Title of the invention: Polyethylene glycol modified product of hepatocyte growth factor or an active fragment thereof
Technical field
[0001]
　The present invention relates to a polyethylene glycol modified product of hepatocyte growth factor or an active fragment thereof.
Background technology
[0002]
　Hepatocyte growth factor is a growth factor having various pharmacological actions, and in addition to the initially found hepatocyte growth factor, anti-apoptotic action, angiogenic action, angiogenic action, anti-organ fibrosis action, and anti-epithelial action. It is known to have a mesenchymal conversion effect, and clinical application to various diseases is being attempted.
[0003]
　However, since the half-life of hepatocyte growth factor in vivo is as short as about 30 minutes, it is suggested that a large amount of hepatocyte growth factor must be frequently administered in order to sustain its pharmacological action (). Non-Patent Document 1). On the other hand, since hepatocyte growth factor has hepatic directionality, it has been reported that when it is administered in a large amount, it induces hepatic hypertrophy due to hepatocyte proliferation as a side effect (Non-Patent Document 2).
[0004]
　As an active fragment which is a natural splicing variant of hepatocyte growth factor, NK1 consisting of N domain and kringle 1 is reported in Non-Patent Document 3, and NK2 consisting of N domain and kringle 1 and kringle 2 is reported in non-patent document 4. .. Further, in Non-Patent Document 5, NK4 consisting of N domain and klingle 1, kringle 2, kringle 3 and kringle 4 is reported as an active fragment produced by a recombinant technique, and these active fragments are in vivo and in. It has been reported that in vivo, it has an agonist activity or an antagonist activity against c-Met, which is a receptor tyrosine kinase of hepatocyte growth factor receptor. In addition, it has been reported that NK1 has hepatic directivity and induces hepatic hypertrophy in vivo (Non-Patent Documents 3 and 6).
[0005]
　So far, polyethylene glycol modified products having the main purpose of prolonging the in vivo half-life of NK4, which is one of the hepatocyte growth factor and its active fragment, have been reported (Patent Documents 1 and 2). Polyethylene glycol is a highly biocompatible polymer polymer, and is widely used as a modifier for extending the in vivo half-life of proteins and reducing immunogenicity.
[0006]
　When a protein is chemically modified with polyethylene glycol, it is known that the physiological activity of the protein is reduced or eliminated depending on the modification position. For example, a modified product obtained by directly chemically modifying hepatocyte growth factor with a plurality of polyethylene glycols can achieve the effect of prolonging the in vivo half-life of hepatocyte growth factor, but its physiological activity is reduced by 30% or more. Is reported in Patent Document 1.
[0007]
　Further, in Non-Patent Document 7, Pseudomonas aeruginosa-derived exotoxin A was chemically modified with polyethylene glycol via a protease-sensitive peptide against rhinovirus-derived 3C protease for the purpose of experimentally controlling drug-derived toxicity. Prodrugs have been reported.
Prior art literature
Patent documents
[0008]
Patent Document 1: International Publication No. 1996/28475
Patent Document 2: Japanese Unexamined Patent Publication No. 2010-174034
Non-patent literature
[0009]
Non-Patent Document 1: Liu K.X. Et al., The American Journal of Physiology, 1998, Vol. 275, p. E835-E842
Non-Patent Document 2: Sakata H. et al . Et al., Cell Growth & Differentiation, 1996, Vol. 7, p. 1513-1523
Non-Patent Document 3: Jakubczak J.L. Et al., Molecular and Cellular Biology, 1998, Vol. 18, No. 3, p. 1275-1283
Non-Patent Document 4: Otsuka T. et al. Et al., Molecular and Cellular Biology, 2000, Vol. 20, No. 6, p. 2055-265
Non-Patent Document 5: Date K. et al . Et al., FEBS Letters, 1997, Vol. 420, No. 1, p. 1-6
Non-Patent Document 6: Ross J. et al. Et al., Gastroenterology, 2012, Vol. 142, p. 897-906
Non-Patent Document 7: Stephan N. et al . Et al., Bioconjugate Chemistry, 2014, Vol. 25, p. 2144-2156
Outline of the invention
Problems to be solved by the invention
[0010]
　However, in Non-Patent Document 7, the functional verification of the prodrug of exotoxin A, which is a toxin protein derived from Pseudomonas aeruginosa, in vivo and ex vivo has not been carried out. In addition, the prodrug of exotoxin A disclosed in Non-Patent Document 7 uses a protease-sensitive peptide at the end of exotoxin A and in the middle of the sequence to suppress the physiological activity of exotoxin A. Glycol is chemically modified, but in general, addition of a peptide in the middle of a protein sequence or chemical modification with polyethylene glycol is difficult to produce, and even after cleavage with a protease, an unnecessary amino acid sequence derived from a protease-sensitive peptide is described above. Since it remains in the middle of the structure of the protein, there is uncertainty from the viewpoint of maintaining the physiological activity originally possessed by the protein. Therefore, it is considered that the technique described in Non-Patent Document 7 is poorly general and cannot be easily applied to other proteins.
[0011]
　In addition, Non-Patent Document 7 does not disclose or suggest any prodrug of hepatocyte growth factor or its active fragment, and the prodrug for the purpose of reducing hepatic directivity derived from hepatocyte growth factor or its active fragment. There is no disclosure or suggestion of in vivo proteases that can be used in design.
[0012]
　Therefore, hepatocyte growth factor or an active fragment thereof can reduce the hepatic orientation derived from the hepatocyte growth factor or an active fragment thereof, and can selectively express the physiological activity of the hepatocyte growth factor or an active fragment thereof in a target disease tissue other than the liver tissue. There is a need to create variants of the factor or its active fragment.
[0013]
　Therefore, an object of the present invention is to provide a modified version of hepatocyte growth factor or an active fragment thereof that solves the above problems.
Means to solve problems
[0014]
　As a result of intensive studies to solve the above problems, the present inventors chemically modified hepatocyte growth factor or an active fragment thereof with polyethylene glycol via a protease-sensitive peptide to obtain hepatocyte growth factor or its active fragment. We have found that hepatocyte growth factor or the physiological activity of the active fragment thereof can be selectively expressed by cleaving the protease-sensitive peptide in the target disease tissue by reducing the hepatic orientation derived from the active fragment. It came to be completed.
[0015]
　That is, the present invention includes the following (1) to (7).
　(1) A polyethylene glycol-modified form of hepatocyte growth factor or an active fragment thereof, in which polyethylene glycol is bound to the end of the hepatocyte growth factor or an active fragment thereof via a protease-sensitive peptide.
　(2) The polyethylene glycol modified product according to (1), wherein the protease-sensitive peptide is an ADAM17-sensitive peptide or a thrombin-sensitive peptide.
　(3) The polyethylene glycol modified product according to (1), wherein the protease-sensitive peptide is an amino acid sequence represented by any one of SEQ ID NOs: 8 to 20 in the sequence listing.
　(4) The polyethylene glycol modified product according to any one of (1) to (3), wherein the polyethylene glycol has a number average molecular weight of 20000 to 100,000.
　(5) The polyethylene glycol modified product according to any one of (1) to (4), wherein the active fragment of the hepatocyte growth factor is NK1.
　(6) The polyethylene glycol modified product according to any one of (1) to (5), wherein the active fragment of the hepatocyte growth factor is the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing.
　(7) A pharmaceutical agent containing the polyethylene glycol modified product according to any one of (1) to (6) as an active ingredient.
Effect of the invention
[0016]
　The polyethylene glycol modified product of the hepatocyte growth factor or the active fragment thereof of the present invention reduces the hepatic directivity inherent in the hepatocyte growth factor or the active fragment thereof, and selectively hepatocytes in a target disease tissue such as a kidney. It is capable of expressing the physiological activity of a growth factor or an active fragment thereof and exerting its pharmacological action.
A brief description of the drawing
[0017]
FIG. 1 is a diagram showing the expression level of the enzymatic activity of ADAM17 protein in kidney tissue and liver tissue of kidney disease model mice and normal mice.
FIG. 2 is a diagram showing the expression level of ADAM17 protein in kidney tissue and liver tissue of kidney disease model mice and normal mice. (A) shows a Western blotting image for ADAM17 protein, and (b) is a figure which quantified the band intensity.
FIG. 3 shows the release of human NK1 which is an active agent from prodrugized human NK1 by protease treatment.
FIG. 4 shows the attenuation of HGF activity in human NK1 by prodrugization of protease-sensitive peptide-added human NK1.
FIG. 5 shows the release of human NK1 which is an active agent from tissue-selective prodrugized human NK1.
FIG. 6 shows the tissue-selective bioactivity of prodrugized human NK1 in vivo.
FIG. 7 is a diagram showing a comparison of activity control of prodrugized human NK1 by different polyethylene glycol modification sites.
Mode for carrying out the invention
[0018]
The polyethylene glycol-modified form of hepatocyte growth factor or its active fragment (hereinafter, also referred to as PEG-modified form of HGF or its active fragment) according to the present invention is used. One molecule of hepatocyte growth factor (HEPATOCYTE GROWTH FACTOR, hereinafter also referred to as HGF) or its active fragment ends (amino end, carboxyl end) with polyethylene glycol (hereinafter, also referred to as PEG) via a protease-sensitive peptide. Are co-located.
[0019]
　One of the embodiments of the PEG-modified form of HGF or an active fragment thereof according to the present invention is one molecule or a plurality of molecules of PEG at the terminal (amino terminal or carboxyl terminal) of HGF or the active fragment thereof via a protease-sensitive peptide. Can be covalently combined. At that time, the binding mode between the terminal of the HGF or the active fragment thereof and the protease-sensitive peptide is not particularly limited, and both may be directly bound or optionally via a spacer sequence. .. The binding mode between the protease-sensitive peptide and the PEG is not particularly limited, and the PEG may be directly bound to the protease-sensitive peptide, or the PEG may be directly bound to the protease-sensitive peptide via an amino acid artificially added to the protease-sensitive peptide. Further, a protease-sensitive peptide for purification or a tag sequence for purification may be contained between the protease-sensitive peptide and an artificially added amino acid or the like. For example, the carboxyl end of a protease-sensitive peptide is covalently bonded to the end of HGF or an active fragment thereof, and the amino end of the protease-sensitive peptide is covalently bonded to PEG, or the amino end of a protease-sensitive peptide is covalently bonded to the end of HGF or an active fragment thereof. Is covalently bonded to the carboxyl end of the protease-sensitive peptide, and PEG is covalently bonded to the carboxyl end of the protease-sensitive peptide. Those in which PEG is covalently bonded are preferable.
[0020]
　Since the amino terminal of HGF or its active fragment is responsible for the dimerization and hepatic directional function of HGF or its active fragment, which is important for controlling the physiological activity of HGF or its active fragment, HGF or its activity. In order to reduce the physiological activity and hepatic directivity of the fragment, it is preferable that one molecule of PEG is covalently bound to the amino terminal of HGF or the active fragment thereof via a protease-sensitive peptide, and the amino of HGF or the active fragment thereof. It is more preferable that the carboxyl end of the protease-sensitive peptide is covalently attached to the terminal and one molecule of PEG is co-linked to the amino end of the protease-sensitive peptide, and the ADAM17-sensitive peptide or trombine-sensitive to the amino end of HGF or an active fragment thereof. It is more preferable that the carboxyl end of the peptide is covalently attached and one molecule of PEG is covalently attached to the amino end of the ADAM17-sensitive peptide or the thrombin-sensitive peptide, and the amino end of NK1 which is one of the active fragments of HGF. It is more preferable that the carboxyl end of the ADAM17-sensitive peptide or the thrombin-sensitive peptide is co-linked, and one molecule of PEG having a number average molecular weight of 20,000 to 100,000 is co-linked to the amino-terminal of the ADAM17-sensitive peptide or the thrombin-sensitive peptide, preferably NK1. The carboxyl end of the ADAM17-sensitive peptide or the thrombin-sensitive peptide is covalently attached to the amino end of the ADAM17-sensitive peptide, and one molecule of a 4-branched PEG having a number average molecular weight of 70,000 to 90000 is co-bonded to the amino end of the ADAM17-sensitive peptide or the thrombin-sensitive peptide. Is most preferable. In addition, in order to control the cleavage efficiency by protease, an arbitrary spacer sequence can be added between HGF or an active fragment thereof and a protease-sensitive peptide.
[0021]
　As another embodiment of the PEG-modified form of HGF or an active fragment thereof according to the present invention, one molecule of PEG covalently bonded via a protease-sensitive peptide in the middle of the sequence of HGF or an active fragment thereof can be mentioned. Be done.
[0022]

　HGF consists of N domain, kringle 1, kringle 2, kringle 3, kringle 4 and SPH domain from the amino terminal side, and N domain, kringle 1, kringle 2, kringle 3 and kringle 4 are α The chain constitutes, and the SPH domain constitutes the β chain. HGF is biosynthesized as a single-stranded protease at the time of expression, but after the secretory signal sequence is removed when it is secreted, the arginine residue at the 494th position and the 495th position from the starting methionine are extracellular. Is processed by a protease between the arginine residues of the arginine residue to form a heterodimeric chain in which the α chain and the β chain are bound by a disulfide bond to form an active form (Miyazawa K. et al., The Journal of Biological Chemistry, 1996, Vol. 271, No. 7, p. 3615-3618). As used herein, the term HGF means an active form of HGF having physiological activity.
[0023]
　HGF has various pharmacological actions such as hepatocyte growth factor, anti-apoptotic action, angiogenesis action, vasodilatory action, anti-organ fibrosis action, and anti-epithelial-mesenchymal transition action. It is known to be induced by binding to the kinase c-Met. When HGF binds to c-Met, a plurality of tyrosine residues inside the cells of c-Met are phosphorylated, and the signal molecule is bound to the phosphorylated tyrosine residue to activate the signal pathway. At that time, Y1234 / 1235 is known as a particularly important phosphorylation site.
[0024]
　In the case of human HGF, it is biosynthesized as a secretory protein consisting of 728 amino acid residues (including a secretory signal sequence (31 amino acid residues from the starting methionine)) (GenBank accession number: M29145), and when secreted, a secretory signal is produced. It becomes a protein consisting of 697 amino acid residues from which the sequence has been removed (SEQ ID NO: 1).
[0025]
　The above HGF is not only one having the same amino acid sequence as the amino acid sequence of naturally occurring HGF (hereinafter, also referred to as natural HGF), but also lacks one or several amino acids in the amino acid sequence of natural HGF. HGF containing an amino acid variant of HGF having a lost, substituted or added (or inserted) amino acid sequence and having physiological activity as HGF, and further modified sugar chain portion of natural HGF and It also includes HGF having no sugar chain moiety. As the amino acid variant of HGF, a mutant having 90% or more sequence identity with the amino acid sequence of natural HGF is preferable, a mutant having 95% or more sequence identity is more preferable, and 98% or more sequence identity is the same. Variants having sex are more preferred. Examples of amino acid variants of HGF include HGF lacking 5 amino acid residues in Klingle 1 (a naturally occurring naturally occurring mutant, hereinafter also referred to as defective HGF) (Kinosaki M. et al., FEBS). Letters, Vol. 434, 1998, p.165-170), which has been reported to have higher specific activity than human HGF (SEQ ID NO: 1) in certain cell types.
[0026]
　Conservative amino acid substitutions generally refer to substitutions between amino acids that have similar chemical, electrical (or polar / hydrophobic) or structural properties. Such substitutions can suppress significant changes in the conformation of the polypeptide containing native HGF, so that the polypeptide can be retained without significantly impairing its activity. In some cases, it can be more active than the natural form. Specific examples of such amino acid substitutions include substitutions between acidic amino acids (eg, aspartic acid (D) and glutamic acid (E)), substitutions between basic amino acids (eg, histidine (H), lysine (K) and arginine (R)). )), Substitution between aromatic amino acids (eg phenylalanine (F), tyrosine (Y) and tryptophan (W)), substitution between hydrophilic amino acids (eg cysteine ​​(C), aspartic acid (D), glutamic acid (E)) Substitution between histidine (H), lysine (K), aspartin (N), glutamine (Q), arginine (R), serine (S) and threonine (T)), hydrophobic amino acids (eg, alanine (A), phenylalanine) (F), isoleucine (I), leucine (L), norleucine (Nle), methionine (M), valine (V), tryptophan (W) and tyrosine (Y)) and the like.
[0027]
　The above-mentioned HGF also includes recombinant HGF produced by gene recombination technology based on the amino acid sequence or base sequence of natural HGF.
[0028]
　As used herein, "sequence identity" refers to the identity between two sequences that can be determined using algorithms such as BLAST, FASTA, etc., and generally refers to the two sequences, including gaps or. When aligned for maximum coincidence without gaps, it can be calculated as a percentage of the number of matched amino acids to the total number of amino acids (including gaps) (Altschul S. et al., Journal of Algorithm). Biologic, 1990, Vol. 215, No. 3, p.403-410; Altschul S. et al., Nuclear Acids Research, 1997, Vol. 25, No. 17, p.3389-3402).
[0029]
　As used herein, "several" refers to an integer of 2-10, i.e. 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[0030]
　An active fragment of HGF contains a part of the structure of HGF, binds to c-Met, and acts as an antagonist against a protein or c-Met that exerts physiological activity (agonist activity) as HGF (antagonist). A protein that exerts its activity.
[0031]
　Examples of the above-mentioned active fragment of HGF include NK1 or NK2, which are natural splicing variants of HGF, or NK4 produced by a recombinant technique. NK1 consists of the N-domain of HGF on the amino-terminal side and Kringle 1, and NK2 consists of the N-domain of HGF on the amino-terminal side, Kringle 1 and Kringle 2. Each of these has been reported to act as an agonist or antagonist against c-Met in vivo (Jakubczak JL et al., Molecular and Cellular Biology, Vol. 18, No. 3, 1998, p. 1275-1283; Otsuka T. et al., Molecular and Cellular Biology, Vol. 20, No. 6, 2000, p. 2055-2065). In addition, NK1 has hepatic directivity and has been reported to induce hepatic hypertrophy in vivo (Jakubczak J.L. et al., Molecular and Cellular Biology, 1998, Vol. 18, No. 3, p. .1275-1283; Ross J. et al., Gastroenterology, 2012, Vol. 142, p. 897-906). NK4 consists of the amino-terminal N-domain of HGF, kringle 1, kringle 2, kringle 3 and kringle 4, and has been reported to act as an antagonist against c-Met (Date K. et al., FEBS Letters). , Vol. 420, No. 1, 1997, p. 1-6).
[0032]
　The above-mentioned active fragment of HGF is not only one having the same amino acid sequence as the amino acid sequence derived from natural HGF, but also one or several amino acids are deleted, substituted or substituted in the amino acid sequence derived from the amino acid sequence of natural HGF. A mutant having an added amino acid sequence and exhibiting physiological activity (agonist activity) as HGF or antagonistic activity against c-Met was also included, and the sugar chain portion derived from natural HGF was further modified. , Or mutants that do not have a sugar chain moiety derived from natural HGF. As the mutant of the active fragment of HGF, a mutant having 90% or more sequence identity with the amino acid sequence derived from the natural HGF is preferable, and a mutant having 95% or more sequence identity is more preferable, and 98% or more. Variants having the sequence identity of are even more preferred. For example, as NK1 highly active mutants, 1K1 (Liesa D. et al., The EMBO Journal, 2001, Vol. 20, No. 20, p. 5543-5555) and M2.2 (Jones DS 2nd. Et al., Proceedings of the National Academic of Sciences of the United States of America, 2011, Vol. 108, No. 32, p. 13035-13040).
[0033]
　The HGF or active fragment thereof contained in the PEG-modified form of HGF or an active fragment thereof according to the present invention includes natural HGF (including variants such as defective HGF), NK1 (including variants), and NK2 (variants). It is preferably NK4 (including a variant), more preferably NK1 (including a variant) or NK2 (including a variant), and NK1 (including a variant). More preferably, it is human NK1 consisting of the amino acid sequence represented by SEQ ID NO: 2. The amino acid sequence represented by SEQ ID NO: 2 does not include a human HGF-derived secretory signal sequence (MWVTKLLPALLQHVLLHLLLPIAIPYAEG: SEQ ID NO: 3).
[0034]
　The above-mentioned HGF or an active fragment thereof includes an amino acid sequence derived from a mammal, and is preferably an amino acid sequence derived from a human, a cat or a dog, and more preferably an amino acid sequence derived from a human.
[0035]
　For HGF or an active fragment thereof, for example, a nucleic acid sequence (DNA) encoding HGF or an active fragment thereof is designed by using a known gene recombination technique, and is transiently or stably introduced into cells and expressed. You can get it with.
[0036]
　In addition, 1 to 20 amino acids may be added as a spacer sequence between HGF or an active fragment thereof and a protease-sensitive peptide by using a known gene recombination technique. An HGF or an active fragment thereof having a spacer sequence added between the HGF or an active fragment thereof and a protease-sensitive peptide is continuously linked to a nucleic acid sequence (DNA) encoding the HGF or the active fragment thereof by using, for example, a known gene recombination technique. Then, a nucleic acid sequence (DNA) encoding an arbitrarily selected spacer sequence is added, and a nucleic acid sequence (DNA) encoding a protease-sensitive peptide is continuously added, and the nucleic acid sequence is transiently or stably introduced into the cell. , Can be obtained by expressing. Examples of amino acids that can be used in the spacer sequence include aspartic acid (D), glutamic acid (E), histidine (H), lysine (K), arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan ( W), alanine (A), cysteine ​​(C), aspartic acid (N), glutamine (Q), serine (S), threonine (T), isoleucine (I), leucine (L), norleucine (Nle), methionine ( M), valine (V) and tryptophan (W) can be mentioned. The spacer sequence to be used is appropriately selected in consideration of the efficiency of cleavage of the protease-sensitive peptide and the efficiency of controlling the activity of HGF or an active fragment thereof by PEG modification.
[0037]

　The protease-sensitive peptide used in the present invention means a peptide whose expression is observed in a target disease tissue other than liver tissue and which is cleaved by a protease whose expression in liver tissue is relatively low. ..
[0038]
　Proteases corresponding to protease-sensitive peptides have different relative differences in enzyme activity required by the target disease tissue, so the relative enzyme between the liver tissue and the target disease tissue by methods such as Western blotting and immunohistochemistry. It is necessary to evaluate the difference in activity and select the appropriate one. For example, when the target disease tissue is kidney tissue as one form thereof, the relative difference between the enzyme activity in the liver tissue and the enzyme activity in the kidney tissue needs to be 3 times or more, and the protease needs to be 10 times or more. Is preferable, and a protease having a concentration of 100 times or more is more preferable.
[0039]
　By selecting the protease-sensitive peptide as described above in the PEG-modified form of HGF or an active fragment thereof according to the present invention, the PEG-modified form is chemically modified with PEG in liver tissue to obtain HGF or an active fragment thereof. In the target disease tissue, these protease-sensitive peptides correspond to the proteases, while the side effects derived from HGF or its active fragment in the liver tissue can be expected to be reduced because the activity of the disease is attenuated and the hepatic directivity is reduced. By liberating PEG, it can be expected that HGF or an active fragment thereof will be released and its physiological activity will be expressed. A fragment of the protease-sensitive peptide cleaved by the corresponding protease may remain in HGF or an active fragment thereof.
[0040]
　Proteases that are expressed in target disease tissues other than liver tissue include, for example, ADAM17 (Melenhorst WB et al., American Journal of Physiologic, 2009, 2009, which has been reported to be expressed in the kidneys of patients with renal disease. Vol. 297, No. 3, p. 781-790), Hepatocyte graft factor activator (HGFA), its upstream factor Trombin (David CJH et al., American Journal of Physiology, 2001, Vol. 15, 2001). No. 4, p1383-1393) or Matrix MetalloProtease (Junwei Y. et al., The Journal of Clinical Investment, 2002, Vol. 110, p.1525-1538) and the like.
[0041]
　ADAM17 is synthesized intracellularly as an inactive precursor, and then cleaved by Furin to become a mature product, exhibiting activity to cleave various proteins (Aldo B. et al., Journal of Biological Chemistry, 2003, Vol. 278, p. 25933-25939; Belen S.J. et al., Journal of Biological Chemistry, 2007, Vol. 282, p. 8325-8331). Unless otherwise stated herein, ADAM17 represents a mature ADAM17.
[0042]
　The protease-sensitive peptide contained in the PEG-modified form of HGF or an active fragment thereof according to the present invention is an existing endogenous peptide that has been reported to be cleaved by the corresponding protease, or an artificial peptide prepared by using a gene recombination technique. Can be used. Protease-sensitive peptides are selected for stability, cleavage efficiency in target tissues and species cross-reactivity. Since the present inventors have found that the expression of ADAM17 and thrombin in liver tissue is relatively lower than that in target disease tissues other than liver tissue, the protease-sensitive peptide is an ADAM17-sensitive peptide or a thrombin-sensitive peptide. Is preferable. Here, the ADAM17-sensitive peptide means a peptide cleaved by ADAM17, and the thrombin-sensitive peptide means a peptide cleaved by thrombin.
[0043]
　Among the ADAM17-sensitive peptides, examples of the bioendogenous peptide include those derived from TNF-alpha (SPLAQAVRSSSR, SEQ ID NO: 8) and those derived from L-selectin (QETNRSFSK, SEQ ID NO: 10). Examples of artificial peptides include Artificial ADAM-17 clearable sequence (PRAAAVKSP, SEQ ID NO: 9) (Caesu C. l. et al., Biochemical Journal, 2009, Vol. 424, p. 79-88). As the ADAM17-sensitive peptide, the peptide having the amino acid sequence represented by SEQ ID NO: 8 to 16 is preferable, and the peptide having the amino acid sequence represented by SEQ ID NO: 9 is more preferable.
[0044]
　Among the thrombin-sensitive peptides, examples of the endogenous peptide in the living body include those derived from Prothrombin (FNPRTFGS, SEQ ID NO: 19) and those derived from HGFA (LRPRIIGG, SEQ ID NO: 20). Examples of artificial peptides include Artificial Thrombin cleavable sequence (TFLTPRGVRLG, SEQ ID NO: 17 and TFPPRSFLG, SEQ ID NO: 18) (Maike G. et al., PLos ONE, 2012, Vol. 7, p. E31756-3171; The Journal of Neuroscience, 2012, Vol. 32, No. 22, p.7622-7631) and the like. As the thrombin-sensitive peptide, a peptide having an amino acid sequence represented by SEQ ID NO: 17 to 20 is preferable, and a peptide having an amino acid sequence represented by SEQ ID NO: 17 is more preferable.
[0045]
　One or more protease-sensitive peptides can be used. By using a protease-sensitive peptide having different cleavage activities for the same protease or a protease-sensitive peptide for different proteases in combination, it is possible to chemically modify a plurality of sites with PEG and control the cleavage activity. ..
[0046]
　For the protease-sensitive peptide, for example, using a known gene recombination technique, a nucleic acid sequence (DNA) encoding a protease-sensitive peptide sequence is designed in succession with a nucleic acid sequence (DNA) encoding HGF or an active fragment thereof, and a cell is used. It can be obtained as HGF or an active fragment thereof to which a protease-sensitive peptide is added by transiently or stably introducing it into the nucleic acid and expressing it.
[0047]
　The above-mentioned protease-sensitive peptide may be one to which an artificial sequence such as a tag sequence is added for the purpose of purification or the like. Examples of the tag sequence include a 6 × His tag, a HAT tag, a c-Myc tag, a FLAG tag, a DYKDDDDK tag, a Strep tag, an HA tag, a GST tag, and an MBP tag. Furthermore, in order to remove these tag sequences after purification of HGF or an active fragment thereof to which a protease-sensitive peptide has been added, in addition to the protease-sensitive peptide, a protease-sensitive peptide for purification, a spacer sequence for purification, etc. The sequence can be optionally inserted between the protease sensitive peptide and the tag sequence. In this case, the HGF or an active fragment thereof to which the protease-sensitive peptide obtained by purification and removal of the tag sequence is added contains a cleavage fragment of a sequence such as a protease cleavage peptide for purification or a spacer sequence for purification. May be.
[0048]
　The above-mentioned protease-sensitive peptide may be one in which a natural amino acid such as cysteine ​​or an unnatural amino acid such as azidophenylalanine is inserted for the purpose of PEG modification or the like. Insertion of the above amino acid into a protease-sensitive peptide is performed, for example, by using a known gene recombination technique to obtain a nucleic acid sequence (DNA) encoding an amino acid to be continuously added to a nucleic acid sequence (DNA) encoding the protease-sensitive peptide sequence. It can be carried out by designing, transiently or stably introducing it into cells, and expressing it. The amino acid insertion site for the purpose of PEG modification or the like is preferably the terminal (amino terminal or carboxyl terminal) of the protease-sensitive peptide.
[0049]
　なお、上記のプロテアーゼ感受性ペプチドは、プロテアーゼ感受性ペプチドと、精製等を目的としたタグ配列等の人工配列又はＰＥＧ修飾等の目的で挿入されたアミノ酸と、の間に、公知の遺伝子組換え技術を用いてスペーサー配列として１～２０個のアミノ酸が付加されたものであってもよい。プロテアーゼ感受性ペプチドと、精製等を目的としたタグ配列等の人工配列又はＰＥＧ修飾等の目的で挿入されたアミノ酸と、の間にスペーサー配列が付加されたプロテアーゼ感受性ペプチドは、例えば、公知の遺伝子組み換え技術を用いてプロテアーゼ感受性ペプチドをコードする核酸配列（ＤＮＡ）に連続して任意に選択したスペーサー配列をコードする核酸配列（ＤＮＡ）を付加し、さらに連続して精製等を目的としたタグ配列等の人工配列又はＰＥＧ修飾等を目的としたアミノ酸を挿入し、細胞に一過性又は安定的に導入し、発現させることで、取得することができる。上記スペーサー配列に使用できるアミノ酸としては、例えば、アスパラギン酸（Ｄ）、グルタミン酸（Ｅ）、ヒスチジン（Ｈ）、リジン（Ｋ）、アルギニン（Ｒ）、フェニルアラニン（Ｆ）、チロシン（Ｙ）、トリプトファン（Ｗ）、アラニン（Ａ）、システイン（Ｃ）、アスパラギン（Ｎ）、グルタミン（Ｑ）、セリン（Ｓ）、スレオニン（Ｔ）、イソロイシン（Ｉ）、ロイシン（Ｌ）、ノルロイシン（Ｎｌｅ）、メチオニン（Ｍ）、バリン（Ｖ）及びトリプトファン（Ｗ）が挙げられる。この際、任意で付加されるスペーサー配列は、プロテアーゼ感受性ペプチドの末端に付加されていることが好ましく、ＨＧＦ又はその活性断片のアミノ末端に付加されたプロテアーゼ感受性ペプチドのアミノ末端にスペーサー配列のカルボキシル末端が付加されており、該スペーサー配列のアミノ末端に精製等を目的としたタグ配列等の人工配列又はＰＥＧ修飾等の目的で挿入されたアミノ酸が付加されていることがより好ましい。上記スペーサー配列は、プロテアーゼ感受性ペプチドの切断効率や、ＰＥＧ修飾によるＨＧＦ又はその活性断片の活性制御の効率を考慮して適宜選択される。
[0050]

　PEG is a highly biocompatible polymer polymer containing water-soluble poly (ethylene oxide), and by chemically modifying a protein with PEG, physical stability and thermal stability with respect to the protein are obtained. , It is known to add clinical usefulness such as resistance to proteolytic enzymes, improvement of solubility, reduction of distribution volume in vivo, improvement of retention in blood (Inada et al., Journalal). of Bioact and Compact Polymers, Vol. 5, 1990, p.343; Delgado et al., Critical Reviews in Protein Systems, Vol. 9, 1992, 1992, p. 1993, p.91).
[0051]
　As the PEG used for producing a PEG-modified form of HGF or an active fragment thereof according to the present invention, those known in the art can be used. Typically, it comprises a repeating unit structure "-(CH 2 CH 2 O) n- " and the structure of the end groups or the entire PEG can change as described below. For example, "-CH 2 CH 2 -O (CH 2 CH 2 O) n -CH 2 CH 2- " and "-(OCH 2 CH 2 ) n " depending on the presence or absence of substitution of the oxygen atom at the terminal. O- "can be included. The PEG used to prepare a PEG-modified form of HGF or an active fragment thereof according to the present invention has a capped end, and as a specific example thereof, a functional group having a reactivity at one end of the PEG (for example, maleimide). The one capped with (base) can be mentioned. Further, the other end of the PEG may be capped, or may be modified to increase hydrophilicity, for example, by introducing a hydroxyl group. When PEG having a branched structure is used, at least one end may be capped with a reactive functional group such as the maleimide group described above, and each end of the other branched chain may be modified. good. In the present invention, in order to chemically modify HGF or an active fragment thereof to which a protease-sensitive peptide is added, a functional group is used so that the PEG terminal exhibits a covalent bond reactivity with HGF or an active fragment thereof to which a protease-sensitive peptide is added. Activated PEG can be used.
[0052]
　The structure of PEG contained in the PEG-modified form of HGF or an active fragment thereof according to the present invention is not particularly limited, and there is no problem with a linear structure or a branched structure, but the hepatic directivity of HGF or an active fragment thereof is reduced. It is preferable to have a branched structure in terms of causing it to occur. Examples of the PEG having a branched structure include 2-branched PEG, 3-branched PEG, and 4-branched PEG, and the number of branches includes more than that, but among them, 4-branched PEG. Is preferable. More specifically, a structure in which two branches, three branches or four branches are branched from the end of the linker portion up to the functional group, a structure in which the PEG chain is further branched from the PEG chain branched from the end of the linker portion up to the functional group, and the like. A structure in which the PEG chain is branched from the end of the linker moiety up to the functional group and the middle of the linker moiety can be mentioned, and the number average molecular weight of each branched PEG chain may be the same or different. Among them, a structure in which the PEG chain is bifurcated from the linker moiety up to the functional group and the PEG chain is further bifurcated from each branched PEG chain is preferable, and the PEG chain is bifurcated from the linker moiety up to the functional group, respectively. The most preferable structure is a structure having a total of four PEG chains having the same number average molecular weight, in which the PEG chain is further branched into two from the branched PEG chain. As the PEG having the most preferable structure, for example, SUNBRIGHT GL4-800MA (NOF CORPORATION) of Yuka Sangyo Co., Ltd. can be mentioned. The preferred embodiment relating to the structure of PEG and the preferred embodiment relating to the number average molecular weight of PEG can be arbitrarily combined. For example, it is a 4-branched type, PEG having a number average molecular weight of 20000 to 100,000, the PEG chain is branched into two from the linker portion up to the functional group, and the PEG chain is further branched into two from each branched PEG chain, the same number. Examples thereof include PEG having a structure having a total of four PEG chains having an average molecular weight and having a number average molecular weight of 70,000 to 90,000.
[0053]
　PEG can be synthesized by ring-opening polymerization of ethylene oxide according to a commercially available product, or a known method or a method similar thereto (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, p. 138-). 161).
[0054]
　The number average molecular weight of PEG contained in the PEG-modified form of HGF or an active fragment thereof according to the present invention is preferably 20000 to 100,000, more preferably 40,000 to 100,000, and further preferably 60,000 to 100,000 in order to attenuate HGF activity. Most preferably 70,000 to 90,000. It is known that the effect of chemically modifying PEG to prolong the in vivo half-life of proteins and the like correlates with the number average molecular weight of PEG (Sundqvist T. et al., Computers and Biomedical Research, 1988, Vol. 21). , No. 2, p.110-116), in general, if the number average molecular weight is 20000 or more, a sufficient effect of extending the in vivo half-life can be expected.
[0055]
　プロテアーゼ感受性ペプチドを付加したＨＧＦ又はその活性断片をＰＥＧで化学修飾するために、ＰＥＧの末端を活性化する必要がある。末端が活性化したＰＥＧの例としては、Ｎ－ヒドロキシスクシンイミドエステル、ニトロベンゼンスルホネートエステル、マレイミド、オルトピリジルジスルフィド、ビニルスルフォン、ヨードアセトアミド、カルボン酸、アジド、ホスフィン又はアミン構造で末端が活性化されたＰＥＧが挙げられ、これらは、市販品、又は、公知の方法に従い合成できる。例えば、各種反応性官能基により活性化されたＰＥＧとして、油化産業株式会社のＳＵＮＢＲＩＧＨＴ（登録商標、日油株式会社）シリーズが挙げられる。本明細書において、プロテアーゼ感受性ペプチドを付加したＨＧＦ又はその活性断片との共有結合反応性を示すようＰＥＧ末端に付加された反応性官能基を単に「官能基」、これら反応性官能基を末端に持つＰＥＧを「官能基により活性化されたＰＥＧ」と記載する。官能基は修飾部位により適宜選択されるが、例えば、アミノ基に対しては、官能基としてＮ－ヒドロキシスクシンイミド（ＮＨＳ）エステル基を用いることで選択的な修飾が可能である。また、システイン残基の側鎖に存在するチオール基は、官能基としてマレイミド基を用いた選択的な修飾が可能である。そして、グルタミン酸の側鎖及びカルボキシル末端に存在するカルボキシル基に対しては、アミノ基等を用いて修飾が可能である。また、アジドフェニルアラニン等の非天然型アミノ酸に含まれるアジド基に対しては、トリアリルホスフィンを用いることで選択的な修飾が可能である。これらの場合、ＰＥＧで化学修飾されたプロテアーゼ感受性ペプチドを付加したＨＧＦ又はその活性断片、すなわち、本発明に係るＨＧＦ又はその活性断片のＰＥＧ修飾体には、ＰＥＧとプロテアーゼ感受性ペプチドとの共有結合反応により生じた官能基が含まれていてもよく、例えば、システインに含まれるチオール基とマレイミド基との共有結合反応の場合にはスクシンイミド基が含まれる。
[0056]
　The preferred embodiments of HGF or an active fragment thereof, preferred embodiments of protease-sensitive peptides, and preferred embodiments of PEG can be arbitrarily combined.
[0057]
In
　the PEG-modified product of HGF or an active fragment thereof according to the present invention, as described above, PEG is covalently bonded to the terminal (amino terminal, carboxyl terminal) of HGF or the active fragment thereof via a protease-sensitive peptide. There is. Examples of the production method include a method of covalently binding PEG with HGF or an active fragment thereof to which a protease-sensitive peptide is added.
[0058]
　An example of a method for producing HGF or an active fragment thereof to which a protease-sensitive peptide is added is given below. The HGF or its active fragment and the protease-sensitive peptide can be expressed as a single molecule of protein without preparing them separately. For example, the HGF or its active fragment to which the protease-sensitive peptide is added can be expressed by using the gene recombination technique described later. By designing a nucleic acid sequence (DNA) encoding a protease-sensitive peptide sequence in succession with a nucleic acid sequence (DNA) encoding HGF or an active fragment thereof, and transiently or stably introducing and expressing it in a cell. , Can be obtained. A nucleic acid sequence (DNA) containing an arbitrary spacer sequence is between HGF or an active fragment thereof and a protease-sensitive peptide, or an artificial sequence such as a tag sequence for purification or PEG modification with a protease-sensitive peptide, etc. A spacer sequence can also be added by designing it so that it is continuously added between the amino acid inserted for the purpose of.
[0059]
　In the production of HGF or an active fragment thereof to which the protease-sensitive peptide has been added, for example, HGF or HGF to which the protease-sensitive peptide has been added, which is obtained by expressing the HGF with the protease-sensitive peptide added using gene recombination technology. It is preferable to use the active fragment. At that time, the protease-sensitive peptide is preferably directly bonded to the amino terminus of HGF or an active fragment thereof, and the carboxyl terminus of the protease-sensitive peptide is preferably directly bonded to the amino terminus of HGF or an active fragment thereof.
[0060]
　Purification or concentration of HGF or an active fragment thereof to which a protease-sensitive peptide is added can be carried out by using a known method, for example, ion exchange, gel filtration, chromatography using a hydrophobic carrier or an affinity carrier, and the like. Alternatively, an unreacted HGF or an active fragment thereof or a functional group-activated PEG or a by-product is removed by a combination thereof to purify or concentrate the HGF or an active fragment thereof to which a protease-sensitive peptide is added. Can be done.
[0061]
　HGF or its active fragment can be extracted from tissues, protein synthesis using gene recombination technology, biological production using recombinant cells expressing HGF or its active fragment (or natural cells expressing HGF), etc. It can also be obtained by using a known method of. Further, as the HGF or an active fragment thereof, a commercially available HGF or an active fragment thereof can also be used.
[0062]
　It is known that HGF or an active fragment thereof can be expressed using prokaryotic cells or eukaryotic cells. For example, NK1 is one of the active fragments of HGF using a known gene recombination technique. It can be obtained by expressing NK1 by transiently or stably introducing a nucleic acid sequence (DNA) encoding NK1 into cells.
[0063]
　For gene recombination technology, for example, Molecular Cloning, 2nd ed. , Cold Spring Harbor Laboratory (1989). An example of the above technique will be described below.
[0064]
　Prepare DNA encoding HGF or an active fragment thereof. The DNA can be obtained by selecting it from a cDNA library prepared from human tissues or cells, taking it out, and subjecting it to a DNA amplification method such as a PCR method. Alternatively, the DNA can be chemically synthesized using, for example, a DNA synthesizer utilizing the phosphoramidite method.
[0065]
　The above DNA is incorporated into an appropriate vector to prepare an expression vector. Such vectors contain elements such as regulatory sequences necessary to express (and secrete, if necessary) the DNA. Specific examples of elements include translation start codons and stop codons, promoters, enhancers, terminators, ribosome binding sites (or Shine dalgarno sequences), selectable marker sequences, signal sequences, etc., and necessary elements are inserted into the vector. NS.
[0066]
　The promoter, promoters suitable depending on the host cell is selected, for example, the genus Escherichia (Escherichia) promoter suitable bacterial cell is a prokaryote, for example a trp promoter, lac promoter, recA promoter, .lambda.P L be a promoter , SPO1 promoter, SPO2 promoter, penP promoter and the like are suitable promoters for cells of Bacillus bacteria. Suitable promoters for yeast cells are, for example, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX promoter and the like. Suitable promoters for plant cells include, for example, the cauliflower mosaic virus (CaMV) promoter. Suitable promoters for insect cells are, for example, P10 promoter, polyhedrin promoter and the like. Suitable promoters for mammalian cells include, for example, Rous sarcoma virus, polyomer virus, chicken head virus, adenovirus, bovine papillomavirus, trisarcoma virus, cytomegalovirus (SMV), Simian virus 40 (SV40), vaccinia virus and the like. Viral promoters, melotionine promoters, heat shock promoters and the like.
[0067]
　Examples of the selection marker include HIS3 gene, LEU2 gene, TRP1 gene, URA3 gene, dihydrofolate reductase gene (methotrexate (MTX) resistance), ampicillin resistance gene, neomycin resistance gene, canamycin resistance gene and the like.
[0068]
　As the expression vector, a vector suitable for the host cell is selected, and for example, a plasmid, a phage, a cosmid, a viral vector, an artificial chromosome (for example, BAC, YAC, etc.) or the like is usually used. Examples of prokaryotic vectors include Escherichia coli-derived plasmids, such as pCR-based plasmids, pBR-based plasmids, and pUC-based plasmids, and Bacillus subtilis-derived plasmids, such as pUB110, pTP5, and pC194. The yeast vector is a yeast-derived plasmid, for example, a pSH-based plasmid or the like. The vector for plant cells is a binary vector or the like. Regarding vectors for mammalian cells, commercially available vectors such as pBK-CMV, pcDNA3.1, pZeoSV (Invitrogen, Stragene), and viral vectors (for example, adenovirus, adeno-associated virus, poxvirus, simple herpesvirus, lentivirus) , Sendai virus, vaccinia virus, SV40, etc.).
[0069]
　Examples of host cells include prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, plant cells, and animal cells (for example, mammalian cells, insect cells, etc.). When transforming or transfecting the above expression vector into a host cell, known methods such as an electroporation method, a microinjection method, a cell fusion method, a DEAE dextran method, a calcium phosphate method, and a particle gun method can be used.
[0070]
　As a method for binding PEG to HGF or an active fragment thereof to which a protease-sensitive peptide is added, for example, HGF to which the above-mentioned protease-sensitive peptide is added or an active fragment thereof is covalently bound to PEG activated by a functional group. A method, in other words, a method of chemically modifying HGF or an active fragment thereof to which the above-mentioned protease-sensitive peptide is added with PEG activated by a functional group can be mentioned.
[0071]
　To chemically modify HGF or an active fragment thereof to which the above protease-sensitive peptide is added, amino acids contained in the protease-sensitive peptide, or cysteines or unnatural amino acids artificially introduced into the protease-sensitive peptide (for example, Amino group, thiol group, carboxyl group, azide group and the like contained in azidophenylalanine) can be used. Known gene recombination techniques can be used for chemical modification of HGF or an active fragment thereof to which the above protease-sensitive peptide is added by PEG (US Pat. No. 4,917,888, 1987/00056, 1999). / 55377, Bioorganic & Medical Chemistry Letters, Vol. 14, 2004, p.5743-5745). For example, when cysteine ​​is artificially inserted into a protease-sensitive peptide added to the amino end or carboxyl end of HGF or an active fragment thereof, the thiol group present in the side chain of the cysteine ​​residue is an intramolecular or intermolecular disulfide. Since no bond is formed, it is possible to perform site-selective chemical modification of the thiol group with PEG having a maleimide group as a functional group. Further, as a method of inserting an unnatural amino acid into the terminal of the protease-sensitive peptide in HGF or an active fragment thereof to which a protease-sensitive peptide is added, a method of introducing azidophenylalanine, azido-Z-lysine or the like by codon modification has been reported. (Japanese Patent Laid-Open No. 2009-207490), it is also possible to perform site-selective chemical modification of the azide group contained in these unnatural amino acids with a PEG having triarylphosphine as a functional group. .. The protease-sensitive peptide can also be used as a site for biotin labeling in cells using a known biotin-ligase expression system (Avidity) and chemically modified with PEG by utilizing the interaction with avidin.
[0072]
　Among them, amino acids (natural type, non-natural type) showing selective reactivity to functional groups are artificially inserted into the terminal of a protease-sensitive peptide added to the terminal (amino terminal, carboxyl terminal) of HGF or an active fragment thereof. The method of Insertion of amino acids (natural type, non-natural type) showing selective reactivity to functional groups can be carried out using the above-mentioned gene recombination technique. In particular, when preparing a PEG-modified form of HGF or an active fragment thereof according to the present invention, it is more preferable to artificially insert a cysteine ​​residue, an azidophenylalanine residue or an azido-Z-lysine residue, and cysteine. It is more preferred to artificially insert the residue. In this case, it is preferable to use PEG activated by a maleimide group.
[0073]
　Purification or concentration of a PEG-modified product of HGF or an active fragment thereof according to the present invention can be carried out by using a known method after chemically modifying with PEG via a protease-sensitive peptide. For example, unreacted HGF or PEG in which active fragments or functional groups thereof are activated by methods such as ion exchange, gel filtration, chromatography using a hydrophobic carrier or an affinity carrier, or a combination thereof, or a sub The product can be removed to purify or concentrate the PEG-modified form of HGF or an active fragment thereof.
[0074]

　The PEG-modified form of HGF or an active fragment thereof according to the present invention can be expected to have an effect of prolonging the in vivo half-life by PEG modification, and further, the PEG-modified form in a target disease tissue expressing a specific protease. By cleaving the protease-sensitive peptide contained in HGF, the physiological activity inherent in HGF or an active fragment thereof can be expressed, so that side effects on the liver are reduced in medicines (for example, organs such as renal fibrosis). It can be used as an active ingredient of a therapeutic or prophylactic agent for fibrosis.
[0075]
　The physiological activity of the PEG-modified form of the above HGF or an active fragment thereof can be easily measured using cultured cells. For example, the pharmacological actions of natural HGF such as c-Met phosphorylation-inducing action, cell proliferation action, cell migration action, and anti-apoptotic action can be used as indicators (Rubin JS et al., The Journal of Biological Chemistry). , 2001, Vol. 276, No. 35, p. 32977-32983; Lietha D. et al., The EMBO Journal, 2001, Vol. 20, No. 20, p. 5543-5555; Liu Y. et al., American Journal. ofPhysiology, 1999, Vol. 277, No. 4, p.624-633).
[0076]
　Further, the behavior of the release of the active HGF or the active fragment thereof from the PEG-modified form of the above-mentioned HGF or the active fragment thereof is simplified by using, for example, the change in the molecular weight of the PEG-modified form and the active form by SDS electrophoresis as an index. It is possible to measure.
[0077]
　The in vivo half-life of the PEG-modified form of the above HGF or its active fragment is calculated, for example, by measuring the blood concentration when intravenously, intraperitoneally, subcutaneously or intradermally administered to a model animal. can. In particular, it can be easily measured by radiolabeling the PEG-modified form of the above HGF or an active fragment thereof.
[0078]
　A drug containing the PEG-modified form of HGF or an active fragment thereof as an active ingredient is a disease (acute inflammatory disease, chronic inflammatory disease, acute ischemic disease or chronic) that can utilize the pharmacological action of HGF or the active fragment thereof. It can be used for the treatment of ischemic diseases, etc.). As a specific disease, for example, it can be used for the treatment of muscle atrophic lateral sclerosis, organ fibrosis, diabetes, spinal cord injury, peritoneal adhesion, transplantation treatment, wound healing, neuropathic pain and the like.
[0079]
　In a drug containing a PEG-modified form of HGF or an active fragment thereof as an active ingredient, for example, when the protease-sensitive peptide is an ADAM17-sensitive peptide, it is compared with the liver in which side effects of HGF or the active fragment thereof are a concern. It can be used as a therapeutic or prophylactic agent for diseases (for example, renal fibrosis) that develop in an organ (for example, the liver) in which the expression level of ADAM17 is high. In addition, in a healthy person, even if the expression level of ADAM17 is equal to or lower than that of the liver, the expression level of ADAM17 is increased due to the lesion, and the expression level of ADAM17 is higher than that of the liver. If there is, a drug containing the PEG-modified form of HGF or an active fragment thereof as an active ingredient can also be used as a therapeutic agent for the disease.
[0080]
　The above-mentioned medicines can be used as useful therapeutic agents for mammals (eg, mice, rats, hamsters, rabbits, dogs, cats, monkeys, cows, sheep or humans), especially humans. When the above-mentioned drug is used as a useful therapeutic agent for humans, it is preferable that the above-mentioned HGF or an active fragment thereof is based on an amino acid sequence derived from human HGF.
[0081]
　As an administration form of the above-mentioned drug, the PEG-modified form of the above-mentioned HGF or an active fragment thereof, which is an active ingredient, may be administered orally or parenterally as it is or in combination with a pharmaceutically acceptable carrier. can. It is preferably administered by subcutaneous, intramuscular or intravenous injection.
[0082]
　Dosage forms for oral administration of the above-mentioned pharmaceuticals include, for example, tablets, pills, capsules, granules, syrups, emulsions or suspensions, which can be produced by known methods. It can contain carriers or excipients commonly used in the pharmaceutical field and, if necessary, additives. Carriers and excipients for tablets include, for example, lactose, maltose, saccharose, starch or magnesium stearate. Examples of the additive include a binder, a disintegrant, a preservative, a delayed release agent, a colorant, a flavoring agent, a stabilizer, a dissolving agent, a thickener, an emulsifier and the like.
[0083]
　Dosage forms for parenteral administration of the above drugs include, for example, injections, eye drops, ointments, poultices, suppositories, nasal absorbents, pulmonary absorbents, transdermal absorbents or topical sustained release agents. These can be produced by known methods. In the solution preparation, for example, the PEG-modified form of the above HGF or an active fragment thereof, which is an active ingredient, is dissolved in a sterile aqueous solution used for an injection or suspended and emulsified in an extract, and embedded in liposomes. Can be prepared in the state. The solid preparation can be prepared as a freeze-dried product by adding mannitol, trehalose, sorbitol, lactose, glucose or the like as an excipient to the PEG-modified form of the above-mentioned HGF or an active fragment thereof, which is an active ingredient. Further, this can be powdered and used. Further, these powders can be mixed with polylactic acid, glycolic acid or the like to be solidified and used. The gelling agent is prepared by dissolving, for example, a PEG-modified product of the above-mentioned HGF or an active fragment thereof, which is an active ingredient, in a thickener such as glycerin, PEG, methyl cellulose, carboxymethyl cellulose, hyaluronic acid or chondroitin sulfate, or a polysaccharide. can do. In addition, the above additives can be added to the formulation, if necessary.
[0084]
　The above-mentioned medicine is appropriately determined according to the age, body weight, target disease, symptom, administration form, administration route, molecular weight of PEG, etc. of the patient, but is generally 0.001 mg to 100 mg / kg / dose. It can be administered in the range of 0.01 mg to 10 mg / kg / dose once / month to once a day, preferably once a month to once a week.
[0085]
　The above-mentioned drugs may be used in combination or in combination with other drugs in order to supplement or enhance the therapeutic or preventive effect or reduce the dose.
[0086]
　In another aspect, the PEG-modified form of HGF or an active fragment thereof according to the present invention comprises a PEG-modified form of HGF or an active fragment thereof by utilizing the morphogenesis-inducing action of HGF or an active fragment thereof. It can be used as a differentiation promoting inducer. Examples of the cells include epithelial cells and neural stem cells.
Example
[0087]
　Hereinafter, the PEG-modified form of HGF or an active fragment thereof according to the present invention will be specifically described with reference to Examples, but the present invention is not limited to these examples.
[0088]
(Example 1) Protease-sensitive peptide-added expression of human NK1: Protease-sensitive peptide was added to
　the amino end of human NK1 from which the secretory signal sequence had been removed from its carboxyl end (SEQ ID NO: 9 as ADAM17-sensitive peptide, as thrombin-sensitive peptide SEQ ID NO: 17), DNA encoding human NK1 in which a 6 × His tag and a cysteine ​​residue are sequentially added to the amino terminal of the protease-sensitive peptide (that is, CHHHHH (SEQ ID NO: 4) is added in addition to the protease-sensitive peptide). It was prepared by artificial synthesis (Fasmac) and inserted into the expression vector Cold-shock protease / pColdIV (Takara Bio). The prepared expression plasmid was gene-introduced into the host Escherichia coli SHuffle T7 Express (New England Biolabs).
[0089]
　In the same manner, a protease-sensitive peptide was added to the carboxyl terminus of human NK1 from which the secretory signal sequence had been removed from its amino terminus (SEQ ID NO: 9 as ADAM17-sensitive peptide), and a cysteine ​​residue and 6 were added to the carboxyl terminus of the protease-sensitive peptide. A DNA encoding human NK1 to which the × His tag was added in order (that is, HHHHHHC (SEQ ID NO: 5) was added in addition to the protease-sensitive peptide) was also introduced. Hereinafter, human NK1 to which a 6 × His tag and a cysteine ​​residue are added in the order represented by SEQ ID NO: 4 or 5 in addition to a protease-sensitive peptide (that is, an ADAM17-sensitive peptide or a thrombin-sensitive peptide) is collectively referred to as a protease-sensitive peptide. It is described as peptide-added human NK1.
[0090]
　The above Escherichia coli was applied to LB agar medium containing kanamycin sulfate or carbenicillin, cultured at 37 ° C. for 18 to 22 hours, and subjected to first-stage selective culture. The obtained colonies were inoculated into LB medium containing kanamycin sulfate or carbenicillin, cultured, and subjected to the second stage selective culture. This culture broth was used as a seed culture broth.
[0091]
　Kanamycin sulfate or carbenicillin seed culture, 0.8% Glucose, 0.7% Glycerol, 2% Bacto-Tryptone, 1% casamino Acid, 1% Yeast Extract, 100 mmol / L Potassium Phosphate, 10 mmol / L MgSO 4 and 0 The cells were cultured in a culture solution containing 5.5% NaCl. A glass flask or a mini jar fermenter was used for culturing. After confirming that the turbidity of the culture solution had risen to an appropriate value, Cold-Shock was applied at 15 ° C. or lower. IPTG was added to the culture medium at the same time as Cold-Shock. The culture was terminated 1.5 to 3 hours after Cold-Shock, the culture broth was collected, and centrifugation was performed to obtain an E. coli sediment expressing a protease-sensitive peptide-added human NK1.
[0092]
(Example 2) Purification of protease-sensitive peptide-added human NK1: From
　the Escherichia coli sedimentation obtained in Example 1, protease-sensitive peptide-added human NK1 was purified by the following method.
[0093]
　The E. coli sediment expressing the protease-sensitive peptide-added human NK1 obtained in Example 1 was suspended in 50 mmol / L Tris-HCl, 5 mmol / L EDTA, 150 mmol / L NaCl, 10 mmol / L CaCl 2 , and 10 mmol / L MgCl 2. After crushing using a high-pressure tabletop homogenizer (Niro Soavi), the crushed solution was purified by centrifugation.
[0094]
　A protease-sensitive peptide-added human NK1 was bound to the heparin carrier by passing a centrifugation-cleaned Escherichia coli lysate through a heparin carrier (HiTrappeparin; GE Healthcare) previously equilibrated with PBS (−). Subsequently, a buffer in which the NaCl concentration contained in PBS (−) was changed in the concentration range of 150 mmol / L to 2000 mmol / L was passed in ascending order of the NaCl concentration to obtain each eluted fraction. Each eluted fraction was subjected to SDS electrophoresis to confirm the protease-sensitive peptide-added human NK1 eluted fraction.
[0095]
　Next, a nickel carrier (complete (registered trademark, Roche Diagnostics Co., Ltd.) His-Tag Purification Resin; Roche) equilibrated with PBS (-) containing 300 mmol / L NaCl in advance was obtained by heparin resin purification. Protease-sensitive peptide-added human NK1 was bound to the resin by passing the protease-sensitive peptide-added human NK1 eluted fraction. Subsequently, a buffer in which imidazole (concentration range of 0 mmol / L to 500 mmol / L) was added to PBS (−) containing 500 mM NaCl was passed in ascending order of imidazole concentration to obtain each eluted fraction. Each eluted fraction was subjected to SDS electrophoresis to confirm the protease-sensitive peptide-added human NK1 eluted fraction. The obtained protease-sensitive peptide-added human NK1 elution fraction was concentrated using a centrifugal ultrafiltration device (Amicon Ultra-15, MWCO = 10000; Merck Millipore) to obtain a protease-sensitive peptide-added human NK1.
[0096]
(Example 3) Synthesis of prodrugized human NK1:
　One molecule of protease-sensitive peptide added One molecule of PEG is shared by a protease-sensitive peptide in the amino-terminal cysteine ​​residue or carboxyl-terminal cysteine ​​residue of human NK1. The bound human NK1 (hereinafter, also referred to as prodrugized human NK1) was synthesized by the following method.
[0097]
　The protease-sensitive peptide-added human NK1 obtained in Example 2 was solvent-substituted with 300 mmol / L NaCl, 2 mmol / L EDTA-containing 100 mmol / L phosphate buffer (pH 6.0), and the molar ratio was relative to the protease-sensitive peptide-added human NK1. PEG activated by a functional group was added so as to be 40-fold. Protease-sensitive peptide-added human NK1 was covalently bound to PEG by incubation overnight at 25 ° C. to synthesize prodrugized human NK1. Examples of the PEG activated by the above functional groups include SUNBRIGHT GL2-400MA (bifurcated type, number average molecular weight 40,000, oilification industry), SUNBRIGHT ME-400MA (linear type, number average molecular weight 40,000, oilification industry) and SUNBRIGHT GL4-800MA (4-branched type, number average molecular weight 80000, oilification industry) was used. Human NK1 to which the ADAM17-sensitive peptide (SEQ ID NO: 9) was added to the amino terminus was chemically modified with the above three types of PEG (hereinafter, collectively referred to as ADAM17 cleavage-type prodrugized human NK1, and the structure of PEG used for the modification together. And the number average molecular weight). Human NK1 to which the ADAM17-sensitive peptide (SEQ ID NO: 9) was added to the carboxyl terminus was chemically modified with PEG having a 4-branched type and a number average molecular weight of 80,000. Human NK1 to which a thrombin-sensitive peptide (SEQ ID NO: 17) was added to the amino terminus was chemically modified with PEG having a bifurcated type and a number average molecular weight of 40,000 (hereinafter, thrombin-cleaving prodrugized human NK1).
[0098]
　Next, the reaction solution containing the prodrugized human NK1 was diluted 10-fold with PBS (-) containing no NaCl, and then passed through an ion exchange carrier (SP-Sepharose6 Fast Flow; GE Healthcare). PEG in which the functional groups of the reaction were activated was removed. The prodrugized human NK1 adsorbed on the ion exchange carrier was eluted with 1.0 mol / L NaCl-containing PBS (-) and then passed through a heparin carrier (Heparin-Sepharose Fast Flow; GE Healthcare) to collect the pass-through solution. The mixture was concentrated using a centrifugal ultrafiltration device (Amicon Ultra-4, MWCO = 30000; Merck Millipore) to obtain the target protein, prodrugized human NK1.
[0099]
(Example 4) Measurement
　of purity of prodrugized human NK1 and contamination rate of protease-sensitive peptide-added human NK1: Purity of prodrugized human NK1 and contamination of precursor protease-sensitive peptide-added human NK1 not chemically modified with PEG. The rate was measured by reverse phase chromatography using high performance liquid chromatography.
[0100]
　A reverse phase column (Intrada WP-RP; Imtaket) was connected to high performance liquid chromatography (LC-10AD system; Shimadzu Corporation), and ADAM17 cleavage type prodrugized human NK1 (3 types) obtained in Example 3 was injected. did. The acetonitrile ratio was increased over time from 0.1% trifluoroacetic acid-containing distilled water (0% acetonitrile) to 100%, and a peak was detected at a wavelength of 280 nm. The peak with a relative retention time of about 0.65 minutes was designated as the protease-sensitive peptide-added human NK1 with respect to the main peak, the prodrugized human NK1, and the purity of the prodrugized human NK1 and the contamination rate of the protease-sensitive sequence-added human NK1 were measured. Calculated.
[0101]
　As a result, the purity of ADAM17 cleavage type prodrugized human NK1 by chemical modification using PEG having a bifurcated type and a number average molecular weight of 40,000 was 98.9% (protease-sensitive peptide-added human NK1 contamination rate 1.1%). there were. The purity of ADAM17-cleaving prodrugized human NK1 by chemical modification using linear PEG having a number average molecular weight of 40,000 was 97.3% (protease-sensitive peptide-added human NK1 contamination rate was 2.7%). The purity of ADAM17-cleaving prodrugized human NK1 by chemical modification using 4-branched PEG having a number average molecular weight of 80,000 was 97.9% (protease-sensitive peptide-added human NK1 contamination rate 2.1%). Therefore, it was confirmed that all of the prodrugized human NK1s could be obtained with high purity.
[0102]
(Example 5) Measurement of ADAM17 activity expression level in
　kidney disease model mouse tissue : The activity expression level of ADAM17 in kidney disease model mouse tissue was measured using an ADAM17 activity measurement kit (SENSOLYTER520 TACE; ANASPEC). The measurement was performed under two cases under each condition.
[0103]
　A unilateral ureteral ligation model mouse (hereinafter referred to as UUO mouse) was prepared as a kidney disease model mouse. The ureters of male ICR strain mice (Charles River Laboratories, Japan) were ligated with silk thread. The incision site was sutured, and after breeding for 1, 7, 10 and 14 days, the kidney and liver of the mouse were removed. As untreated mice (hereinafter referred to as normal mice), the kidneys and livers of the mice after breeding on the 0th and 14th days were removed. A solvent obtained by adding 0.1% Triton-X100 to Component C attached to SENSOLYTER520 TACE (Anaspec) was added to the tissue pieces, and the tissue was disrupted using a handy microhomogenizer (hist colon). The crushed tissue was allowed to stand at 4 ° C. or on ice for 15 minutes and centrifuged at 4 ° C. and 2000 × g for 15 minutes to obtain a supernatant.
[0104]
　The total protein content of the centrifugation supernatant was measured using the Protein Assay (Bio-Rad) reagent.
[0105]
　The ADAM17 activity in the centrifugal supernatant was detected by measuring the fluorescence at 535 nm when excited at 485 nm, and the ADAM17 activity per 1 μg of protein amount was calculated.
[0106]
　The results are shown in FIG. The vertical axis shows the ADAM17 activity per 1 μg of protein in each example. “Day 0”, “Day 1”, “Day 7”, “Day 10” and “Day 14” on the horizontal axis indicate the tissues of mice bred for 0, 1, 7, 10 and 14 days, respectively. "Normal" indicates a normal mouse, and "UUO" indicates a UUO mouse.
[0107]
　Both UUO and normal mice showed more ADAM17 activity in kidney tissue than in liver tissue. In addition, since the activity of ADAM17 increased with the lapse of days after ureteral ligation, it was shown that the activity of ADAM17 increased in the kidney tissue with advanced kidney disease as compared with the liver tissue and the normal kidney tissue. Therefore, it has been shown that ADAM17 is useful as a target molecule used for controlling its activity in prodrugization of HGF or an active fragment thereof.
[0108]
(Example 6) Measurement of expression level of ADAM17 protein in
　kidney disease model mouse tissue : Expression level of ADAM17 protein in kidney disease model mouse tissue was evaluated by Western blotting. The evaluation was performed under 2 cases under each condition.
[0109]
　From the UUO mice and normal mice prepared by the same method as in Example 5, the UUO mice were bred 7 and 14 days after the ureteral ligation, and the normal mice were bred on the day when the ureteral ligation of the UUO mice was performed (that is, 0 days). ), The kidney and liver were removed, and a handy microhomogenizer (hist colon) was added to the tissue piece by adding 0.1% Triton-X100 to Component C attached to SENSOLYTE® 520 TACE (Anasec). The tissue was crushed using. The crushed tissue was allowed to stand at 4 ° C. or on ice for 15 minutes and centrifuged at 4 ° C. and 2000 × g for 15 minutes to obtain a supernatant.
[0110]
　Western blotting was performed using the obtained supernatant. Anti-ADAM17 antibody-Activation site (abcam) was used as the primary antibody for the detection of ADAM17 precursor and ADAM17 mature product, anti-Rab IgG-HRP (CST) was used as the secondary antibody, and Human was used as the primary antibody for the detection of GAPDH. / Mouse / Rat GAPDH / G3PH Antibodies (R & D Systems) and donkey antigoat IgG-HRP (Santa Cruz) were used as secondary antibodies.
[0111]
　Images taken using the ChemiDoc® XRS + system (Bio-RadRAD) were analyzed and the band strength of the ADAM17 precursor or ADAM17 mature product divided by the band strength of GAPDH was calculated. It was used as an index of the expression level of ADAM17 protein in tissues.
[0112]
　The results are shown in FIG. (A) shows the Western blotting image of each example, and (b) shows the average value calculated from the expression level of ADAM17 protein in the tissue calculated from the band intensity of each example. “Day 0”, “Day 7” and “Day 14” in the figure indicate mice bred for 0, 7 and 14 days, respectively. "Normal" indicates a normal mouse, and "UUO" indicates a UUO mouse. The vertical axis of (b) shows the expression level of ADAM17 protein in the tissue.
[0113]
　In kidney tissue, a decrease in the expression level of ADAM17 precursor protein and an increase in the expression level of ADAM17 mature protein showing ADAM17 activity were observed over time after ureteral ligation.
[0114]
　From these results, it was clarified that the expression level of ADAM17 mature protein was increased in the kidney tissue with advanced kidney disease as compared with the liver tissue and the normal kidney tissue.
[0115]
　From the above, it was shown that ADAM17 is useful as a target molecule used for controlling the activity of HGF or its active fragment in prodrugization.
[0116]
(Example 7) Evaluation of activity release by protease treatment of prodrugized human NK1: The
　amount of human NK1 (active substance) released by protease treatment of prodrugized human NK1 was evaluated by SDS electrophoresis.
[0117]
　The prodrugized human NK1 (ADAM17 cleavage type prodrugized human NK1 (3 types) and thrombin cleaved prodrugized human NK1 (1 type)) obtained in Example 3 was attached to SENSOLYTE 520 TACE (Anasec). Was diluted 10-fold with. ADAM17 (TACE, His Tag, Human Recombinant; Calbiochem) was added to ADAM17 cleavage-type prodrugized human NK1 so that the reaction molar ratio was prodrugized human NK1: ADAM17 = 5: 1. Thrombin (Thrombin from human plasma; Sigma) was added to the thrombin-cleaving prodrugized human NK1 so that the reaction molar ratio was prodrugized human NK1: thrombin = 1:10. As a control of the experiment, the following operation was similarly performed on the prodrugized human NK1 to which the corresponding protease was not added. After incubating at 37 ° C. for 1 hour, the reaction solution was TCA-precipitated, and SDS electrophoresis was performed to confirm the band position obtained by CBB staining.
[0118]
　The results are shown in FIG. In the figure, "-" indicates a sample without protease addition, and "+" indicates a sample with protease addition. Further, "thrombin cleavage type" indicates thrombin cleavage type prodrugized human NK1, and "ADAM17 cleavage type" indicates ADAM17 cleavage type prodrugized human NK1. The molecular weight indicates a number average molecular weight.
[0119]
　It was shown that by treating each prodrugized human NK1 with the corresponding protease (ADAM17 or thrombin), the protease-sensitive peptide contained in each prodrugized human NK1 was cleaved and the active human NK1 was released. Was done.
[0120]
(Example 8) Evaluation of activity attenuation by chemically modifying NK1 with PEG:
　The degree of attenuation of HGF activity of ADAM17 cleavage-type prodrugized human NK1 was determined by the HGF receptor present on the cell surface of human lung epithelial cell line A549. It was evaluated by the In-Cell ELISA method using the amount of phosphorylation induced in the body as an index.
[0121]
　A549 cells were suspended in 10% FCS-containing MEM medium (Nacalai Tesque) , seeded on a 96-well plate for imaging (Becton Dickinson) at a density of 1.5 × 10 4 / well, and cultured overnight. After the cells became about 70% confluent, the medium was replaced with a serum-free MEM medium (Nacalai Tesque), and the cells were cultured for 16 hours or more to achieve a serum starvation state. The prodrugized human NK1 treated with ADAM17 or the prodrugized human NK1 not treated with ADAM17 in Example 7 was added to the cells in a serum starved state, and the cells were reacted at 37 ° C. for 10 minutes. In this example, ADAM17 cleavage type prodrugized NK1 (3 types) obtained in Example 3 by amino-terminal modification was used as the prodrugized human NK1.
[0122]
　The cells were fixed with PBS containing 4% formalin (-), subsequently permeated through the cell membrane with PBS containing 0.3% Triton-X and 0.6% hydrogen peroxide (-), and then blocked with 10% BSA. Processed. Anti-pYcMet (CST) as a primary antibody and anti-Rab IgG-HRP (CST) as a secondary antibody were added to the cells after the blocking treatment and reacted, and then 1 × QuantaRed Enhanced Chemifluorescent HRP Substrate (Thermo Fisher Scientific). Fick) was added. After incubation at room temperature, the fluorescence intensity (RFU) at a wavelength of 590 nm was measured using a plate reader (PerkinElmer) and used as the amount of HGF receptor phosphorylation induced.
[0123]
　From the obtained amount of HGF receptor phosphorylation induced, EC50 of the concentration reaction curve of the sample not treated with ADAM17 was calculated using Prism4 (GraphPad, 4-parameter approximation). Next, as the degree of HGF activity attenuation by PEG modification, the EC50 (nM) of the sample not treated with ADAM17 has the same signal intensity as the EC50 value of the sample not treated with ADAM17, and the sample treated with ADAM17 has the same signal intensity. The value divided by the concentration (nM) (activity fluctuation value (hereinafter, y)) was calculated.
[0124]
　Next, the CBB-stained image obtained in Example 7 was analyzed using the ChemiDoc XRS + system (Bio-Rad), and the cleavage efficiency (hereinafter, w) of the ADAM17-treated prodrugized human NK1 was determined from the band intensity as follows. Calculated according to equation (1). Further, using the purity (%, hereinafter, v) of the prodrugized NK1 obtained in Example 4, the protease-sensitive peptide-added human NK1 (that is, ADAM17 due to poor purification) was obtained by the following formulas (2) to (5), respectively. HGF activity (hereinafter, preNK1) by the sample not added), HGF activity by prodrug human NK1 (hereinafter, Pro), prodrugization cleaved by ADAM17 contained in the sample to which ADAM17 was added. HGF activity by human NK1 (hereinafter, Pro · cut) and HGF activity by prodrugized human NK1 that was not cleaved (that is, uncut) by ADAM17 (hereinafter, Pro · noncut) were defined. At this time, the relative activity (%) of the prodrugized NK1 when the activity of the protease-sensitive peptide-added human NK1 was set to 100 was expressed as x (algebra).

　Cleavage efficiency of prodrugized human NK1 (%, w) = 100- (band strength of prodrugized human NK1 in sample to which ADAM17 was added / band strength of prodrugized human NK1 in sample to which ADAM17 was not added) × 100 ... Formula (1)

　Prodrug-sensitive peptide-added activity by human NK1 contained in the sample to which ADAM17 was not added (preNK1) = (100-v) / 100 × 1 ... Formula (2)

　ADAM17 was added. Activity by prodrugized human NK1 contained in non-prodrug sample (Pro) = (v / 100) × (x / 100) ・ ・ ・ Equation (3)

　Activity by cleaved prodrugized human NK1 contained in the sample to which ADAM17 was added (Pro · cut) = (v / 100) × (w / 100) × 1 ... Formula (4) In

　the sample to which ADAM17 was added Activity by the included uncleaved prodrugized human NK1 (Pro · noncut) = (v / 100) × (100-w) / 100 × (x / 100) ・ ・ ・ Equation (5)
[0125]
　Here, the activity fluctuation value (y) can be described as in the formula (6) using the formulas defined by the formulas (1) to (5).

　Activity fluctuation value (y) = (preNK1 + Pro · cut + Pro · noncut) / (preNK1 + Pro) ・ ・ ・ Equation (6)
[0126]
　By resolving Eq. (6) for algebra x, the following Eq. (7) can be obtained. By substituting the measured values ​​of v, w and y into the formula (7), the relative activity (%) x of the prodrugized NK1 is calculated.

　x = {10 4 x (100-v) + 10 2 x v x w-10 4 x y x (100-v)} / ​​{v x (10 2 x y) -v x (100-w)} ...・ Equation (7)
[0127]
　The results are shown in FIG. The vertical axis shows the fluorescence intensity (RFU) representing the amount of HGF receptor phosphorylation induced, and the horizontal axis shows the treatment concentration (nmol / L) of prodrugized human NK1. In the figure, "ADAM17-" indicates a sample of prodrugized human NK1 not treated with ADAM17, and "ADAM17 +" indicates a sample of prodrugized human NK1 treated with ADAM17. Further, "ADAM17 cleavage type" indicates ADAM17 cleavage type prodrugized human NK1. The molecular weight indicates a number average molecular weight.
[0128]
　In the ADAM17 cleavage type prodrugized human NK1 chemically modified with any of the PEGs, the prodrugized human NK1 (ADAM17-) not treated with ADAM17 was compared with the prodrugized human NK1 (ADAM17 +) treated with ADAM17. It was shown that the activity was low and the HGF activity was attenuated by chemical modification with PEG. Further, compared with the protease-sensitive peptide-added human NK1 not chemically modified with PEG, the ADAM17 cleavage type prodrug-modified human NK1 by chemical modification using PEG having a bifurcated type and a number average molecular weight of 40,000 has an HGF activity of 80.1. ADAM17 cleavage-type prodrug NK1 by chemical modification using PEG with a% attenuation and a linear type with a number average molecular weight of 40,000 has a HGF activity attenuated by 70.7% and a 4-branched type with a number average molecular weight of 80,000. The chemical modification of ADAM17 cleavage-type prodrug NK1 used attenuated HGF activity by 97.1%. Of the three types of PEG, the prodrugized human NK1 by chemical modification using a 4-branched type PEG having a number average molecular weight of 80,000 has the most attenuated HGF activity, and the HGF activity is the human NK1 released by ADAM17 treatment. It was 1/30 of.
[0129]
(Example 9) Evaluation of release of active substance from tissue-selective prodrugized human NK1:
　Prodrugized human NK1 was exposed to a tissue disruption solution, and the amount of released human NK1 (active substance) was subjected to SDS electrophoresis. Evaluated by. In this example, the 4-branched type, amino-terminal modified ADAM17 cleavage type prodrugized human NK1 obtained in Example 3 by modification with PEG having a number average molecular weight of 80,000 was used. The evaluation was performed in 2 or 3 cases under each condition.
[0130]
　UUO mice were prepared in the same manner as in Example 5 using male mice of the C57BL / 6J strain (Charles River Laboratories, Japan). After ureteral ligation and breeding for 16 days, the kidneys and livers of UUO mice and normal mice were excised, respectively.
[0131]
　50 mmol / L Tris-HCl, 10 mmol / L CaCl 2 and 0.05% Brij35-containing buffer solution having a pH of 7 was added to the kidney and liver of the removed mice, respectively , and the tissue disrupted solution was added in the same manner as in Example 6. I got the Qing dynasty. The amount of protein in the supernatant of the tissue disruption solution was measured using Protein Assay (Bio-Rad).
[0132]
　ADAM17 cleavage type prodrugized human NK1 was added to the supernatant of each tissue disruption solution and incubated at 37 ° C. for 30 minutes.
[0133]
　Western blotting was performed using the incubated sample. HGFα (B-3) mouse monoclonal IgG (Santa Cruz) was used as the primary antibody, and Peroxidase-conjugate AffiniPure Donkey Anti-Mouse IgG (Jackson) was used as the secondary antibody. Since the band of prodrugized NK1 could not be detected by this method, the protease-sensitive peptide-added human NK1 contained in the prodrugized human NK1 (that is, the precursor mixed due to poor purification) and the ADAM17 cleavage type The band of human NK1 released from prodrugized human NK1 was detected and used as an index for evaluation.
[0134]
　The Western blotting image of each example is shown in FIG. A band of the active human NK1 was detected in the sample of ADAM17 cleavage type prodrugized human NK1 mixed with the renal lysate, and the band intensity was determined by the protease-sensitive peptide-added human NK1 contained in the prodrugized human NK1. That is, it was stronger than the precursor mixed due to poor purification), indicating that it was an active substance released from the prodrugized human NK1. In addition, since the band strength of human NK1 was stronger in the UUO renal lysate with high ADAM17 activity, it was shown that more human NK1 was released. On the other hand, in the ADAM17 cleavage type prodrugized human NK1 mixed with the liver crushed solution, the band of the active human NK1 was not detected. Therefore, it was shown that ADAM17 cleavage type prodrugized human NK1 releases human NK1 which is an active substance selectively in kidney tissue.
[0135]
　From the above, ADAM17 cleavage type prodrugized human NK1 has reduced HGF activity in liver tissue and shows higher HGF activity than liver tissue in kidney tissue in which ADAM17 is highly expressed, and is expected to have a medicinal effect in kidney tissue. It was shown that it can be done and side effects in liver tissue can be reduced.
[0136]
(Example 10) Measurement of activity
　of prodrugized human NK1 in vivo: The amount of tissue-selective c-Met phosphorylation induced by ADAM17-cleaving prodrugized human NK1 in vivo was measured by ADAM17-cleaving human being It was evaluated by a western blotting method using a tissue disruption solution of mice administered with NK1 or protease (ADAM17) -sensitive peptide-added human NK1. In this example, the bifurcated type obtained in Example 3, the amino-terminal modified ADAM17 cleavage type prodrugized human NK1 obtained by chemical modification using PEG having a number average molecular weight of 40,000, and the protease obtained in Example 2 ( ADAM17) Sensitive peptide-added human NK1 was used.
[0137]
　UUO mice were prepared in the same manner as in Example 5, and 14 days after ureteral ligation, a protease (ADAM17) -sensitive peptide-added human NK1 (PEG-unmodified) or PEG was bifurcated and had a number average molecular weight of 40,000. Human NK1 was administered via the tail vein at a dose of 400 μg / kg. As a control of the experiment, the following operations were similarly performed on normal mice and UUO mice to which the solvent of prodrugized human NK1 (PBS (-) containing 0.3 mol / L NaCl) was administered. The kidneys and liver were removed from the mice 0.18 hours after administration.
[0138]
　The kidney and liver of the mouse were disrupted using a tabletop multisample cell disruptor (Shakemaster Neo; biomedical science), respectively, and RIPA Buffer containing a phosphatase inhibitor and a protease inhibitor was added and allowed to stand on ice for 2 hours. Then, the supernatant was obtained by centrifugation, and the amount of protein was measured by the same method as in Example 8.
[0139]
　Western blotting was performed using the supernatant. Anti-total cMet (CST) was used as the primary antibody for detection of Total c-Met, anti-Ms IgG-HRP (Jackson) was used as the secondary antibody, and anti-pYcMet was used as the primary antibody for detection of phosphorylated c-Met. (CST), anti-Rab IgG-HRP (CST) was used as the secondary antibody.
[0140]
　Images taken using the ChemiDoc XRS + system (Bio-RadRAD) were analyzed, and the value obtained by dividing the band intensity of phosphorylated c-Met by the band intensity of Total c-Met was calculated. From the calculated value, a relative value when the value in the kidney of the UUO mouse solvent-administered group was set to 1 was calculated, and used as an evaluation index as the amount of HGF receptor phosphorylation induced.
[0141]
　The results are shown in FIG. The vertical axis shows the value of the amount of HGF receptor phosphorylation induced. "Normal + solvent" on the horizontal axis indicates a solvent administration group for normal mice, "UUO + solvent" indicates a solvent administration group for UUO mice, and "UUO + protease-sensitive peptide-added human NK1" indicates protease sensitivity to UUO mice. The peptide-added human NK1 administration group is shown, and "UUO + prodrugized human NK1" indicates a prodrugized human NK1 administration group for UUO mice.
[0142]
　In the kidney tissue of UUO mice, the amount of HGF receptor phosphorylation induced was equivalent between the protease-sensitive peptide-added human NK1 administration group and the prodrugized human NK1 administration group, whereas in the liver tissue, the prodrugized human NK1 was used. The amount of HGF receptor phosphorylation induced was attenuated by 69% as compared with the protease-sensitive peptide-added human NK1. Therefore, the prodrugized human NK1 selectively exerts physiological activity in the target disease tissue (that is, the kidney which is a tissue highly expressing ADAM17) in vivo, and the hepatic orientation inherent in the protease-sensitive peptide-added human NK1 is reduced. Showed that there is.
[0143]
(Example 11) Comparison of activity control of
　prodrugized human NK1 by difference in PEG modification site: PEG modification to the protease-sensitive peptide-added human NK1 obtained in Example 2 in which an ADAM17-sensitive peptide was added to the amino terminal. The effect of the difference in the position of HGF on the regulation of HGF activity was evaluated by the In-Cell ELISA method using the amount of HGF receptor phosphorylation induced on the cell surface of human lung epithelial cell line A549 as an index. In this example, the amino terminus of human NK1 obtained in Example 3 is bifurcated via an ADAM17-sensitive peptide (SEQ ID NO: 9) and chemically modified with PEG having a number average molecular weight of 40,000 to form an ADAM17 cleavage type prodrug. Human NK1 (hereinafter referred to as amino-terminal modified prodrugized human NK1) and PEG obtained in Example 3 in which the carboxyl terminus of human NK1 is bifurcated via an ADAM17-sensitive peptide (SEQ ID NO: 9) and has a number average molecular weight of 40,000. ADAM17 cleavage type prodrugized human NK1 chemically modified with (hereinafter, carboxyl-terminal modified prodrugized human NK1) was used.
[0144]
　A549 cells were suspended in 10% FCS-containing MEM medium (Nacalai Tesque) , seeded on a 96-well plate for imaging (Becton Dickinson) at a density of 1.5 × 10 4 / well, and cultured overnight. After the cells became about 70% confluent, the medium was replaced with a serum-free MEM medium (Nacalai Tesque), and the cells were cultured for 16 hours or more to achieve a serum starvation state. Prodrugized human NK1 or protease-sensitive peptide-added human NK1 was added to serum-starved cells and reacted at 37 ° C. for 10 minutes.
[0145]
　The cells were fixed with PBS containing 4% formalin (-), subsequently permeated through the cell membrane with PBS containing 0.3% Triton-X and 0.6% hydrogen peroxide (-), and then blocked with 10% BSA. Processed. Anti-pYcMet (CST) as a primary antibody and anti-Rab IgG-HRP (CST) as a secondary antibody were added to the cells after the blocking treatment and reacted, and then 1 × QuantaRed Enhanced Chemifluorescent HRP Substrate (Thermo Fisher Scientific). Fick) was added. After incubation at room temperature, the fluorescence intensity (RFU) at a wavelength of 590 nm was measured using a plate reader (PerkinElmer) and used as the amount of HGF receptor phosphorylation induced.
[0146]
　From the obtained amount of HGF receptor phosphorylation induced, protease-sensitive peptide-added human NK1 without PEG modification in each of prodrugized human NK1 in which protease-sensitive peptide and PEG were added to the amino-terminal or carboxyl-terminal of human NK1 The relative value was calculated when the value of was set to 100.
[0147]
　The results are shown in FIG. The vertical axis shows the value of the amount of HGF receptor phosphorylation induced, and shows the relative value when the value at the time of treatment with the protease-sensitive peptide-added human NK1 is 100. The legend "amino-terminal modification" indicates a sample in which a protease-sensitive peptide and PEG are added to the amino-terminal of human NK1, and "carboxyl-terminal modification" indicates a sample in which a protease-sensitive peptide and PEG are added to the carboxyl-terminal of human NK1. The sample is shown.
[0148]
　The amount of HGF receptor phosphorylation induced (relative value) of prodrugized human NK1 is 41 for amino-terminal modified prodrugized human NK1, whereas it is 58 for carboxyl-terminal modified prodrugized human NK1, and the amino terminal is amino-terminal. Modified prodrugized human NK1 was more attenuated by chemical modification with PEG as compared to carboxyl-terminal modified prodrugized human NK1. Therefore, it was shown that the PEG-modified form of HGF or an active fragment thereof of the present invention can further attenuate its activity by modifying the amino terminus of HGF or an active fragment thereof.
Industrial applicability
[0149]
　The polyethylene glycol modified product of the hepatocyte growth factor or the active fragment thereof of the present invention can selectively release the hepatocyte growth factor or the active fragment thereof in the target disease tissue, and thus exhibits high activity in the target disease tissue. It can be used as a drug with reduced side effects in liver tissue derived from hepatocyte growth factor or an active fragment thereof.
[0150]
　SEQ ID NO: 1: Amino acid sequence of human HGF not containing secretory signal sequence
　SEQ ID NO: 2: Amino acid sequence of human NK1 not containing
　secretory signal sequence
　SEQ ID NO: 3: Amino acid terminal of human HGF-derived secretory signal SEQ ID NO: 4: Amino end of human NK1 Amino acid
　SEQ ID NO: 5 of cysteine ​​residue and 6 × His tag added to: Amino acid SEQ ID NO: 6 of 6 × His tag and cysteine ​​residue added to the carboxyl end of human NK1:
　Base sequence of human HGF gene SEQ ID NO:
　7 : Amino acid sequence of human HGF
　SEQ ID NO : 8 to 16: Amino acid sequence of ADAM17-sensitive peptide
　SEQ ID NO : 17 to 20: Amino acid sequence of thrombin-sensitive peptide
The scope of the claims
[Claim 1]
　A polyethylene glycol-modified form of hepatocyte growth factor or an active fragment thereof, in which polyethylene glycol is bound to the end of hepatocyte growth factor or an active fragment thereof via a protease-sensitive peptide.
[Claim 2]
　The polyethylene glycol modified product according to claim 1, wherein the protease-sensitive peptide is an ADAM17-sensitive peptide or a thrombin-sensitive peptide.
[Claim 3]
　The polyethylene glycol modified product according to claim 1, wherein the protease-sensitive peptide is an amino acid sequence represented by any one of SEQ ID NOs: 8 to 20 in the sequence listing.
[Claim 4]
　The polyethylene glycol modified product according to any one of claims 1 to 3, wherein the polyethylene glycol has a number average molecular weight of 20000 to 100,000.
[Claim 5]
　The polyethylene glycol modified product according to any one of claims 1 to 4, wherein the active fragment of the hepatocyte growth factor is NK1.
[Claim 6]
　The polyethylene glycol modified product according to any one of claims 1 to 5, wherein the active fragment of the hepatocyte growth factor is the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing.
[Claim 7]
　A medicament containing the polyethylene glycol modified product according to any one of claims 1 to 6 as an active ingredient.