measured and calculated) and weight. The results are shown in Table 2.

[0082] <q p> The bronchial embolization material was dried in a circulating air dryer at 100°C under atmospheric pressure for 3 hours, and then at 40°C under atmospheric pressure for 15 hours to obtain a dried product (DP). The weight of the dried product (DP) was designated as P. Water having a weight 1,000 times that of P was added to a plastic cup, and the dried product (DP) was immersed in the water and allowed to stand at 25°C under 1 atmosphere for 72 hours, after which it was removed. This was dried in a circulating air dryer at 100°C under atmospheric pressure for 3 hours, and then at 40°C under atmospheric pressure for 15 hours to obtain the weight of the product (DQ). The weight of the product (DQ) was designated as Q. Q / P was calculated using the above P and Q. The results are shown in Table 2.

[0083] <r p> The bronchial embolization material was dried in a circulating air dryer at 100°C under atmospheric pressure for 3 hours, and then at 40°C under atmospheric pressure for 15 hours to obtain a dried product (DP). The weight of the dried product (DP) was designated as P. Water having a weight 1,000 times that of P was added to a plastic cup, and the dried product (DP) was immersed in the water and allowed to stand at 25°C under 1 atmosphere for 2 hours, after which it was removed. This was dried in a circulating air dryer at 100°C under atmospheric pressure for 3 hours, and then at 40°C under atmospheric pressure for 15 hours to obtain the weight of the product (DR). The weight of the product (DR) was designated as R. R / P was calculated using the above P and R. The results are shown in Table 2.

[0084] [Table 2]

[0085] <Evaluation Examples 1 to 15 and Comparative Evaluation Examples 1 to 3: Evaluation of Bronchial Embolization Materials> Adult beagle dogs (1-2 years old, weighing 9-11 kg) underwent thoracotomy under general anesthesia, and lung resection (resection of the areas listed in Table 3) was performed. The bronchial embolization material listed in Table 3 was fixed to the bronchial stump at four points using absorbable sutures, without suturing the stump together. After confirming that there was no significant air leakage, the surgical wound was sutured. Two, four, and eight weeks after surgery, the lumen was observed using a bronchoscope to evaluate the degree of absorption of the bronchial embolization material, the degree of embolization, the presence or absence of deviation, the presence or absence of air leakage, and the presence or absence of subcutaneous emphysema. In addition, the animals were sacrificed 8 weeks after surgery, and tissues, including the trachea and the bronchial stump where the embolization material had been placed, were collected to evaluate the degree of bronchial embolization and repair, as well as the degree of granulation tissue formation, histologically. The histological evaluation of the degree of bronchial embolization and repair, and the degree of granulation tissue formation was also performed on two other adult beagle dogs, sacrificing them at 2 or 4 weeks after the injection instead of 8 weeks after the injection. The results are shown in Table 3.

[0086] [Evaluation of the degree of bronchial obstruction] The lumen was observed using a bronchoscope and evaluated according to the following criteria. A higher score for the degree of bronchial embolization indicates a more excellent bronchial embolization effect. 1: Bronchial tubes are closed 0: Bronchial tubes are not closed

[0087] [Absorption of bronchial embolization material] The lumen was observed using a bronchoscope and evaluated according to the following criteria. 1: Absorption of bronchial embolization material 0: No absorption of bronchial embolization material

[0088] [Whether or not there is deviation] The lumen was observed using a bronchoscope and evaluated according to the following criteria. 1: No deviation of the bronchial embolization material 0: Dislodged bronchial embolization material

[0089] [Air leaks] The lumen was observed using a bronchoscope and evaluated according to the following criteria. 1: No air leakage 0: Air leak

[0090] [Evaluation of the occurrence of subcutaneous emphysema] The body surface of the dog was visually observed and evaluated according to the following criteria. 1: No subcutaneous emphysema 0: Subcutaneous emphysema present

[0091] [Evaluation of the degree of bronchial repair] The collected tissues were stained with hematoxylin and eosin and observed under a light microscope. The degree of inflammation, vascular proliferation, nerve regeneration, and epithelial regeneration were evaluated according to the following criteria. <Inflammation> 3: Mild inflammation 2: Moderate inflammation 1: Severe inflammation <Vascular proliferation> 3: Good playback 2: Medium reproduction 1: No growth <Nerve regeneration> 3: Good playback 2: Medium reproduction 1: Almost no playback <Epithelial regeneration> 3: Good playback 2: Medium reproduction 1: Almost no playback

[0092] [Evaluation of the degree of granulation] Tissue sections were prepared from the collected tissues, stained with hematoxylin and eosin, and images of the tissue sections were obtained using an optical microscope. The images of the tissue sections were analyzed using ImageJ (National Institutes of Health, USA), and the degree of granulation formation was evaluated using the following formula: Granulation degree (%) = granulation regeneration length (mm) / bronchial defect length (mm) × 100 The values ​​for "granulation regeneration length" and "bronchial defect length" were obtained by analyzing tissue section images using ImageJ.

[0093] [Table 3] [Industrial Applicability]

[0094] The bronchial embolization material of the present invention can be used as an embolization material against air leaks from the stumps of the bronchi or lungs following lung resection, and has the effect of promoting tissue repair and replacement, making it suitable as a therapeutic material.< / r> < / q>

Claims

1. A bronchial embolization material containing a protein (A), the protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y'), the total number of the polypeptide chains (Y) and (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 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; 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; The protein (A) satisfies the following relational formula (1): The density of the bronchial embolization material is 90 to 300 mg / cm ３ and The bronchial embolization material satisfies the following relational formula (2): the protein (A) is a protein (A11-2-1) having a polypeptide chain (S1-1) which is the (GAGAGS) 4 sequence (5) shown in SEQ ID NO:5, and a polypeptide chain (Y'11) which is the (GVGVP) 4 GKGVP(GVGVP) 3 sequence (6) shown in SEQ ID NO:6; The protein (A) is a bronchial embolization material that has been treated with an organic solvent. 0.50≦[total number of amino acids constituting the amino acid sequence (X) contained in the protein (A) and amino acids constituting the amino acid sequence (X′) contained in the protein (A)] / [total number of amino acids constituting the protein (A)]≦0.80 (1) [In the relational formula (1), the amino acid sequence (X') is an amino acid sequence in which 60% or less of the amino acids in the amino acid sequence (X) are substituted with lysine and / or arginine.] 0.01≦Q / P (2) [In the relational formula (2), P is the weight of the dried product (DP) obtained by drying the bronchial embolization material at 100°C under 1 atmosphere for 3 hours, and then drying at 40°C under 1 atmosphere for 15 hours; Q is the weight of the dried product (DP) obtained by immersing the dried product (DP) in water having a weight 1,000 times that of P at 25°C under 1 atmosphere for 72 hours, removing the dried product, drying it at 100°C under 1 atmosphere for 3 hours, and then drying it at 40°C under 1 atmosphere for 15 hours (DQ).]

2. The bronchial embolization material according to claim 1 , wherein the protein (A) satisfies the following relational formula (3): 0.3≦R / P (3) [In the relational formula (3), P is the weight of the dried product (DP) obtained by drying the bronchial embolization material at 100°C under 1 atmosphere for 3 hours, and then drying at 40°C under 1 atmosphere for 15 hours; R is the weight of the dried product (DR) obtained by immersing the dried product (DP) in water having a weight 1,000 times that of P at 25°C under 1 atmosphere for 2 hours, removing the dried product, drying it at 100°C under 1 atmosphere for 3 hours, and then drying it at 40°C under 1 atmosphere for 15 hours.]

3. 3. The bronchial embolization material according to claim 1 or 2, wherein the protein (A) 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.

4. 4. The bronchial embolization material according to claim 3, wherein a ratio of the number of said GAGAGS sequence (2) to the total number of said amino acid sequence (X) and said amino acid sequence (X') in one molecule of protein (A) (GAGAGS sequence (2):total of amino acid sequence (X) and amino acid sequence (X')) is 1:1.5 to 1:

20.

5. The bronchi embolization material according to any one of claims 1 to 4, wherein the protein (A) is a protein (A11) having a polypeptide chain (Y'1) in which one amino acid in a sequence of 2 to 200 consecutive amino acids in the GVGVP sequence (4) is substituted with lysine.

6. 6. The bronchial embolization 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).

7. A bronchial embolization material containing a protein (A), 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 90% or more to these amino acid sequences; The density of the bronchial embolization material is 90 to 300 mg / cm 3 ; The bronchial embolization material satisfies the following relational formula (2): The protein (A) is a bronchial embolization material that has been treated with an organic solvent. 0.01≦Q / P (2) [In the relational formula (2), P is the weight of the dried product (DP) obtained by drying the bronchial embolization material at 100°C under 1 atmosphere for 3 hours, and then drying at 40°C under 1 atmosphere for 15 hours; Q is the weight of the dried product (DP) obtained by immersing the dried product (DP) in water having a weight 1,000 times that of P at 25°C under 1 atmosphere for 72 hours, removing the dried product, drying it at 100°C under 1 atmosphere for 3 hours, and then drying it at 40°C under 1 atmosphere for 15 hours (DQ).]

Images