WO2020226038A1 - Antifibrotic agent - Google Patents
Antifibrotic agent Download PDFInfo
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- WO2020226038A1 WO2020226038A1 PCT/JP2020/016590 JP2020016590W WO2020226038A1 WO 2020226038 A1 WO2020226038 A1 WO 2020226038A1 JP 2020016590 W JP2020016590 W JP 2020016590W WO 2020226038 A1 WO2020226038 A1 WO 2020226038A1
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- amino acid
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- polypeptide
- cytoglobin
- fibrosis
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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Definitions
- the present invention relates to an antifibrotic agent.
- liver cancer develops on the basis of non-alcoholic steatohepatitis (NASH) associated with hepatitis B and C virus infections, heavy drinking and diabetic obesity. That is, liver cancer develops from chronic inflammation and fibrotic liver as a base, and develops at an annual rate of 8% regardless of the cause, and once liver cancer occurs, recurrence and intrahepatic metastasis are repeated.
- NASH non-alcoholic steatohepatitis
- Cirrhosis is a condition in which the liver parenchyma is replaced with extracellular matrix protein such as type I collagen and the number of functional hepatocytes decreases.
- the hepatic parenchyma is replaced by myofibroblasts (MFBs) in which hepatic stellate cells (HSCs) whose main function is vitamin A storage are activated and their traits are changed. It is a pathological condition to be treated.
- Transforming growth factor- ⁇ Transforming growth factor (TGF) - ⁇ ) and connective tissue growth factor (CTGF) are involved in this transformation, and these factors continuously activate hepatic stellate cells.
- TGF Transforming growth factor
- CTGF connective tissue growth factor
- Non-Patent Documents 1 and 2 It has been reported that an increase in myofibroblasts in the parenchyma is a factor that reduces hepatocellular function and contributes to the development of liver cancer (Non-Patent Documents 1 and 2). Therefore, suppression of hepatic stellate cell activation and control of myofibroblasts are thought to lead to the development of therapeutic methods for liver fibrosis and liver cancer.
- Cytoglobin In proteome analysis in fibroblasts before and after fibrosis, cytoglobin (Cygb) was found in hepatic stellate cells (HSC). Cytoglobin is also known to be expressed in the pancreas (pancreatic stellate cells) of extrahepatic organs and fibroblasts near the renal tubular epithelium (Non-Patent Document 3). Cytoglobin-deficient mice (Cygb -/- ) have been prepared for the purpose of clarifying the role of cytoglobin in the liver pathology (Patent Document 1). As a result, the cytoglobin-deficient mice are treated with diethylnitrosamine, which is a liver carcinogen.
- Non-Patent Document 4 Diethylnitrosamine, DEN is easily carcinogenic to administration and that oxidative stress enhancement is involved in the process. Furthermore, it has been found that overexpression of cytoglobin suppresses hepatic stellate cell activation (Non-Patent Document 5).
- Non-Patent Document 6 hypoxic conditions in tissues and cells induce cytoglobin
- Non-Patent Document 7 exposure of liver stellate cells to arundic acid induces cytoglobin
- Non-Patent Document 8 It has also been reported that when human stellate cells in the liver are exposed to Fibroblast growth factor 2, cytoglobin is induced (Non-Patent Document 8).
- a liver disease treating / preventing agent containing menatetrenone (vitamin K-II) as an active ingredient can suppress cell proliferation of hepatocellular carcinoma and suppress the occurrence of portal vein invasion after hepatocellular carcinoma treatment ( Patent Documents 2 to 4), menatetrenone can suppress the carcinogenesis of liver cancer derived from chronic liver disease, liver cirrhosis, or hepatitis C viral liver cirrhosis (Patent Document 5), and has proliferative activity or antiproliferative activity.
- Patent Document 6 A proposal for using a related 1,4-naphthoquinone compound as an anticancer agent has been reported.
- hepatitis C the promotion of comprehensive hepatitis countermeasures promoted by the Ministry of Health, Labor and Welfare has made it possible to obtain a cure rate of 95% or more even in cases of high virus type 1B.
- liver fibrosis There is currently no direct treatment for liver fibrosis. As mentioned above, some have been reported as therapeutic or prophylactic agents for liver diseases, but these agents are therapeutic agents for suppressing carcinogenesis or once carcinogenesis, and are liver fibers that are a pre-carcinogenesis stage. No treatment or prevention of fibrosis has been considered. In addition, although a high cure rate has been obtained for hepatitis C, the virus elimination rate is low when the symptoms progress to liver cirrhosis. Even if the virus is eliminated, the residual activated stellate cells cannot restore liver function and cause cancer. In particular, since liver cirrhosis is a disease with low treatment satisfaction, there is a high unmet medical need for a drug for treating liver cirrhosis. If an effective therapeutic drug for liver cirrhosis is developed, it will have a great impact on society such as improving the QOL of patients and reducing medical expenses, and its medical value is extremely high.
- liver fibrosis which is the mother of liver cancer
- cytoglobin is essential for maintaining the homeostasis of the liver microenvironment and its deficiency is thought to promote liver fibrosis and induce carcinogenesis
- drugs that contribute to the maintenance of cytoglobin in hepatic stellate cells are those of liver fibrosis. It can be a candidate substance as a therapeutic agent.
- arcidic acid and fibroblast growth factor 2 induce cytoglobin.
- arundic acid is a compound whose clinical application to humans has been discontinued.
- fibroblast growth factor 2 is a multifunctional substance, and there are concerns about various undesired actions, so clinical application to humans is expected to be difficult.
- an object of the present invention is to provide an effective drug for suppressing fibrosis.
- the fibrotic reaction can be reduced by exposing cytoglobin itself to cells. Furthermore, it was found that the fibrotic reaction can be further reduced by attaching a specific tag to cytoglobin.
- the present invention has been completed by further studies based on these findings.
- the present invention provides the inventions of the following aspects.
- Item 1. (1) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 1. (2) A polypeptide containing an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and having an ability to suppress fibrosis, and (3) A cytoglobin protein comprising at least one polypeptide having an amino acid sequence having an amino acid sequence identity of 80% or more with respect to the amino acid sequence represented by SEQ ID NO: 1 and having an ability to suppress fibrosis, and the above-mentioned An antifibrinolytic agent comprising a modified cytoglobin protein as an active ingredient, which comprises at least one tag of oligohistidine and TAT peptide bound to the cytoglobin protein.
- Item 2. The antifibrotic agent according to Item 1, wherein the oligohistidine has a length of 3 to 10 amino acid residues.
- Item 3. Item 2. The antifibrotic agent according to Item 1 or 2, wherein the tag is oligohistidine.
- Item 4. Item 2. The antifibrotic agent according to any one of Items 1 to 3, wherein the tag is bound to the N-terminal of the cytoglobin protein.
- Item 4. The antifibrotic agent according to any one of Items 1 to 4, wherein the polypeptide has a length of 152 to 190 amino acid residues.
- Item 6. The polypeptide (11) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
- Item 2. The antifibrotic agent according to any one of Items 1 to 5, wherein the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 is 80% or more, and at least one of the polypeptides capable of suppressing fibrosis. .. Item 7.
- the polypeptide (21) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
- Item 9 The polypeptide (41) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4.
- the anti-fibrotic agent described in Crab. Item 11.
- Item 13 Item 2.
- Item 14. (1) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 1.
- a polypeptide containing an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and having an ability to suppress fibrosis and (3) A cytoglobin protein comprising at least one polypeptide having an amino acid sequence having an amino acid sequence identity of 80% or more with respect to the amino acid sequence represented by SEQ ID NO: 1 and having an ability to suppress fibrosis, and the above-mentioned Use of modified cytoglobin proteins, including at least one tag of oligohistidine and TAT peptide bound to cytoglobin protein, for the production of anti-fibrotic agents.
- Item 15 A polypeptide containing an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and having an ability to suppress fibrosis
- a cytoglobin protein comprising at least one polypeptide having an amino acid sequence having an amino acid sequence identity of 80% or more with respect
- a polypeptide containing the amino acid sequence shown in SEQ ID NO: 1. (2) A polypeptide containing an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and having an ability to suppress fibrosis, and (3) A cytoglobin protein comprising at least one polypeptide having an amino acid sequence having an amino acid sequence identity of 80% or more with respect to the amino acid sequence represented by SEQ ID NO: 1 and having an ability to suppress fibrosis, and the above-mentioned
- a method for treating a fibrotic disease comprising the step of administering an antifibrinolytic agent comprising a modified cytoglobin protein containing at least one tag of oligohistidine and TAT peptide bound to the cytoglobin protein as an active ingredient.
- the antifibrotic agent of the present invention is effective in preventing or treating tissue fibrosis. Furthermore, it is also effective in preventing cancer because it suppresses the suppression of fibrosis, which is the base of cancer.
- the relationship between the exposure time and the expression level of the fibrosis-related protein when the cytoglobin His-rhCYGB having an oligohistidine tag obtained in Test Example 1 was exposed to HHSteC cells is shown.
- the relationship between the dose and the expression level of the fibrosis-related protein when the cytoglobin His-rhCYGB having an oligohistidine tag obtained in Test Example 1 was exposed to HHSteC cells is shown.
- the relationship between the dose and the transcription amount of the fibrosis-related gene when the cytoglobin His-rhCYGB having an oligohistidine tag obtained in Test Example 1 was exposed to HHSteC cells is shown.
- the measurement results of the AST and ALT levels in serum when the cytoglobin His-rhCYGB having an oligohistidine tag obtained in Test Example 2 was administered to a liver fibrosis mouse model are shown.
- the transcription amount of the fibrosis-related gene when the cytoglobin His-rhCYGB having an oligohistidine tag obtained in Test Example 2 was administered to a liver fibrosis mouse model is shown.
- the results of tissue staining of hepatic stellate cells of liver tissue when cytoglobin His-rhCYGB having an oligohistidine tag obtained in Test Example 2 was administered to a liver fibrosis mouse model are shown.
- the relationship between the exposure time and the expression level of the fibrosis-related protein when the cytoglobin TAT-rhCYGB having a TAT peptide obtained in Test Example 3 was exposed to HHSteC cells is shown.
- the relationship between the dose and the expression level of the fibrosis-related protein when the cytoglobin TAT-rhCYGB having a TAT peptide obtained in Test Example 3 was exposed to HHSteC cells is shown.
- the relationship between the dose and the transcription amount of the fibrosis-related gene when the cytoglobin TAT-rhCYGB having a TAT peptide obtained in Test Example 3 was exposed to HHSteC cells is shown.
- the relationship between the exposure time and the transcription amount of the fibrosis-related gene when the cytoglobin 6R-rhCYGB having a 6R peptide tag obtained in Test Example 6 was exposed to HHSteC cells is shown (Comparative Example).
- the expression level of the fibrosis-related protein when the cobalt-substituted cytoglobin Co-CYGB obtained in Test Example 7 was exposed to HHSteC cells is shown (reference example).
- the transcription amount of the fibrosis-related gene when the cobalt-substituted cytoglobin Co-CYGB obtained in Test Example 7 was exposed to HHSteC cells is shown (reference example).
- 6 is a visible absorption spectrum showing the iron heme activity of various cytoglobins obtained in Test Example 8.
- the relationship between the dose and the expression level of the fibrosis-related protein when the cytoglobin protein pp4 having an oligohistidine tag obtained in Test Example 8 was exposed to HHSteC cells is shown.
- the relationship between the dose and the transcription amount of the fibrosis-related gene when the cytoglobin protein pp4 having an oligohistidine tag obtained in Test Example 8 is exposed to HHSteC cells is shown.
- the relationship between the dose and the expression level of the fibrosis-related protein when the cytoglobin protein pp5 having an oligohistidine tag obtained in Test Example 8 was exposed to HHSteC cells is shown.
- the relationship between the dose and the expression level of the fibrosis-related protein when the cytoglobin protein pp6 having an oligohistidine tag obtained in Test Example 8 was exposed to HHSteC cells is shown.
- the relationship between the dose and the expression level of the fibrosis-related protein when the cytoglobin protein pp7 having an oligohistidine tag obtained in Test Example 8 was exposed to HHSteC cells is shown.
- the relationship between the dose and the expression level of the fibrosis-related protein when the cytoglobin protein pp1 having an oligohistidine tag obtained in Test Example 8 was exposed to HHSteC cells is shown.
- the relationship between the dose and the transcription amount of the fibrosis-related gene when the cytoglobin protein pp1 having an oligohistidine tag obtained in Test Example 8 is exposed to HHSteC cells is shown.
- the relationship between the dose and the expression level of the fibrosis-related protein when the cytoglobin protein pp2 having an oligohistidine tag obtained in Test Example 8 was exposed to HHSteC cells is shown.
- the relationship between the dose and the expression level of the fibrosis-related protein when the cytoglobin protein pp3 having an oligohistidine tag obtained in Test Example 8 was exposed to HHSteC cells is shown.
- the serum AST value and ALT value when cytoglobin having an oligohistidine tag is administered to the hepatitis / liver fibrosis model obtained in Test Example 9 are shown.
- the amount of fibrosis-related gene transcription obtained when cytoglobin having an oligohistidine tag is administered to the hepatitis / liver fibrosis model obtained in Test Example 9 is shown.
- the results of tissue staining of liver tissue obtained when cytoglobin having an oligohistidine tag was administered to the hepatitis / liver fibrosis model obtained in Test Example 9 are shown.
- the antifibrotic agent of the present invention is characterized by containing a modified cytoglobin protein modified by a specific tag as an active ingredient.
- a modified cytoglobin protein modified by a specific tag as an active ingredient.
- the cytoglobin protein in the active ingredient of the antifibrotic agent of the present invention means to include both full-length cytoglobin and a partial protein (cytoglobin fragment) of full-length cytoglobin.
- the cytoglobin protein is at least one of the following polypeptides (1) to (3).
- a polypeptide containing an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted, and having an ability to suppress fibrosis and
- the amino acid sequence shown in SEQ ID NO: 1 corresponds to the amino acid sequence of a partial protein (18th to 170th of SEQ ID NO: 5 described later) showing antifibrotic ability in human-derived cytoglobin (SEQ ID NO: 5 described later).
- Cytoglobin (SEQ ID NO: 5 described later) has a 6-coordination structure (heme binding activity) such that the imidazole groups of histidine residues at positions 81 and 113 sandwich the heme, and binds to oxygen, nitric oxide, etc.
- the cytoglobin protein contains the 81st and 113th positions (heme binding sites).
- the cytoglobin protein does not exhibit the antifibrotic effect of the active ingredient in the present invention only by including both heme binding sites, and the expression of the effect is the amino acid sequence of positions 18 to 170 including the heme binding site. Needs.
- the polypeptides shown in (2) and (3) above are variants of the polypeptide shown in (1) above.
- the modification of the introduced amino acid may include only one modification (for example, substitution only) from substitutions, additions, insertions, and deletions.
- the above modifications eg, substitution and insertion
- the number of amino acids to be introduced, substituted, added, inserted or deleted may be one or several, for example, 1 to 32, preferably 1 to 16.
- the number is preferably 1 to 10, more preferably 1 to 8, more preferably 1 to 3, still more preferably 1 or 2, and particularly preferably 1.
- sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 may be 80% or more, preferably 85% or more, preferably 90% or more, still more preferably 95. % Or more, particularly preferably 99% or more.
- sequence identity is defined as BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)].
- sequence identity is defined as BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)].
- NCBI National Center for Biotechnology Information
- conservative substitution is a preferred embodiment of the introduced amino acid substitution.
- conservative substitution means replacing one or several amino acid residues with another chemically similar amino acid residue.
- one hydrophobic residue may be replaced by another hydrophobic residue
- one polar residue may be replaced by another polar residue having the same charge, and the like.
- Functionally similar amino acids capable of making such substitutions are known in the art for each amino acid.
- non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine.
- polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine.
- positively charged (basic) amino acids include arginine, histidine, and lysine.
- negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
- “having an inhibitory ability on fibrosis” means having an inhibitory ability on fibrosis equivalent to that of the polypeptide shown in (1).
- the transcription amount of the fibrosis-related gene transcription amount to mRNA
- the transcription amount of the polypeptide of (1) above and the transcription amount of the fibrosis-related gene are equivalent (that is, the transcription amount of (1) above).
- the transcription amount of the fibrosis-related gene by the polypeptide is 100%, it is about 80 to 120%).
- Examples of fibrosis-related genes include ⁇ Sma, Col1a1, Col3a1, and Tfg ⁇ -1.
- the cytoglobin protein is not particularly limited as long as it is a polypeptide containing the amino acid sequence shown by SEQ ID NO: 1 showing antifibrotic ability, but from the viewpoint of obtaining higher antifibrotic ability, 152 to 190 amino acids. It is preferably a residue-length polypeptide. Specific examples of cytoglobin proteins include the following.
- Specific examples 1 of the cytoglobin protein include at least one of the following polypeptides (11) to (13).
- (11) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
- (12) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and has an inhibitory ability to suppress fibrosis, and (13).
- polypeptide (11) is a partial protein (18th to 170th of SEQ ID NO: 5 described later) that exhibits antifibrotic ability in human-derived cytoglobin (SEQ ID NO: 5 described later).
- the relationship between the polypeptides shown in (12) and (13) and the polypeptide shown in (11) is the relationship between the polypeptides shown in (2) and (3) and the polypeptide shown in (1). Similar to relationships.
- Specific examples 2 of the cytoglobin protein include at least one of the following polypeptides (21) to (23).
- (21) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
- (22) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 and which has the ability to suppress fibrosis.
- (23) A polypeptide having a sequence identity of 80% or more with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 and having an ability to suppress fibrosis.
- the above-mentioned polypeptide (21) consists of an amino acid sequence (4th to 176th positions of SEQ ID NO: 5 described later) containing a portion showing antifibrotic ability in human-derived cytoglobin (SEQ ID NO: 5 described later).
- the relationship between the polypeptides shown in (22) and (23) and the polypeptide shown in (21) above is the relationship between the polypeptides shown in (2) and (3) and the polypeptide shown in (1). Similar to relationships.
- Specific examples 3 of the cytoglobin protein include at least one of the following polypeptides (31) to (33).
- (31) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3.
- (32) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 and which has the ability to suppress fibrosis.
- (33) A polypeptide having a sequence identity of 80% or more with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 and having an ability to suppress fibrosis.
- polypeptide (31) consists of an amino acid sequence (18th to 190th positions of SEQ ID NO: 5 described later) containing a portion showing antifibrotic ability in human-derived cytoglobin (SEQ ID NO: 5 described later).
- the relationship between the polypeptides shown in (32) and (33) and the polypeptide shown in (31) is the relationship between the polypeptides shown in (2) and (3) and the polypeptide shown in (1). Similar to relationships.
- Specific examples 4 of the cytoglobin protein include at least one of the following polypeptides (41) to (43).
- (41) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4.
- (42) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4 and which has the ability to suppress fibrosis.
- (43) A polypeptide having a sequence identity of 80% or more with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4 and having an ability to suppress fibrosis.
- polypeptide (41) consists of an amino acid sequence (4th to 190th positions of SEQ ID NO: 5 described later) containing a portion showing antifibrotic ability in human-derived cytoglobin (SEQ ID NO: 5 described later).
- the relationship between the polypeptides shown in (42) and (43) and the polypeptide shown in (41) is the relationship between the polypeptides shown in (2) and (3) and the polypeptide shown in (1). Similar to relationships.
- Specific examples 5 of the cytoglobin protein include at least one of the following polypeptides (51) to (53).
- (51) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5.
- (52) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5 and which has the ability to suppress fibrosis.
- (53) A polypeptide having a sequence identity of 80% or more with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5 and having an ability to suppress fibrosis.
- the polypeptide of (51) above is a human-derived cytoglobin (SEQ ID NO: 5).
- the relationship between the polypeptides shown in (52) and (53) and the polypeptide shown in (51) is the relationship between the polypeptides shown in (2) and (3) and the polypeptide shown in (1). Similar to relationships.
- the amino acid residue located at the N-terminal is the 2nd to 17th (or 3rd to 15th, 3rd to 13th, 3rd to 3rd) of SEQ ID NO: 5. It is preferable that it is not one of the 11th, 3rd to 9th, 3rd to 7th, especially 3rd to 5th). More preferably, among these polypeptides, those shown as Specific Example 1 of Cytoglobin Protein, Specific Example 3 of Cytoglobin Protein, and Specific Example 5 of Cytoglobin Protein can be mentioned, and particularly preferably, of Cytoglobin Protein. Specific examples 5 include those shown.
- the above cytoglobin protein is modified by a specific tag.
- the present inventors have found that the cytoglobin protein itself permeates the cell membrane, but by having the specific tag, the cell membrane permeability of the cytoglobin protein can be further improved.
- the tag used in the antifibrotic agent of the present invention is oligohistidine or TAT peptide. Oligohistidine and TAT peptides are peptides having cell membrane penetrating ability. Among the known peptides having cell membrane penetrating ability, oligohistidine and TAT peptide exhibit particularly excellent cell membrane penetrating ability when bound to cytoglobin protein.
- Oligohistidine is a peptide consisting of histidine residues.
- the length of the oligohistidine is not particularly limited, and can be appropriately set according to the degree of drug efficacy required for the symptom to which the antifibrotic agent of the present invention is applied.
- the length of oligohistidine includes 3 to 10 amino acid residues, preferably 4 to 8 amino acid residues, and more preferably 5 to 6 amino acid residues.
- the TAT peptide is known as an arginine-rich basic peptide derived from the Transactivator of transcription protein (Tat protein) involved in the transcriptional regulation of the human immunodeficiency virus.
- the TAT peptide is a peptide that constitutes an RNA-binding region of Tat protein, that is, a sequence essential for binding of Tat protein's nuclear localization signal (NLS) to RNA.
- the length of the Tat peptide includes 9 to 15 amino acid residues.
- Specific examples of the Tat protein include the protein shown in SEQ ID NO: 6.
- the TAT peptide derived from the Tat protein set forth in SEQ ID NO: 6 has the sequence RKKRRQRRR (SEQ ID NO: 7) at positions 49 to 57 corresponding to the RNA binding region of the Tat protein set forth in SEQ ID NO: 6.
- the sequence of the entire TAT peptide is derived from the Tat protein.
- the TAT peptide contains positions 49 to 57 of the Tat protein shown in SEQ ID NO: 6, and at the N-terminal, positions 45 to 48, 46 to 48, and 47 to 48 of the Tat protein shown in SEQ ID NO: 6.
- amino acid residue at position or 48 and / or the amino acid residue at position 58-60, 58-59, or 58 at the C-terminus can be contained.
- the amino acid residue other than the RNA-binding region may not be derived from the Tat peptide.
- TAT peptide used in the present invention include the following.
- RKKRRQRRR SEQ ID NO: 7; peptide consisting of positions 49-57 of the Tat protein shown in SEQ ID NO: 6)
- GRKKRRQRRRAHQ SEQ ID NO: 8; peptide consisting of positions 48-60 of the Tat protein shown in SEQ ID NO: 6)
- GRKKRRQRRRA SEQ ID NO: 9; peptide consisting of positions 48-58 of the Tat protein shown in SEQ ID NO: 6)
- YGRKKRRQRRR SEQ ID NO: 10; peptide consisting of positions 47-57 of the Tat protein shown in SEQ ID NO: 6)
- -GRKKRRQRRRPPQ SEQ ID NO: 11
- GRKKRRQRRRP SEQ ID NO: 12
- the TAT peptide has a more excellent effect of improving the cell membrane permeability of cytokines.
- oligohistidine is inferior to TAT peptide in improving the cell membrane permeability of cytoglobin protein, but cytoglobin protein having oligohistidine has antifibrinolytic ability at a lower dose than cytoglobin protein having TAT peptide. It is preferable in that it can be exerted.
- the reason why the cytoglobin protein having an oligohistidine tag is more effective than the cytoglobin protein having a TAT peptide tag regardless of the cell membrane permeability is not clear, but at least it has an oligohistidine tag.
- the location of the cytoglobin protein and the cytoglobin protein having a TAT peptide tag in the cell after permeating the cell membrane is significantly different from each other.
- the binding site of the tag in the cytoglobin protein is not particularly limited, but the N-terminal can be mentioned from the viewpoint of obtaining a preferable cell membrane penetrating ability of the cytoglobin protein and / or a preferable antifibrotic ability. ..
- the tag may be directly bound to the cytoglobin protein, or may be indirectly bound via several amino acid residues, for example, 1 to 5 amino acid residues.
- the tag-modified cytoglobin protein is an amino acid residue that may intervene between the cytoglobin protein sequence, the tag sequence, and the cytoglobin protein sequence and the tag sequence. Alternatively, it may further have 1 to 5 amino acid residues on the opposite side of the tag sequence from the cytoglobin protein side and / or on the opposite side of the tag sequence of the cytoglobin protein.
- the antifibrotic agent of the present invention is a polypeptide in which the above-mentioned polypeptides (1) to (3) and the above-mentioned tag are bound, and the production method thereof is not particularly limited.
- the above-mentioned polypeptides (1) to (3) and the above-mentioned tag may be prepared separately, and then both may be bound to each other, or the above-mentioned polypeptides (1) to (3) and the above-mentioned tag may be combined. It may be prepared as a fusion polypeptide.
- the polypeptides, tags, and fusion polypeptides of (1) to (3) above can be biologically prepared using host cells by various gene recombination techniques available to those skilled in the art.
- polypeptides (1) to (3), tags, and fusion polypeptides can also be prepared by an organic chemical method utilizing a peptide synthesis reaction such as a solid phase synthesis method available to those skilled in the art. It can also be prepared by automatic synthesis using a peptide synthesizer.
- the antifibrotic agent of the present invention may contain other pharmacologically active ingredients in addition to the above-mentioned active ingredients, depending on the type of disease to be treated.
- the antifibrotic agent of the present invention contains, if necessary, a pharmaceutically acceptable carrier or additive in order to prepare a desired dosage form and formulation form. May be good.
- a pharmaceutically acceptable carrier or additive include diluents, excipients, binders, disintegrants, lubricants, suspending agents, solubilizers, stabilizers, sweeteners, colorants, flavoring agents, and odorants. Examples thereof include agents, surfactants, moisturizers, preservatives, pH adjusters, buffers, thickeners and the like.
- the dosage form of the anti-fibrotic agent dosage forms the present invention is not particularly limited, it may be appropriately set depending on the dosage forms and the like.
- Specific examples of the dosage form of the antifibrinolytic agent of the present invention include liquid preparations such as injections, syrups, cell suspensions, and liposome preparations; tablets, hard capsules, soft capsules, granules, powders, etc. Examples include solid preparations such as pills.
- it when it is used as an injection, it may be in the form of a powder for preparation at the time of use (for example, lyophilized powder) which is dissolved in physiological saline or the like before use.
- the antifibrotic agent of the present invention is used by applying it to a disease that is expected to have a preventive or therapeutic effect by suppressing tissue fibrosis in vivo.
- the antifibrotic agent of the present invention can be used for the prevention or treatment of fibrosis, and can also be used for the prevention of cancer based on fibrotic tissue.
- the antifibrotic agent of the present invention can be used for the prevention or treatment of fibrosis of various tissues, and specific examples of the tissues include liver (hepatic stellate cell), pancreas (pancreatic stellate cell), kidney (urinary tubule). Fibrotic cells in the vicinity of the epithelium), brain, thoracic gland, lung, breast, heart, stomach, intestine, skin, bone marrow and the like, preferably liver, pancreas, kidney, and more preferably liver.
- the antifibrotic agent of the present invention can be used for the prevention or treatment of various fibrotic diseases, and specific examples of fibrotic diseases include liver fibrosis, liver cirrhosis, and pancreatic cystic fibrosis.
- fibrotic diseases include liver fibrosis, liver cirrhosis, and pancreatic cystic fibrosis.
- the antifibrotic agent of the present invention can also be used for prevention of various cancers, and specific examples of cancers include liver cancer, pancreatic cancer, kidney cancer, brain cancer, thoracic adenocarcinoma, lung cancer, breast cancer, gastric cancer, and the like. Colorectal cancer, colon cancer, skin cancer, osteosarcoma, chondrosarcoma can be mentioned, preferably liver cancer, pancreatic cancer, kidney cancer, and more preferably liver cancer.
- the organism to be administered may be any organism that is required to suppress fibrosis, such as humans, rats, hamsters, guinea pigs, mice, cows, sheep, pigs, goats, monkeys, and rabbits. Mammals and the like, preferably humans.
- Administration method Examples of the administration form of the antifibrotic agent of the present invention include parenteral administration such as local administration, subcutaneous administration, intraperitoneal administration, intramuscular administration, intravenous administration, transrectal administration, and intradermal administration; oral administration. However, it may be appropriately set according to the type of disease to be applied.
- the dose of the antifibrotic agent of the present invention may be appropriately set according to the type of disease to be applied, the age, sex, body weight, degree of symptom, administration form, etc. of the subject to be administered. For example, per day. , 8 to 10 mg / 60 kg, preferably 9 to 10 mg / 60 kg, more preferably about 9.5 to 9.7 mg / 60 kg, can be administered once or in several divided doses.
- Test Example 1 Effect of His-rhCYGB on HHSteC cells and primary cultured human stellate cells (in vitro)
- a fusion polypeptide His-rhCYGB in which an oligohistidine tag consisting of 6 histidine residues was bound to the N-terminal of cytoglobin shown in SEQ ID NO: 5 was prepared, and for the prepared His-rhCYGB, HHSteC cells were prepared. And the effect on primary cultured human stellate cells was examined.
- His-rhCYGB was cleaned up using fast protein liquid chromatography (FPLC), followed by further cleanup and desalting using a Sephadex G25 column.
- FPLC fast protein liquid chromatography
- the purified His-rhCYGB had no endotoxin contamination, and its toxicity to cells was ruled out.
- the presence of heme was confirmed by the pyridine hemochromogen assay.
- the prepared His-rhCYGB has the sequence shown in SEQ ID NO: 13.
- HHSteC cells from the cell line (purchased from ScienCell Research Laboratories) were used in the experiment.
- Intracellular CYGB and fibrosis-related proteins were confirmed by Western blotting at 1 hour, 4 hours, 8 hours, 24 hours and 48 hours.
- a Western blot image is shown in FIG. As shown in FIG.
- the intracellular CYGB concentration exposed to His-rhCYGB increased in a time-dependent manner.
- the expressed ⁇ SMA decreased in a time-dependent manner.
- the expressed type I collagen (COL1A1) also decreased in a time-dependent manner and disappeared after 48 hours.
- GAPDH is a housekeeping protein. Western blotting was independently tested three times to confirm reproducibility.
- the solution contained in the solution was prepared, exposed to HHSteC cells for 48 hours, and intracellular CYGB was confirmed by Western blot.
- a Western blot image is shown in FIG.
- the intracellular CYGB concentration exposed to His-rhCYGB increased in a dose-dependent manner and reached a plateau at 40 ⁇ g / mL and above.
- the expressed ⁇ SMA and type I collagen (COL1A1) decreased in a dose-dependent manner.
- Western blotting was independently tested three times to confirm reproducibility.
- a phosphate buffer solution containing no His-rhCYGB (0 ⁇ g / mL) and a solution containing His-rhCYGB at a concentration of 5 ⁇ g / mL, 10 ⁇ g / mL, 20 ⁇ g / mL, or 40 ⁇ g / mL in the phosphate buffer solution.
- Test Example 2 Effect of His-rhCYGB on liver fibrosis model (in vivo)
- TAA thioacetamide
- TAA model TAA was intraperitoneally administered twice a week for 10 weeks. During the dosing period, the TAA dose was gradually increased from 50 mg / kg to 400 mg / kg. In addition to TAA, physiological saline was intravenously administered from the tail vein twice a week for 5 weeks from the 6th week to the 10th week (control).
- TAA-CY2 model TAA was intraperitoneally administered for 10 weeks in the same manner as in (i) above, and during the 2 weeks from 9th to 10th week, in addition to TAA, His-rhCYGB was 2 mg / kg.
- TAA was intraperitoneally administered for 10 weeks in the same manner as in (i) above, and for 5 weeks from the 6th week to the 10th week, His-rhCYGB was administered at a dose of 2 mg / kg in addition to TAA. It was administered intravenously from the tail vein twice a week. The dose was determined based on the results of the pilot study.
- FIG. 4 shows the measurement results of AST and ALT levels in serum in the above three groups.
- serum AST and ALT increased to 157.9 and 130.1 IU / L, respectively, by TAA administration, while those values were obtained in the group (TAA-CY2) in which His-rhCYGB was administered for 2 weeks.
- H & E hematoxylin and eosin staining
- SiR saliva red
- astrocyte activation marker ⁇ SMA neutrophil marker Neu
- macrophage marker CD68 astrocyte activation marker CD68
- Test Example 3 Effect of TAT-rhCYGB on HHSteC cells (in vitro)
- a fusion polypeptide TAT-rhCYGB in which the TAT peptide tag shown in SEQ ID NO: 10 was bound to the N-terminal of cytoglobin shown in SEQ ID NO: 5 was prepared, and the prepared TAT-rhCYGB had an effect on stellate cells. was verified.
- TAT-rhCYGB A TEV protease recognition sequence and a TAT site were introduced into the vector used in Test Example 1 to induce a fusion protein in the same manner as in Test Example 1. Since the derived fusion protein has not only a TAT tag but also a His tag, the His tag was removed by treating the fusion protein with TEV protease. As a result, TAT-rhCYGB in which the TAT peptide tag was bound to cytoglobin was obtained.
- the prepared TAT-rhCYGB has the sequence shown in SEQ ID NO: 14.
- HHSteC cells from the cell line were used in the experiment.
- a solution containing 40 ⁇ g / mL TAT-rhCYGB in a phosphate buffer solution was prepared and exposed to HHSteC cells for 0 to 48 hours before exposure (-) and 0.25 hours and 0.5 hours after the start of exposure.
- Intracellular CYGB and fibrosis-related proteins were measured at 1 hour, 4 hours, 8 hours, 24 hours and 48 hours. The results are shown in FIG. As shown in FIG. 7, the intracellular CYGB concentration exposed to TAT-rhCYGB increased in a time-dependent manner, and the expressed ⁇ SMA and type I collagen (COL1A1) decreased in a time-dependent manner.
- GAPDH is a housekeeping protein.
- a phosphate buffer solution contained in was prepared, exposed to HHSteC cells for 48 hours, and intracellular CYGB was measured. The results are shown in FIG. As shown in FIG. 8, the intracellular CYGB concentration exposed to TAT-rhCYGB increased in a dose-dependent manner. In addition, the expressed ⁇ SMA and type I collagen (COL1A1) decreased in a dose-dependent manner.
- the solution to be included in the solution was prepared, exposed to HHSteC cells for 48 hours, and the mRNA levels of ⁇ Sma, Col3a1 and Tgf ⁇ -1 (Transforming growth factor ⁇ -1) were measured. The results are shown in FIG. As shown in FIG. 9, the mRNA expression levels of ⁇ Sma, Col3a1 and Tgf ⁇ -1 (Transforming growth factor ⁇ -1) in the cells exposed to TAT-rhCYGB decreased in a dose-dependent manner.
- Test Example 4 Comparison of cell permeability (in vitro)
- the wild-type cytoglobin CYGB having no tag having the amino acid sequence of SEQ ID NO: 5; Native-CYGB
- the cytoglobin having the oligohistidine tag prepared in Test Example 1 His-rhCYGB
- the cell membrane permeability of cytoglobin (TAT-rhCYGB) having a TAT peptide tag prepared in Test Example 3 was verified.
- wild-type cytoglobin (Native-CYGB) and cytoglobin having an oligohistidine tag (His-rhCYGB) are prepared, and each of them is prepared into a solution containing 40 ⁇ g / mL in physiological saline, and each solution is prepared.
- HHSteC cells were exposed for 48 hours. Intracellular CYGB expression at 48 hours after exposure was analyzed by Western blot. The result is shown in FIG. As shown in FIG. 10, cell membrane permeability was observed in Native-CYGB itself. Furthermore, it was found that the cell membrane permeability was improved by tagging cytoglobin. The cell membrane permeability of His-rhCYGB was improved by 170% based on the cell membrane permeability of Native-CYGB.
- a solution containing 40 ⁇ g / mL of cytoglobin (His-rhCYGB) having an oligohistidine tag and cytoglobin (TAT-rhCYGB) having both a TAT peptide tag in physiological saline was prepared.
- Solution was exposed to HHSteC cells for 48 hours. Intracellular before exposure (-) and at 0.25 hours, 0.5 hours, 1 hour, 4 hours, 8 hours, 24 hours and 48 hours after exposure.
- the CYGB expression of CYGB was analyzed by Western blot. The results are shown in FIG. 11. As shown in FIG.
- the His-rhCYGB having an oligohistidine tag and the TAT-rhCYGB having a TAT peptide tag are TAT-rhCYGB.
- TAT-rhCYGB was superior in cell membrane permeability because the intracellular concentration reached the plateau more rapidly (specifically, at 0.25 hours).
- His-rhCYGB which is inferior in cell membrane permeability, is more fibrotic-related at a lower dose. It can suppress gene transcription.
- Test Example 5 Tag-rhCYGB (in vitro) in HHSteC cells
- cytoglobin His-rhCYGB
- TAT-rhCYGB cytoglobin having a TAT peptide tag prepared in Test Example 3
- His-rhCYGB was labeled with Alexa Fluor, prepared at 10 ⁇ g / mL in physiological saline, and exposed to HHSteC cells for 48 hours. The exposed cells were subjected to marker multiple staining for lysosomes and endoplasmic reticulum. In addition, nucleic acids were stained with DAPI. The results are shown in FIG. 12 (a). As shown in FIG. 12 (a), His-rhCYGB incorporated into HHSteC cells was found to be localized in lysosomes but not in the endoplasmic reticulum.
- His-rhCYGB was labeled with Alexa Fluor 488, prepared at 10 ⁇ g / mL in physiological saline, and exposed to HHSteC cells for 24 hours.
- endosome organelle markers LysoTracker, Molecular Probes, USA; 100 nM; 15 minutes, 37 ° C, 5% CO 2
- mitochondrial organelle markers MitoTracker, Molecular Probes; 100 nM; 15 minutes, 37).
- His-rhCYGB incorporated into HHSteC cells is localized in mitochondria, early endosomes, anaphase endosomes, and lysosomes, but is not present in the endoplasmic reticulum.
- TAT-rhCYGB Existence mode of TAT-rhCYGB in HHSteC cells
- Two groups of HHSteC cells were prepared, one was a control (No treatment) group and the other was a TAT-rhCYGB administration group.
- 10 ⁇ g / mL TAT-rhCYGB was exposed for 48 hours.
- CYGB, SMA, and DAPI staining were performed on each of the control group and the TAT-rhCYGB administration group.
- CYGB is stained red
- ⁇ SMA is stained green
- DAPI DAPI nuclear counterstain
- Test Example 6 Effect of 6R-CYGB on HHSteC cells (in vitro) (Comparative example)
- a fusion polypeptide 6R-rhCYGB in which a comparative 6R peptide tag (GRRRRRRRAS: SEQ ID NO: 15) was bound to the N-terminal of the cytoglobin shown in SEQ ID NO: 5 was prepared, and the prepared 6R-rhCYGB was prepared. The effect on stellate cells was examined.
- GRRRRRRRAS comparative 6R peptide tag
- FIG. 14 also shows the results of 6R-CYGB of Test Example 1 at 48 hours. As shown in FIG. 14, the intracellular CYGB concentration exposed to 6R-rhCYGB increased in a time-dependent manner, but the amount of ⁇ SMA and type I collagen (COL1A1) expressed did not change. GAPDH is a housekeeping protein.
- 6R-rhCYGB-free (-) phosphate buffer solution and 6R-rhCYGB are contained in physiological saline at a concentration of 5 ⁇ g / mL, 10 ⁇ g / mL, 20 ⁇ g / mL, 40 ⁇ g / mL, or 80 ⁇ g / mL.
- a phosphate buffer solution was prepared, exposed to HHSteC cells for 48 hours, and intracellular CYGB was measured. The results are shown in FIG. As shown in FIG. 15, the intracellular CYGB concentration exposed to 6R-rhCYGB increased in a dose-dependent manner, but the amount of ⁇ SMA expressed and type I collagen (COL1A1) changed regardless of the dose. I didn't.
- 6R-rhCYGB-free (0 ⁇ g / mL) phosphate buffer solution and 6R-rhCYGB in phosphate buffer solution at concentrations of 5 ⁇ g / mL, 10 ⁇ g / mL, 20 ⁇ g / mL, 40 ⁇ g / mL, or 80 ⁇ g / mL.
- a solution containing the mixture was prepared, exposed to HHSteC cells for 48 hours, and the amount of ⁇ Sma mRNA transcribed was measured. The results are shown in FIG. As shown in FIG. 16, the mRNA transcription level of ⁇ Sma in cells exposed to 6R-rhCYGB did not change regardless of dose.
- Test Example 7 Iron heme binding and antifibrotic effect of cytoglobin (reference example)
- Co-CYGB which replaces iron ions contained in the heme of wild-type cytoglobin with cobalt ions, on the antifibrosis of HHSteC cells was examined.
- Cobalt substitution of cytoglobin The iron ion contained in the heme bound to cytoglobin was replaced with cobalt ion as follows.
- an apoprotein (Apo-CYGB) was prepared by removing the auxiliary molecular group of wild-type cytoglobin, and Co-CYGB was prepared by inserting cobalt-protoporphyrin (Co-PPIX) into Apo-CYGB. ..
- Co-CYGB was purified on a PD-10 column and equilibrated with phosphate buffer. The ultraviolet absorption spectrum of Co-CYGB was confirmed by a cobalt peak having a wavelength of 426 nm.
- Co-CYGB was exposed to HHSteC cells at a concentration of 40 ⁇ g / mL for 40 hours, and intracellular CYGB and fibrosis-related proteins were measured (Co-CY-1, Co-CY-2).
- FIG. 17 the same procedure was performed except that the HHSteC cells were not exposed to Co-CYGB (Cont-1, Cont-2), and wild-type cytoglobin was used instead of Co-CYGB.
- PC1, PC2 The results of exposure under the conditions are also shown.
- Co-CY-1 and Co-CY-2 in which heme iron of cytoglobin was replaced with cobalt exposed cytoglobin, unlike PC1 and PC2 exposed to wild-type cytoglobin. Similar to the absent Co-CY-1 and Co-CY-2, the amounts of ⁇ SMA and type I collagen (COL1A1) expressed did not change. GAPDH is a housekeeping protein. From this result, it was shown that the substitution of heme iron with cobalt inhibits the antifibrotic effect of cytoglobin, that is, the iron heme activity of cytoglobin is related to the antifibrotic effect.
- Test Example 8 Various cytoglobin proteins and anti-fibrotic effect In this test example, the anti-fibrotic effect was verified for the His-tag modified product of a partial protein of full-length cytoglobin.
- cytoglobin protein The verified cytoglobin proteins are as follows. -Pp1: 1st to 20th (SEQ ID NO: 17) of full-length cytoglobin (SEQ ID NO: 5) (for comparison) -Pp2: 75th to 95th (SEQ ID NO: 18) of full-length cytoglobin (SEQ ID NO: 5) (for comparison) -Pp3: 75th to 120th (SEQ ID NO: 19) of full-length cytoglobin (SEQ ID NO: 5) (for comparison) -Pp4: 18th to 170th (SEQ ID NO: 1) of full-length cytoglobin (SEQ ID NO: 5) -Pp5: 4th to 176th (SEQ ID NO: 2) of full-length cytoglobin (SEQ ID NO: 5) -Pp6: 18th to 190th (SEQ ID NO: 3) of full-length cytoglobin (SEQ ID NO: 5) -Pp7: 4th to 190th (S
- HHSteC cells from the cell line were used in the experiment.
- a phosphate buffer solution was prepared, exposed to HHSteC cells for 48 hours, and intracellular CYGB was measured. The results are shown in FIG. As shown in FIG. 20, ⁇ SMA expressed in cells exposed to His-pp4 and type I collagen (COL1A1) decreased in a dose-dependent manner.
- GAPDH is a housekeeping protein.
- type I collagen (COL1A1) expressed in cells exposed to His-pp5 to His-pp7 decreased in a dose-dependent manner. Comparing the results of FIGS. 20 and 22 to 24, the COL1A1 reducing effect was particularly high in His-pp5, His-pp7, and His-pp8 (PC; His-CYGB).
- type I collagen (COL1A1) expressed in cells exposed to His-pp1 to His-pp3 did not change regardless of the dose.
- FIG. 26 there was no change in the transcription amount of ⁇ Sma in the cells exposed to His-pp1.
- Group 1 (Cont) was given a normal diet
- Group 2 (DDC4w) was given a 0.1 wt% 3,5-diethoxycarbonyl-1,4-dihydrocoridine-added diet (DDC diet) for 4 weeks. Feeding was performed, and saline was injected into the tail vein twice a week at the 3rd and 4th weeks.
- Group 3 (DDC4w-rhCY2w) was fed a DDC diet for 4 weeks, and in the 3rd and 4th weeks, it was injected into the tail vein twice a day at a frequency of 2 mg / kg body weight.
- Group 2 and Group 3 are DDC-induced mouse cholestasis models (hepatitis / liver fibrosis model). Mice were sacrificed 2 days after the last intraperitoneal injection and serum and liver tissue were collected.
- FIG. 29 The measurement of AST value and ALT value in serum is shown in FIG.
- group 2 serum AST and ALT increased from group 1 (Cont) to 423.3 IU / L and 205.1 IU / L, respectively, and His-rhCYGB was increased for 2 weeks.
- group 3 DDC4w-rhCy2w administered, their values were significantly reduced to 266.9 IU / L and 129.5 IU / L, respectively (p ⁇ 0.05). That is, it was confirmed that the serum AST / ALT level of the hepatitis / liver fibrosis model was significantly reduced by the administration of His-rhCYGB.
- FIG. 30 shows the measurement results of the mRNA transcription levels of the fibrosis-related genes ⁇ Sma, Col1a1, Tgf ⁇ -1, Tgf ⁇ -3 and the inflammatory factors Tnf- ⁇ and Ccl-2. As shown in FIG. 30, the amount of transcription of each mRNA was significantly reduced by administration of His-rhCYGB.
- H & E hematoxylin and eosin staining
- SiR saliva red staining for staining collagen fibers
- stars were used for the liver tissues of Group 1 (Cont), Group 2 (DDC4w), and Group 3 (DDC4w-rhCY2w).
- the cell activation marker ⁇ SMA, neutrophil marker Neu, macrophage marker CD68, and bile duct cell marker CK19 were subjected to DAPI (4,6-diamidino-2-phenylindole) staining for nuclear counterstaining. The results are shown in FIG. As shown in the results of H & E staining and Sirius red staining in FIG.
- SEQ ID NO: 13 is a histidine-tagged cytoglobin.
- SEQ ID NO: 14 is a TAT-tagged site globin.
- SEQ ID NO: 15 is a 6R peptide tag.
- SEQ ID NO: 16 is a 6R peptide-tagged cytoglobin.
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Abstract
The purpose of the present invention is to provide a medicine that is effective for inhibiting hepatic fibrosis. A modified cytoglobin protein comprising: a cytoglobin protein comprising at least one polypeptide selected from among (1) a polypeptide which contains the amino acid sequence represented by SEQ ID NO: 1, (2) a polypeptide which contains an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 1 by substitution, addition, insertion or deletion of one to several amino acid residues and which is capable of inhibiting hepatic fibrosis, and (3) a polypeptide which contains an amino acid sequence showing a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1 and which is capable of inhibiting hepatic fibrosis; and an oligohistidine tag and/or a TAT peptide tag attached to the cytoglobin protein. This modified cytoglobin protein is usable as an active ingredient of an antifibrotic agent.
Description
本発明は、抗線維化剤に関する。
The present invention relates to an antifibrotic agent.
近年、肝癌患者数の増大が問題となっている。２０１２年度国立がん研究センターの報告によると、国内の肝癌死亡者数は男女ともに全癌中４位であり、２０１０年には年間３３０００人にも及んでいる。肝癌は、Ｂ型、Ｃ型肝炎ウイルス感染、多量飲酒や糖尿病肥満と関連する非アルコール性脂肪性肝炎（ＮＡＳＨ）を土台として発生する。即ち、肝癌は慢性炎症と線維化肝を母地として生じ、病因の如何を問わず年率８％で発癌し、一旦肝癌が生じると再発と肝内転移を繰り返す。肝硬変とは、肝実質がＩ型コラーゲンなどの細胞外マトリックス蛋白で置換されて機能的肝細胞が減少する病態である。この線維性肝臓は、生理的状態ではビタミンＡ貯蔵を主機能とする肝星細胞（Ｈｅｐａｔｉｃ ｓｔｅｌｌａｔｅ ｃｅｌｌ、ＨＳＣ）が活性化して形質を変えた筋線維芽細胞（Ｍｙｏｆｉｂｒｏｂｌａｓｔ、ＭＦＢ）で肝実質が置換される病態である。この形質転換には、トランスフォーミング増殖因子－β（Ｔｒａｎｓｆｏｒｍｉｎｇ ｇｒｏｗｔｈ ｆａｃｔｏｒ（ＴＧＦ）－β）や結合組織成長因子（ｃｏｎｎｅｃｔｉｖｅ ｔｉｓｓｕｅ ｇｒｏｗｔｈ ｆａｃｔｏｒ、ＣＴＧＦ）が関与し、これらの因子が肝星細胞の持続活性化や実質での筋線維芽細胞の増加が肝細胞機能を低下させる要因であり、肝癌発症に寄与することが報告されている（非特許文献１及び２）。そのため、肝星細胞の活性化抑制と筋線維芽細胞の制御が肝繊維化及び肝癌の治療法開発に繋がると考えられている。
In recent years, an increase in the number of liver cancer patients has become a problem. According to a report by the National Cancer Center in 2012, the number of deaths from liver cancer in Japan is the fourth highest among all cancers in both men and women, reaching 33,000 per year in 2010. Liver cancer develops on the basis of non-alcoholic steatohepatitis (NASH) associated with hepatitis B and C virus infections, heavy drinking and diabetic obesity. That is, liver cancer develops from chronic inflammation and fibrotic liver as a base, and develops at an annual rate of 8% regardless of the cause, and once liver cancer occurs, recurrence and intrahepatic metastasis are repeated. Cirrhosis is a condition in which the liver parenchyma is replaced with extracellular matrix protein such as type I collagen and the number of functional hepatocytes decreases. In the physiological state, the hepatic parenchyma is replaced by myofibroblasts (MFBs) in which hepatic stellate cells (HSCs) whose main function is vitamin A storage are activated and their traits are changed. It is a pathological condition to be treated. Transforming growth factor-β (Transforming growth factor (TGF) -β) and connective tissue growth factor (CTGF) are involved in this transformation, and these factors continuously activate hepatic stellate cells. It has been reported that an increase in myofibroblasts in the parenchyma is a factor that reduces hepatocellular function and contributes to the development of liver cancer (Non-Patent Documents 1 and 2). Therefore, suppression of hepatic stellate cell activation and control of myofibroblasts are thought to lead to the development of therapeutic methods for liver fibrosis and liver cancer.
線維化前後の線維芽細胞におけるプロテオーム解析において、サイトグロビン（ｃｙｔｏｇｌｏｂｉｎ， Ｃｙｇｂ）が肝星細胞（ＨＳＣ）で見出された。サイトグロビンは肝外臓器の膵臓（膵星細胞）や腎臓の尿細管上皮近傍の線維芽細胞にも発現することが知られている（非特許文献３）。サイトグロビンの肝病態における役割を明らかにする目的でサイトグロビン欠損マウス（Ｃｙｇｂ-/-）を作製されており（特許文献１）、その結果、サイトグロビン欠損マウスが、肝発がん物質であるジエチルニトロサミン（ｄｉｅｔｈｙｌｎｉｔｒｏｓａｍｉｎｅ，ＤＥＮ）投与に対して易発がん性を呈すること、及び、その過程に酸化ストレス亢進が関与することが見出されている（非特許文献４）。さらに、サイトグロビン過剰発現が肝星細胞活性化を抑制することも見出されている（非特許文献５）。
In proteome analysis in fibroblasts before and after fibrosis, cytoglobin (Cygb) was found in hepatic stellate cells (HSC). Cytoglobin is also known to be expressed in the pancreas (pancreatic stellate cells) of extrahepatic organs and fibroblasts near the renal tubular epithelium (Non-Patent Document 3). Cytoglobin-deficient mice (Cygb -/- ) have been prepared for the purpose of clarifying the role of cytoglobin in the liver pathology (Patent Document 1). As a result, the cytoglobin-deficient mice are treated with diethylnitrosamine, which is a liver carcinogen. It has been found that (diethylnitrosamine, DEN) is easily carcinogenic to administration and that oxidative stress enhancement is involved in the process (Non-Patent Document 4). Furthermore, it has been found that overexpression of cytoglobin suppresses hepatic stellate cell activation (Non-Patent Document 5).
サイトグロビンの発現誘導に関しては、更に次の知見が報告されている。例えば、組織や細胞において低酸素状態にするとサイトグロビンが誘導されること（非特許文献６）、肝臓の星細胞をアルンジン酸に暴露させるとサイトグロビンが誘導されること（非特許文献７）、及び、肝臓のヒト星細胞をＦｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ ２に暴露させるとサイトグロビンが誘導されること（非特許文献８）が報告されている。
The following findings have been reported regarding the induction of cytoglobin expression. For example, hypoxic conditions in tissues and cells induce cytoglobin (Non-Patent Document 6), and exposure of liver stellate cells to arundic acid induces cytoglobin (Non-Patent Document 7). It has also been reported that when human stellate cells in the liver are exposed to Fibroblast growth factor 2, cytoglobin is induced (Non-Patent Document 8).
一方、肝疾患の治療又は予防剤としては、次の報告がある。例えば、メナテトレノン（ビタミンＫ－ＩＩ）を有効成分とする肝疾患治療・予防剤が、肝細胞癌の細胞増殖を抑制したり、肝細胞癌治療後の門脈浸潤の発生を抑制したりできること（特許文献２～４）、メナテトレノンが、慢性肝疾患由来、肝硬変由来、又は、Ｃ型肝炎ウイルス性肝硬変由来の肝癌の発癌を抑制し得ること（特許文献５）、アポトーシス促進活性または抗増殖活性に関係する１，４－ナフトキノン系化合物を抗癌剤として使用する提案（特許文献６）が報告されている。
On the other hand, there are the following reports as treatments or preventive agents for liver diseases. For example, a liver disease treating / preventing agent containing menatetrenone (vitamin K-II) as an active ingredient can suppress cell proliferation of hepatocellular carcinoma and suppress the occurrence of portal vein invasion after hepatocellular carcinoma treatment ( Patent Documents 2 to 4), menatetrenone can suppress the carcinogenesis of liver cancer derived from chronic liver disease, liver cirrhosis, or hepatitis C viral liver cirrhosis (Patent Document 5), and has proliferative activity or antiproliferative activity. A proposal for using a related 1,4-naphthoquinone compound as an anticancer agent (Patent Document 6) has been reported.
また、Ｃ型肝炎については、厚生労働省が進める肝炎総合対策の推進により、１Ｂ型高ウイルス症例でも９５％以上の根治率が得られるようになっている。
Regarding hepatitis C, the promotion of comprehensive hepatitis countermeasures promoted by the Ministry of Health, Labor and Welfare has made it possible to obtain a cure rate of 95% or more even in cases of high virus type 1B.
肝線維化を直接治療する方法は今のところ存在しない。上述のとおり、肝疾患の治療又は予防剤としていくつか報告されているが、それらの薬剤は、発癌の抑制、又は、一旦発癌した場合の治療薬であって、発癌の前段階である肝線維化の治療又は予防については一切検討されていない。また、Ｃ型肝炎では高い根治率が得られるようになっているが、肝硬変まで症状が進行した場合はウイルス排除率が低い。たとえウイルスが排除されたとしても、残存活性化星細胞により肝機能を回復できず発癌する。特に肝硬変は治療満足度が低い疾患であるため、肝硬変治療薬に対するアンメット・メディカル・ニーズは高い。有効な肝硬変治療薬が開発されれば、患者のＱＯＬ向上や医療費の削減など社会に与えるインパクトは大きく、医学的価値は極めて高い。
There is currently no direct treatment for liver fibrosis. As mentioned above, some have been reported as therapeutic or prophylactic agents for liver diseases, but these agents are therapeutic agents for suppressing carcinogenesis or once carcinogenesis, and are liver fibers that are a pre-carcinogenesis stage. No treatment or prevention of fibrosis has been considered. In addition, although a high cure rate has been obtained for hepatitis C, the virus elimination rate is low when the symptoms progress to liver cirrhosis. Even if the virus is eliminated, the residual activated stellate cells cannot restore liver function and cause cancer. In particular, since liver cirrhosis is a disease with low treatment satisfaction, there is a high unmet medical need for a drug for treating liver cirrhosis. If an effective therapeutic drug for liver cirrhosis is developed, it will have a great impact on society such as improving the QOL of patients and reducing medical expenses, and its medical value is extremely high.
肝癌の母地となる肝線維化を治療することは、肝硬変の治療や肝癌の予防において非常に有効であると考えられる。サイトグロビンが肝臓の微小環境の恒常性維持に不可欠で、その欠損は肝線維化を促進し発癌誘導させると考えられることから、肝星細胞におけるサイトグロビン維持に寄与する薬剤は、肝線維化の治療剤として候補物質となりうる。上述のとおり、アルシジン酸やｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ ２が、サイトグロビンを誘導することが示されている。しかしながら、アルンジン酸はヒトへの臨床応用が中止になった化合物である。また、ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ ２は多機能性物質であり様々な不所望の作用が懸念されるため、ヒトへの臨床応用には困難が予想される。
Treatment of liver fibrosis, which is the mother of liver cancer, is considered to be very effective in the treatment of liver cirrhosis and the prevention of liver cancer. Since cytoglobin is essential for maintaining the homeostasis of the liver microenvironment and its deficiency is thought to promote liver fibrosis and induce carcinogenesis, drugs that contribute to the maintenance of cytoglobin in hepatic stellate cells are those of liver fibrosis. It can be a candidate substance as a therapeutic agent. As mentioned above, it has been shown that arcidic acid and fibroblast growth factor 2 induce cytoglobin. However, arundic acid is a compound whose clinical application to humans has been discontinued. In addition, fibroblast growth factor 2 is a multifunctional substance, and there are concerns about various undesired actions, so clinical application to humans is expected to be difficult.
そこで本発明は、線維化を抑制するために有効な薬剤を提供することを目的とする。
Therefore, an object of the present invention is to provide an effective drug for suppressing fibrosis.
本発明者らが鋭意研究を重ねたところ、サイトグロビン自体を細胞に暴露させることで、線維化反応を低減できることを見出した。さらに、サイトグロビンに特定のタグを付すことによって、線維化反応をより一層低減できることも見出した。本発明は、これらの知見に基づいて、更に検討を重ねることにより完成したものである。
As a result of diligent research by the present inventors, it was found that the fibrotic reaction can be reduced by exposing cytoglobin itself to cells. Furthermore, it was found that the fibrotic reaction can be further reduced by attaching a specific tag to cytoglobin. The present invention has been completed by further studies based on these findings.
即ち、本発明は、下記に掲げる態様の発明を提供する。
項１． （１）配列番号１で示されるアミノ酸配列を含むポリペプチド、
（２）配列番号１で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されたアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド、及び
（３）配列番号１で示されるアミノ酸配列に対する配列同一性が８０％以上のアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種のポリペプチドからなるサイトグロビンタンパク質と、前記サイトグロビンタンパク質に結合したオリゴヒスチジン及びＴＡＴペプチドの少なくともいずれかのタグと、を含む修飾サイトグロビンタンパク質を有効成分として含む、抗線維化剤。
項２． 前記オリゴヒスチジンの長さが、３～１０アミノ酸残基である、項１に記載の抗線維化剤。
項３． 前記タグがオリゴヒスチジンである、項１又は２に記載の抗線維化剤。
項４． 前記タグが、前記サイトグロビンタンパク質のＮ末端に結合している、項１～３のいずれかに記載の抗線維化剤。
項５． 前記ポリペプチドの長さが、１５２～１９０アミノ酸残基である、項１～４のいずれかに記載の抗線維化剤。
項６． 前記ポリペプチドが、
（１１）配列番号１で示されるアミノ酸配列からなるポリペプチド、
（１２）配列番号１で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（１３）配列番号１で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能をするポリペプチド
の少なくとも１種である、項１～５のいずれかに記載の抗線維化剤。
項７． 前記ポリペプチドが、
（２１）配列番号２で示されるアミノ酸配列からなるポリペプチド、
（２２）配列番号１及び配列番号２で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（２３）配列番号１及び配列番号２で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種である、項１～５のいずれかに記載の抗線維化剤。
項８． 前記ポリペプチドが、
（３１）配列番号３で示されるアミノ酸配列からなるポリペプチド、
（３２）配列番号１及び配列番号３で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（３３）配列番号１及び配列番号３で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種である、項１～５のいずれかに記載の抗線維化剤。
項９． 前記ポリペプチドが、
（４１）配列番号４で示されるアミノ酸配列からなるポリペプチド、
（４２）配列番号１及び配列番号４で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（４３）配列番号１及び配列番号４で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種である、項１～５のいずれかに記載の抗線維化剤。
項１０． 前記ポリペプチドが、
（５１）配列番号５で示されるアミノ酸配列からなるポリペプチド、
（５２）配列番号１及び配列番号５で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（５３）配列番号１及び配列番号５で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種である、項１～５のいずれかに記載の抗線維化剤。
項１１． 肝線維化抑制剤である、項１～１０のいずれかに記載の抗線維化剤。
項１２． 肝硬変治療薬である、項１～１１のいずれかに記載の抗線維化剤。
項１３． 肝癌予防薬である、項１～１２のいずれかに記載の抗線維化剤。
項１４． （１）配列番号１で示されるアミノ酸配列を含むポリペプチド、
（２）配列番号１で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されたアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド、及び
（３）配列番号１で示されるアミノ酸配列に対する配列同一性が８０％以上のアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種のポリペプチドからなるサイトグロビンタンパク質と、前記サイトグロビンタンパク質に結合したオリゴヒスチジン及びＴＡＴペプチドの少なくともいずれかのタグと、を含む修飾サイトグロビンタンパク質の、抗線維化剤の製造のための使用。
項１５． 線維化疾患患者に、（１）配列番号１で示されるアミノ酸配列を含むポリペプチド、
（２）配列番号１で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されたアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド、及び
（３）配列番号１で示されるアミノ酸配列に対する配列同一性が８０％以上のアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種のポリペプチドからなるサイトグロビンタンパク質と、前記サイトグロビンタンパク質に結合したオリゴヒスチジン及びＴＡＴペプチドの少なくともいずれかのタグと、を含む修飾サイトグロビンタンパク質を有効成分として含む、抗線維化剤を投与する工程を含む、線維化疾患の治療方法。 That is, the present invention provides the inventions of the following aspects.
Item 1. (1) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 1.
(2) A polypeptide containing an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and having an ability to suppress fibrosis, and (3) A cytoglobin protein comprising at least one polypeptide having an amino acid sequence having an amino acid sequence identity of 80% or more with respect to the amino acid sequence represented by SEQ ID NO: 1 and having an ability to suppress fibrosis, and the above-mentioned An antifibrinolytic agent comprising a modified cytoglobin protein as an active ingredient, which comprises at least one tag of oligohistidine and TAT peptide bound to the cytoglobin protein.
Item 2. Item 2. The antifibrotic agent according to Item 1, wherein the oligohistidine has a length of 3 to 10 amino acid residues.
Item 3.Item 2. The antifibrotic agent according to Item 1 or 2, wherein the tag is oligohistidine.
Item 4. Item 2. The antifibrotic agent according to any one of Items 1 to 3, wherein the tag is bound to the N-terminal of the cytoglobin protein.
Item 5. Item 4. The antifibrotic agent according to any one of Items 1 to 4, wherein the polypeptide has a length of 152 to 190 amino acid residues.
Item 6. The polypeptide
(11) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
(12) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and has an inhibitory ability to suppress fibrosis, and (13).Item 2. The antifibrotic agent according to any one of Items 1 to 5, wherein the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 is 80% or more, and at least one of the polypeptides capable of suppressing fibrosis. ..
Item 7. The polypeptide
(21) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
(22) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 and which has the ability to suppress fibrosis. (23) Any ofItems 1 to 5, wherein the sequence identity with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 is 80% or more, and at least one of the polypeptides having the ability to suppress fibrosis. The anti-fibrotic agent described in Crab.
Item 8. The polypeptide
(31) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3.
(32) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 and which has the ability to suppress fibrosis. (33) Any ofItems 1 to 5, wherein the sequence identity with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 is 80% or more, and at least one of the polypeptides having the ability to suppress fibrosis. The anti-fibrotic agent described in Crab.
Item 9. The polypeptide
(41) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4.
(42) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4 and which has the ability to suppress fibrosis. (43) Any ofItems 1 to 5, wherein the sequence identity with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4 is 80% or more, and at least one of the polypeptides having the ability to suppress fibrosis. The anti-fibrotic agent described in Crab.
Item 10. The polypeptide
(51) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5.
(52) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5 and which has the ability to suppress fibrosis. (53) Any ofItems 1 to 5, wherein the sequence identity with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5 is 80% or more, and at least one of the polypeptides having the ability to suppress fibrosis. The anti-fibrotic agent described in Crab.
Item 11.Item 2. The antifibrosis agent according to any one of Items 1 to 10, which is a liver fibrosis inhibitor.
Item 12. Item 2. The antifibrotic agent according to any one of Items 1 to 11, which is a therapeutic agent for liver cirrhosis.
Item 13.Item 2. The antifibrotic agent according to any one of Items 1 to 12, which is a liver cancer preventive agent.
Item 14. (1) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 1.
(2) A polypeptide containing an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and having an ability to suppress fibrosis, and (3) A cytoglobin protein comprising at least one polypeptide having an amino acid sequence having an amino acid sequence identity of 80% or more with respect to the amino acid sequence represented by SEQ ID NO: 1 and having an ability to suppress fibrosis, and the above-mentioned Use of modified cytoglobin proteins, including at least one tag of oligohistidine and TAT peptide bound to cytoglobin protein, for the production of anti-fibrotic agents.
Item 15. For patients with fibrotic disease, (1) a polypeptide containing the amino acid sequence shown in SEQ ID NO: 1.
(2) A polypeptide containing an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and having an ability to suppress fibrosis, and (3) A cytoglobin protein comprising at least one polypeptide having an amino acid sequence having an amino acid sequence identity of 80% or more with respect to the amino acid sequence represented by SEQ ID NO: 1 and having an ability to suppress fibrosis, and the above-mentioned A method for treating a fibrotic disease, comprising the step of administering an antifibrinolytic agent comprising a modified cytoglobin protein containing at least one tag of oligohistidine and TAT peptide bound to the cytoglobin protein as an active ingredient.
項１． （１）配列番号１で示されるアミノ酸配列を含むポリペプチド、
（２）配列番号１で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されたアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド、及び
（３）配列番号１で示されるアミノ酸配列に対する配列同一性が８０％以上のアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種のポリペプチドからなるサイトグロビンタンパク質と、前記サイトグロビンタンパク質に結合したオリゴヒスチジン及びＴＡＴペプチドの少なくともいずれかのタグと、を含む修飾サイトグロビンタンパク質を有効成分として含む、抗線維化剤。
項２． 前記オリゴヒスチジンの長さが、３～１０アミノ酸残基である、項１に記載の抗線維化剤。
項３． 前記タグがオリゴヒスチジンである、項１又は２に記載の抗線維化剤。
項４． 前記タグが、前記サイトグロビンタンパク質のＮ末端に結合している、項１～３のいずれかに記載の抗線維化剤。
項５． 前記ポリペプチドの長さが、１５２～１９０アミノ酸残基である、項１～４のいずれかに記載の抗線維化剤。
項６． 前記ポリペプチドが、
（１１）配列番号１で示されるアミノ酸配列からなるポリペプチド、
（１２）配列番号１で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（１３）配列番号１で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能をするポリペプチド
の少なくとも１種である、項１～５のいずれかに記載の抗線維化剤。
項７． 前記ポリペプチドが、
（２１）配列番号２で示されるアミノ酸配列からなるポリペプチド、
（２２）配列番号１及び配列番号２で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（２３）配列番号１及び配列番号２で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種である、項１～５のいずれかに記載の抗線維化剤。
項８． 前記ポリペプチドが、
（３１）配列番号３で示されるアミノ酸配列からなるポリペプチド、
（３２）配列番号１及び配列番号３で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（３３）配列番号１及び配列番号３で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種である、項１～５のいずれかに記載の抗線維化剤。
項９． 前記ポリペプチドが、
（４１）配列番号４で示されるアミノ酸配列からなるポリペプチド、
（４２）配列番号１及び配列番号４で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（４３）配列番号１及び配列番号４で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種である、項１～５のいずれかに記載の抗線維化剤。
項１０． 前記ポリペプチドが、
（５１）配列番号５で示されるアミノ酸配列からなるポリペプチド、
（５２）配列番号１及び配列番号５で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（５３）配列番号１及び配列番号５で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種である、項１～５のいずれかに記載の抗線維化剤。
項１１． 肝線維化抑制剤である、項１～１０のいずれかに記載の抗線維化剤。
項１２． 肝硬変治療薬である、項１～１１のいずれかに記載の抗線維化剤。
項１３． 肝癌予防薬である、項１～１２のいずれかに記載の抗線維化剤。
項１４． （１）配列番号１で示されるアミノ酸配列を含むポリペプチド、
（２）配列番号１で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されたアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド、及び
（３）配列番号１で示されるアミノ酸配列に対する配列同一性が８０％以上のアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種のポリペプチドからなるサイトグロビンタンパク質と、前記サイトグロビンタンパク質に結合したオリゴヒスチジン及びＴＡＴペプチドの少なくともいずれかのタグと、を含む修飾サイトグロビンタンパク質の、抗線維化剤の製造のための使用。
項１５． 線維化疾患患者に、（１）配列番号１で示されるアミノ酸配列を含むポリペプチド、
（２）配列番号１で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されたアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド、及び
（３）配列番号１で示されるアミノ酸配列に対する配列同一性が８０％以上のアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種のポリペプチドからなるサイトグロビンタンパク質と、前記サイトグロビンタンパク質に結合したオリゴヒスチジン及びＴＡＴペプチドの少なくともいずれかのタグと、を含む修飾サイトグロビンタンパク質を有効成分として含む、抗線維化剤を投与する工程を含む、線維化疾患の治療方法。 That is, the present invention provides the inventions of the following aspects.
(2) A polypeptide containing an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and having an ability to suppress fibrosis, and (3) A cytoglobin protein comprising at least one polypeptide having an amino acid sequence having an amino acid sequence identity of 80% or more with respect to the amino acid sequence represented by SEQ ID NO: 1 and having an ability to suppress fibrosis, and the above-mentioned An antifibrinolytic agent comprising a modified cytoglobin protein as an active ingredient, which comprises at least one tag of oligohistidine and TAT peptide bound to the cytoglobin protein.
Item 3.
(11) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
(12) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and has an inhibitory ability to suppress fibrosis, and (13).
Item 7. The polypeptide
(21) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
(22) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 and which has the ability to suppress fibrosis. (23) Any of
(31) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3.
(32) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 and which has the ability to suppress fibrosis. (33) Any of
(41) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4.
(42) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4 and which has the ability to suppress fibrosis. (43) Any of
(51) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5.
(52) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5 and which has the ability to suppress fibrosis. (53) Any of
Item 11.
Item 13.
Item 14. (1) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 1.
(2) A polypeptide containing an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and having an ability to suppress fibrosis, and (3) A cytoglobin protein comprising at least one polypeptide having an amino acid sequence having an amino acid sequence identity of 80% or more with respect to the amino acid sequence represented by SEQ ID NO: 1 and having an ability to suppress fibrosis, and the above-mentioned Use of modified cytoglobin proteins, including at least one tag of oligohistidine and TAT peptide bound to cytoglobin protein, for the production of anti-fibrotic agents.
(2) A polypeptide containing an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and having an ability to suppress fibrosis, and (3) A cytoglobin protein comprising at least one polypeptide having an amino acid sequence having an amino acid sequence identity of 80% or more with respect to the amino acid sequence represented by SEQ ID NO: 1 and having an ability to suppress fibrosis, and the above-mentioned A method for treating a fibrotic disease, comprising the step of administering an antifibrinolytic agent comprising a modified cytoglobin protein containing at least one tag of oligohistidine and TAT peptide bound to the cytoglobin protein as an active ingredient.
本発明の抗線維化剤によると、組織の線維化の予防又は治療に有効である。さらに、癌の母地となる線維化抑制を抑制するため、癌の予防にも有効である。
According to the antifibrotic agent of the present invention, it is effective in preventing or treating tissue fibrosis. Furthermore, it is also effective in preventing cancer because it suppresses the suppression of fibrosis, which is the base of cancer.
本発明の抗線維化剤は、特定のタグにより修飾された修飾サイトグロビンタンパク質を有効成分とすることを特徴とする。以下、本発明の抗線維化剤について詳述する。
The antifibrotic agent of the present invention is characterized by containing a modified cytoglobin protein modified by a specific tag as an active ingredient. Hereinafter, the antifibrotic agent of the present invention will be described in detail.
有効成分
本発明の抗線維化剤の有効成分におけるサイトグロビンタンパク質とは、全長サイトグロビンと、全長サイトグロビンの部分タンパク質（サイトグロビン断片）との両方を含む意である。 Active Ingredient The cytoglobin protein in the active ingredient of the antifibrotic agent of the present invention means to include both full-length cytoglobin and a partial protein (cytoglobin fragment) of full-length cytoglobin.
本発明の抗線維化剤の有効成分におけるサイトグロビンタンパク質とは、全長サイトグロビンと、全長サイトグロビンの部分タンパク質（サイトグロビン断片）との両方を含む意である。 Active Ingredient The cytoglobin protein in the active ingredient of the antifibrotic agent of the present invention means to include both full-length cytoglobin and a partial protein (cytoglobin fragment) of full-length cytoglobin.
サイトグロビンタンパク質は、以下の（１）～（３）の少なくとも１種のポリペプチドである。
（１）配列番号１で示されるアミノ酸配列を含むポリペプチド、
（２）配列番号１で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されたアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド、及び
（３）配列番号１で示されるアミノ酸配列に対する配列同一性が８０％以上のアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド The cytoglobin protein is at least one of the following polypeptides (1) to (3).
(1) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 1.
(2) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide containing an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted, and having an ability to suppress fibrosis, and (3) A polypeptide containing an amino acid sequence having 80% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 and having an ability to suppress fibrosis.
（１）配列番号１で示されるアミノ酸配列を含むポリペプチド、
（２）配列番号１で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されたアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド、及び
（３）配列番号１で示されるアミノ酸配列に対する配列同一性が８０％以上のアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド The cytoglobin protein is at least one of the following polypeptides (1) to (3).
(1) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 1.
(2) In the amino acid sequence shown in SEQ ID NO: 1, a polypeptide containing an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted, and having an ability to suppress fibrosis, and (3) A polypeptide containing an amino acid sequence having 80% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 and having an ability to suppress fibrosis.
配列番号１で示されるアミノ酸配列は、ヒト由来のサイトグロビン（後述配列番号５）において抗線維化能を示す部分タンパク質のアミノ酸配列（後述配列番号５の１８～１７０番目）に相当する。サイトグロビン（後述配列番号５）は、第８１位と第１１３位のヒスチジン残基のイミダゾール基がヘムを挟み込むように６配位構造をとり（ヘム結合活性）、酸素及び一酸化窒素等と結合する機能を有する。本発明における有効成分による抗線維化効果の発現には少なくともヘム結合活性が関与するため、サイトグロビンタンパク質は、当該第８１位及び第１１３位（ヘム結合部位）を含んでいる。一方、サイトグロビンタンパク質は、両方のヘム結合部位を含むだけでは本発明における有効成分による抗線維化効果は発現せず、当該効果の発現には、ヘム結合部位を含め１８～１７０番目のアミノ酸配列を要する。上記（２）及び（３）に示すポリペプチドは、前記（１）に示すポリペプチドの変異体である。
The amino acid sequence shown in SEQ ID NO: 1 corresponds to the amino acid sequence of a partial protein (18th to 170th of SEQ ID NO: 5 described later) showing antifibrotic ability in human-derived cytoglobin (SEQ ID NO: 5 described later). Cytoglobin (SEQ ID NO: 5 described later) has a 6-coordination structure (heme binding activity) such that the imidazole groups of histidine residues at positions 81 and 113 sandwich the heme, and binds to oxygen, nitric oxide, etc. Has the function of Since at least heme-binding activity is involved in the expression of the anti-fibrotic effect of the active ingredient in the present invention, the cytoglobin protein contains the 81st and 113th positions (heme binding sites). On the other hand, the cytoglobin protein does not exhibit the antifibrotic effect of the active ingredient in the present invention only by including both heme binding sites, and the expression of the effect is the amino acid sequence of positions 18 to 170 including the heme binding site. Needs. The polypeptides shown in (2) and (3) above are variants of the polypeptide shown in (1) above.
上記（２）のポリペプチドにおいて、導入されるアミノ酸の改変は、置換、付加、挿入、及び欠失の中から１種類の改変のみ（例えば置換のみ）を含むものであってもよく、２種以上の改変（例えば、置換と挿入）を含んでいても良い。上記（２）のポリペプチドにおいて、導入される置換、付加、挿入又は欠失されるアミノ酸は、１個又は数個であればよく、例えば、１～３２個、好ましくは１～１６個、より好ましくは１～１０個、さらに好ましくは１～８個、一層好ましくは１～３個、より一層好ましくは１又は２個、特に好ましくは１個が挙げられる。
In the above-mentioned polypeptide (2), the modification of the introduced amino acid may include only one modification (for example, substitution only) from substitutions, additions, insertions, and deletions. The above modifications (eg, substitution and insertion) may be included. In the polypeptide of (2) above, the number of amino acids to be introduced, substituted, added, inserted or deleted may be one or several, for example, 1 to 32, preferably 1 to 16. The number is preferably 1 to 10, more preferably 1 to 8, more preferably 1 to 3, still more preferably 1 or 2, and particularly preferably 1.
また、上記（３）のポリペプチドにおいて、配列番号１で示されるアミノ酸配列に対する配列同一性は、８０％以上であればよいが、好ましくは８５％以上、好ましくは９０％以上、更に好ましくは９５％以上、特に好ましくは９９％以上が挙げられる。
Further, in the above-mentioned polypeptide (3), the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 may be 80% or more, preferably 85% or more, preferably 90% or more, still more preferably 95. % Or more, particularly preferably 99% or more.
ここで、上記（３）のポリペプチドおいて、配列番号１で示されるアミノ酸配列に対する配列同一性とは、配列番号１に示すアミノ酸配列と比較して算出される配列同一性である。また、「配列同一性」とは、ＢＬＡＳＴ ＰＡＣＫＡＧＥ［ｓｇｉ３２ ｂｉｔ ｅｄｉｔｉｏｎ，Ｖｅｒｓｉｏｎ ２．０．１２；ａｖａｉｌａｂｌｅ ｆｒｏｍ Ｎａｔｉｏｎａｌ Ｃｅｎｔｅｒ ｆｏｒ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｆｏｒｍａｔｉｏｎ（ＮＣＢＩ）］のｂｌ２ｓｅｑ ｐｒｏｇｒａｍ（Ｔａｔｉａｎａ Ａ．Ｔａｔｓｕｓｏｖａ，Ｔｈｏｍａｓ Ｌ．Ｍａｄｄｅｎ，ＦＥＭＳ Ｍｉｃｒｏｂｉｏｌ．Ｌｅｔｔ．，Ｖｏｌ．１７４，ｐ２４７－２５０，１９９９）により得られるアミノ酸配列の同一性の値を示す。パラメーターは、Ｇａｐ ｉｎｓｅｒｔｉｏｎ Ｃｏｓｔ ｖａｌｕｅ：１１、Ｇａｐ ｅｘｔｅｎｓｉｏｎ Ｃｏｓｔ ｖａｌｕｅ：１に設定すればよい。
Here, in the above-mentioned polypeptide (3), the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 is the sequence identity calculated in comparison with the amino acid sequence shown in SEQ ID NO: 1. In addition, "sequence identity" is defined as BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)]. The value of the identity of the amino acid sequence obtained by Microbiol. Lett., Vol. 174, p247-250, 1999) is shown. The parameters may be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.
また、上記（２）及び（３）のポリペプチドにおいて、配列番号１で示されるアミノ酸配列に対してアミノ酸置換が導入されている場合、導入されるアミノ酸置換の好適な一態様として保存的置換が挙げられる。ここで、「保存的置換」とは、１若しくは数個のアミノ酸残基を、別の化学的に類似したアミノ酸残基で置き換えることを意味する。例えば、ある疎水性残基を別の疎水性残基によって置換する場合、ある極性残基を同じ電荷を有する別の極性残基によって置換する場合などが挙げられる。このような置換を行うことができる機能的に類似のアミノ酸は、アミノ酸毎に当該技術分野において公知である。具体例を挙げると、非極性（疎水性）アミノ酸としては、アラニン、バリン、イソロイシン、ロイシン、プロリン、トリプトファン、フェニルアラニン、メチオニンなどが挙げられる。極性（中性）アミノ酸としては、グリシン、セリン、スレオニン、チロシン、グルタミン、アスパラギン、システインなどが挙げられる。陽電荷をもつ（塩基性）アミノ酸としては、アルギニン、ヒスチジン、リジンなどが挙げられる。また、負電荷をもつ（酸性）アミノ酸としては、アスパラギン酸、グルタミン酸などが挙げられる。
Further, in the above-mentioned polypeptides (2) and (3), when an amino acid substitution is introduced into the amino acid sequence shown in SEQ ID NO: 1, conservative substitution is a preferred embodiment of the introduced amino acid substitution. Can be mentioned. Here, "conservative substitution" means replacing one or several amino acid residues with another chemically similar amino acid residue. For example, one hydrophobic residue may be replaced by another hydrophobic residue, one polar residue may be replaced by another polar residue having the same charge, and the like. Functionally similar amino acids capable of making such substitutions are known in the art for each amino acid. Specific examples include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
上記（２）及び（３）のポリペプチドにおいて、「線維化抑制能を有する」とは、（１）に示すポリペプチドと同等の線維化抑制能を有することをいう。具体的には、線維化関連遺伝子の転写量（ｍＲＮＡへの転写量）を測定した場合に、上記（１）のポリペプチドと線維化関連遺伝子の転写量が同等（即ち、上記（１）のポリペプチドによる線維化関連遺伝子の転写量を１００％とした場合に、８０～１２０％程度であること）であることを意味する。線維化関連遺伝子としては、αＳｍａ、Ｃｏｌ１ａ１、Ｃｏｌ３ａ１、Ｔｆｇβ－１等が挙げられる。
In the above-mentioned polypeptides (2) and (3), "having an inhibitory ability on fibrosis" means having an inhibitory ability on fibrosis equivalent to that of the polypeptide shown in (1). Specifically, when the transcription amount of the fibrosis-related gene (transcription amount to mRNA) is measured, the transcription amount of the polypeptide of (1) above and the transcription amount of the fibrosis-related gene are equivalent (that is, the transcription amount of (1) above). When the transcription amount of the fibrosis-related gene by the polypeptide is 100%, it is about 80 to 120%). Examples of fibrosis-related genes include αSma, Col1a1, Col3a1, and Tfgβ-1.
サイトグロビンタンパク質としては、抗線維化能を示す配列番号１で示されるアミノ酸配列が含まれているポリペプチドであれば特に限定されないが、より高い抗線維化能を得る観点から、１５２～１９０アミノ酸残基の長さのポリペプチドであることが好ましい。具体的なサイトグロビンタンパク質の例としては、以下のものが挙げられる。
The cytoglobin protein is not particularly limited as long as it is a polypeptide containing the amino acid sequence shown by SEQ ID NO: 1 showing antifibrotic ability, but from the viewpoint of obtaining higher antifibrotic ability, 152 to 190 amino acids. It is preferably a residue-length polypeptide. Specific examples of cytoglobin proteins include the following.
（サイトグロビンタンパク質の具体例１）
サイトグロビンタンパク質の具体例１として、以下の（１１）～（１３）の少なくとも１種のポリペプチドが挙げられる。
（１１）配列番号１で示されるアミノ酸配列からなるポリペプチド、
（１２）配列番号１で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（１３）配列番号１で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能をするポリペプチド。 (Specific example 1 of cytoglobin protein)
Specific examples 1 of the cytoglobin protein include at least one of the following polypeptides (11) to (13).
(11) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
(12) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and has an inhibitory ability to suppress fibrosis, and (13). A polypeptide having 80% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 and capable of suppressing fibrosis.
サイトグロビンタンパク質の具体例１として、以下の（１１）～（１３）の少なくとも１種のポリペプチドが挙げられる。
（１１）配列番号１で示されるアミノ酸配列からなるポリペプチド、
（１２）配列番号１で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（１３）配列番号１で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能をするポリペプチド。 (Specific example 1 of cytoglobin protein)
Specific examples 1 of the cytoglobin protein include at least one of the following polypeptides (11) to (13).
(11) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
(12) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and has an inhibitory ability to suppress fibrosis, and (13). A polypeptide having 80% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 and capable of suppressing fibrosis.
上記（１１）のポリペプチドは、ヒト由来のサイトグロビン（後述配列番号５）において抗線維化能を示す部分タンパク質（後述配列番号５の１８～１７０番目）である。上記（１２）及び（１３）に示すポリペプチドと上記（１１）に示すポリペプチドとの関係は、前記（２）及び前記（３）に示すポリペプチドと前記（１）に示すポリペプチドとの関係と同様である。
The above-mentioned polypeptide (11) is a partial protein (18th to 170th of SEQ ID NO: 5 described later) that exhibits antifibrotic ability in human-derived cytoglobin (SEQ ID NO: 5 described later). The relationship between the polypeptides shown in (12) and (13) and the polypeptide shown in (11) is the relationship between the polypeptides shown in (2) and (3) and the polypeptide shown in (1). Similar to relationships.
（サイトグロビンタンパク質の具体例２）
サイトグロビンタンパク質の具体例２として、以下の（２１）～（２３）の少なくとも１種のポリペプチドが挙げられる。
（２１）配列番号２で示されるアミノ酸配列からなるポリペプチド、
（２２）配列番号１及び配列番号２で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（２３）配列番号１及び配列番号２で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド。 (Specific example 2 of cytoglobin protein)
Specific examples 2 of the cytoglobin protein include at least one of the following polypeptides (21) to (23).
(21) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
(22) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 and which has the ability to suppress fibrosis. (23) A polypeptide having a sequence identity of 80% or more with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 and having an ability to suppress fibrosis.
サイトグロビンタンパク質の具体例２として、以下の（２１）～（２３）の少なくとも１種のポリペプチドが挙げられる。
（２１）配列番号２で示されるアミノ酸配列からなるポリペプチド、
（２２）配列番号１及び配列番号２で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（２３）配列番号１及び配列番号２で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド。 (Specific example 2 of cytoglobin protein)
Specific examples 2 of the cytoglobin protein include at least one of the following polypeptides (21) to (23).
(21) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
(22) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 and which has the ability to suppress fibrosis. (23) A polypeptide having a sequence identity of 80% or more with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 and having an ability to suppress fibrosis.
上記（２１）のポリペプチドは、ヒト由来のサイトグロビン（後述配列番号５）において抗線維化能を示す部分を含むアミノ酸配列（後述配列番号５の４～１７６番目）からなる。上記（２２）及び（２３）に示すポリペプチドと上記（２１）に示すポリペプチドとの関係は、前記（２）及び前記（３）に示すポリペプチドと前記（１）に示すポリペプチドとの関係と同様である。
The above-mentioned polypeptide (21) consists of an amino acid sequence (4th to 176th positions of SEQ ID NO: 5 described later) containing a portion showing antifibrotic ability in human-derived cytoglobin (SEQ ID NO: 5 described later). The relationship between the polypeptides shown in (22) and (23) and the polypeptide shown in (21) above is the relationship between the polypeptides shown in (2) and (3) and the polypeptide shown in (1). Similar to relationships.
（サイトグロビンタンパク質の具体例３）
サイトグロビンタンパク質の具体例３として、以下の（３１）～（３３）の少なくとも１種のポリペプチドが挙げられる。
（３１）配列番号３で示されるアミノ酸配列からなるポリペプチド、
（３２）配列番号１及び配列番号３で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（３３）配列番号１及び配列番号３で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド。 (Specific example 3 of cytoglobin protein)
Specific examples 3 of the cytoglobin protein include at least one of the following polypeptides (31) to (33).
(31) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3.
(32) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 and which has the ability to suppress fibrosis. (33) A polypeptide having a sequence identity of 80% or more with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 and having an ability to suppress fibrosis.
サイトグロビンタンパク質の具体例３として、以下の（３１）～（３３）の少なくとも１種のポリペプチドが挙げられる。
（３１）配列番号３で示されるアミノ酸配列からなるポリペプチド、
（３２）配列番号１及び配列番号３で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（３３）配列番号１及び配列番号３で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド。 (Specific example 3 of cytoglobin protein)
Specific examples 3 of the cytoglobin protein include at least one of the following polypeptides (31) to (33).
(31) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3.
(32) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 and which has the ability to suppress fibrosis. (33) A polypeptide having a sequence identity of 80% or more with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 and having an ability to suppress fibrosis.
上記（３１）のポリペプチドは、ヒト由来のサイトグロビン（後述配列番号５）において抗線維化能を示す部分を含むアミノ酸配列（後述配列番号５の１８～１９０番目）からなる。上記（３２）及び（３３）に示すポリペプチドと上記（３１）に示すポリペプチドとの関係は、前記（２）及び前記（３）に示すポリペプチドと前記（１）に示すポリペプチドとの関係と同様である。
The above-mentioned polypeptide (31) consists of an amino acid sequence (18th to 190th positions of SEQ ID NO: 5 described later) containing a portion showing antifibrotic ability in human-derived cytoglobin (SEQ ID NO: 5 described later). The relationship between the polypeptides shown in (32) and (33) and the polypeptide shown in (31) is the relationship between the polypeptides shown in (2) and (3) and the polypeptide shown in (1). Similar to relationships.
（サイトグロビンタンパク質の具体例４）
サイトグロビンタンパク質の具体例４として、以下の（４１）～（４３）の少なくとも１種のポリペプチドが挙げられる。
（４１）配列番号４で示されるアミノ酸配列からなるポリペプチド、
（４２）配列番号１及び配列番号４で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（４３）配列番号１及び配列番号４で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド。 (Specific example 4 of cytoglobin protein)
Specific examples 4 of the cytoglobin protein include at least one of the following polypeptides (41) to (43).
(41) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4.
(42) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4 and which has the ability to suppress fibrosis. (43) A polypeptide having a sequence identity of 80% or more with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4 and having an ability to suppress fibrosis.
サイトグロビンタンパク質の具体例４として、以下の（４１）～（４３）の少なくとも１種のポリペプチドが挙げられる。
（４１）配列番号４で示されるアミノ酸配列からなるポリペプチド、
（４２）配列番号１及び配列番号４で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（４３）配列番号１及び配列番号４で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド。 (Specific example 4 of cytoglobin protein)
Specific examples 4 of the cytoglobin protein include at least one of the following polypeptides (41) to (43).
(41) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4.
(42) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4 and which has the ability to suppress fibrosis. (43) A polypeptide having a sequence identity of 80% or more with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4 and having an ability to suppress fibrosis.
上記（４１）のポリペプチドは、ヒト由来のサイトグロビン（後述配列番号５）において抗線維化能を示す部分を含むアミノ酸配列（後述配列番号５の４～１９０番目）からなる。上記（４２）及び（４３）に示すポリペプチドと上記（４１）に示すポリペプチドとの関係は、前記（２）及び前記（３）に示すポリペプチドと前記（１）に示すポリペプチドとの関係と同様である。
The above-mentioned polypeptide (41) consists of an amino acid sequence (4th to 190th positions of SEQ ID NO: 5 described later) containing a portion showing antifibrotic ability in human-derived cytoglobin (SEQ ID NO: 5 described later). The relationship between the polypeptides shown in (42) and (43) and the polypeptide shown in (41) is the relationship between the polypeptides shown in (2) and (3) and the polypeptide shown in (1). Similar to relationships.
（サイトグロビンタンパク質の具体例５）
サイトグロビンタンパク質の具体例５として、以下の（５１）～（５３）の少なくとも１種のポリペプチドが挙げられる。
（５１）配列番号５で示されるアミノ酸配列からなるポリペプチド、
（５２）配列番号１及び配列番号５で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（５３）配列番号１及び配列番号５で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド。 (Specific example 5 of cytoglobin protein)
Specific examples 5 of the cytoglobin protein include at least one of the following polypeptides (51) to (53).
(51) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5.
(52) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5 and which has the ability to suppress fibrosis. (53) A polypeptide having a sequence identity of 80% or more with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5 and having an ability to suppress fibrosis.
サイトグロビンタンパク質の具体例５として、以下の（５１）～（５３）の少なくとも１種のポリペプチドが挙げられる。
（５１）配列番号５で示されるアミノ酸配列からなるポリペプチド、
（５２）配列番号１及び配列番号５で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（５３）配列番号１及び配列番号５で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド。 (Specific example 5 of cytoglobin protein)
Specific examples 5 of the cytoglobin protein include at least one of the following polypeptides (51) to (53).
(51) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5.
(52) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5 and which has the ability to suppress fibrosis. (53) A polypeptide having a sequence identity of 80% or more with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5 and having an ability to suppress fibrosis.
上記（５１）のポリペプチドは、ヒト由来のサイトグロビン（配列番号５）である。上記（５２）及び（５３）に示すポリペプチドと上記（５１）に示すポリペプチドとの関係は、前記（２）及び前記（３）に示すポリペプチドと前記（１）に示すポリペプチドとの関係と同様である。
The polypeptide of (51) above is a human-derived cytoglobin (SEQ ID NO: 5). The relationship between the polypeptides shown in (52) and (53) and the polypeptide shown in (51) is the relationship between the polypeptides shown in (2) and (3) and the polypeptide shown in (1). Similar to relationships.
これらのポリペプチドの中でもより高い抗線維化能を得る観点から、Ｎ末端に位置するアミノ酸残基が、配列番号５の２～１７番目（又は、３～１５番目、３～１３番目、３～１１番目３～９番目、３～７番目、特に３～５番目）のいずれかであるもの以外であることが好ましい。より好ましくは、これらのポリペプチドの中でも、サイトグロビンタンパク質の具体例１、サイトグロビンタンパク質の具体例３、サイトグロビンタンパク質の具体例５として示したものが挙げられ、特に好ましくは、サイトグロビンタンパク質の具体例５として示したものが挙げられる。
From the viewpoint of obtaining higher antifibrotic ability among these polypeptides, the amino acid residue located at the N-terminal is the 2nd to 17th (or 3rd to 15th, 3rd to 13th, 3rd to 3rd) of SEQ ID NO: 5. It is preferable that it is not one of the 11th, 3rd to 9th, 3rd to 7th, especially 3rd to 5th). More preferably, among these polypeptides, those shown as Specific Example 1 of Cytoglobin Protein, Specific Example 3 of Cytoglobin Protein, and Specific Example 5 of Cytoglobin Protein can be mentioned, and particularly preferably, of Cytoglobin Protein. Specific examples 5 include those shown.
本発明の抗線維化剤は、上記のサイトグロビンタンパク質が、特定のタグによって修飾されている。本発明者らにより、サイトグロビンタンパク質自体が細胞膜を透過することが見出されたが、当該特定のタグを有することにより、サイトグロビンタンパク質の細胞膜透過性を一層向上させることができる。本発明の抗線維化剤で用いられるタグは、オリゴヒスチジン又はＴＡＴペプチドである。オリゴヒスチジン及びＴＡＴペプチドは、細胞膜透過能を有するペプチドである。公知の細胞膜透過能を有するペプチドの中でも、オリゴヒスチジン及びＴＡＴペプチドは、サイトグロビンタンパク質に結合した場合に、特に優れた細胞膜透過能を発揮する。
In the antifibrotic agent of the present invention, the above cytoglobin protein is modified by a specific tag. The present inventors have found that the cytoglobin protein itself permeates the cell membrane, but by having the specific tag, the cell membrane permeability of the cytoglobin protein can be further improved. The tag used in the antifibrotic agent of the present invention is oligohistidine or TAT peptide. Oligohistidine and TAT peptides are peptides having cell membrane penetrating ability. Among the known peptides having cell membrane penetrating ability, oligohistidine and TAT peptide exhibit particularly excellent cell membrane penetrating ability when bound to cytoglobin protein.
オリゴヒスチジンは、ヒスチジン残基からなるペプチドである。オリゴヒスチジンの長さとしては特に限定されず、本発明の抗線維化剤の適用対象の症状に対して求められる薬効の程度に応じ、適宜設定することができる。例えば、オリゴヒスチジンの長さとしては、３～１０アミノ酸残基が挙げられ、好ましくは４～８アミノ酸残基が挙げられ、より好ましくは５～６アミノ酸残基が挙げられる。
Oligohistidine is a peptide consisting of histidine residues. The length of the oligohistidine is not particularly limited, and can be appropriately set according to the degree of drug efficacy required for the symptom to which the antifibrotic agent of the present invention is applied. For example, the length of oligohistidine includes 3 to 10 amino acid residues, preferably 4 to 8 amino acid residues, and more preferably 5 to 6 amino acid residues.
ＴＡＴペプチドは、ヒト免疫不全ウイルスの転写制御に係わるTransactivator of transcriptionタンパク質（Ｔａｔタンパク質）由来のアルギニンに富む塩基性ペプチドとして知られいる。具体的には、ＴＡＴペプチドは、Ｔａｔタンパク質のＲＮＡ結合領域、つまり、Ｔａｔタンパク質の核移行シグナル（Nuclear Localization Signal: ＮＬＳ）とＲＮＡとの結合に必須の配列を構成するペプチドである。Ｔａｔペプチドの長さとしては、９～１５アミノ酸残基が挙げられる。Ｔａｔタンパク質の具体例としては配列番号６で示されるタンパク質が挙げられる。配列番号６で示されるＴａｔタンパク質に由来するＴＡＴペプチドは、配列番号６で示されるＴａｔタンパク質のＲＮＡ結合領域に相当する４９～５７位の配列RKKRRQRRR（配列番号７）を有する。
The TAT peptide is known as an arginine-rich basic peptide derived from the Transactivator of transcription protein (Tat protein) involved in the transcriptional regulation of the human immunodeficiency virus. Specifically, the TAT peptide is a peptide that constitutes an RNA-binding region of Tat protein, that is, a sequence essential for binding of Tat protein's nuclear localization signal (NLS) to RNA. The length of the Tat peptide includes 9 to 15 amino acid residues. Specific examples of the Tat protein include the protein shown in SEQ ID NO: 6. The TAT peptide derived from the Tat protein set forth in SEQ ID NO: 6 has the sequence RKKRRQRRR (SEQ ID NO: 7) at positions 49 to 57 corresponding to the RNA binding region of the Tat protein set forth in SEQ ID NO: 6.
ＴＡＴペプチドがＴａｔタンパク質のＲＮＡ結合領域以外のアミノ酸残基を有する場合、そのようなＴＡＴペプチド全体の配列がＴａｔタンパク質に由来していることが好ましい。例えば、ＴＡＴペプチドが配列番号７に示される９アミノ酸残基を超える長さを有する場合も、ＴＡＴペプチド全体の配列が配列番号６に示すＴａｔタンパク質に由来していることが好ましい。具体的には、ＴＡＴペプチドは、配列番号６に示すＴａｔタンパク質の４９～５７位を含み、且つ、Ｎ末端に配列番号６に示すＴａｔタンパク質の４５～４８位、４６～４８位、４７～４８位、又は４８位のアミノ酸残基並びに／若しくはＣ末端に５８～６０位、５８～５９位、又は５８位のアミノ酸残基を含むことができる。上記の他、本発明においては、ＴＡＴペプチドがＴａｔタンパク質のＲＮＡ結合領域以外のアミノ酸残基を有する場合、ＲＮＡ結合領域以外のアミノ酸残基がＴａｔペプチドに由来していなくてもよい。
When the TAT peptide has an amino acid residue other than the RNA-binding region of the Tat protein, it is preferable that the sequence of the entire TAT peptide is derived from the Tat protein. For example, even when the TAT peptide has a length exceeding the 9 amino acid residues shown in SEQ ID NO: 7, it is preferable that the entire sequence of the TAT peptide is derived from the Tat protein shown in SEQ ID NO: 6. Specifically, the TAT peptide contains positions 49 to 57 of the Tat protein shown in SEQ ID NO: 6, and at the N-terminal, positions 45 to 48, 46 to 48, and 47 to 48 of the Tat protein shown in SEQ ID NO: 6. The amino acid residue at position or 48 and / or the amino acid residue at position 58-60, 58-59, or 58 at the C-terminus can be contained. In addition to the above, in the present invention, when the TAT peptide has an amino acid residue other than the RNA-binding region of Tat protein, the amino acid residue other than the RNA-binding region may not be derived from the Tat peptide.
本発明で用いられるＴＡＴペプチドの具体例としては、以下が挙げられる。
・RKKRRQRRR（配列番号７；配列番号６に示すＴａｔタンパク質の４９～５７位からなるペプチド）
・GRKKRRQRRRAHQ（配列番号８；配列番号６に示すＴａｔタンパク質の４８～６０位からなるペプチド）
・GRKKRRQRRRA（配列番号９；配列番号６に示すＴａｔタンパク質の４８～５８位からなるペプチド）
・YGRKKRRQRRR（配列番号１０；配列番号６に示すＴａｔタンパク質の４７～５７位からなるペプチド）
・GRKKRRQRRRPPQ（配列番号１１）
・GRKKRRQRRRP（配列番号１２） Specific examples of the TAT peptide used in the present invention include the following.
RKKRRQRRR (SEQ ID NO: 7; peptide consisting of positions 49-57 of the Tat protein shown in SEQ ID NO: 6)
GRKKRRQRRRAHQ (SEQ ID NO: 8; peptide consisting of positions 48-60 of the Tat protein shown in SEQ ID NO: 6)
GRKKRRQRRRA (SEQ ID NO: 9; peptide consisting of positions 48-58 of the Tat protein shown in SEQ ID NO: 6)
YGRKKRRQRRR (SEQ ID NO: 10; peptide consisting of positions 47-57 of the Tat protein shown in SEQ ID NO: 6)
-GRKKRRQRRRPPQ (SEQ ID NO: 11)
・ GRKKRRQRRRP (SEQ ID NO: 12)
・RKKRRQRRR（配列番号７；配列番号６に示すＴａｔタンパク質の４９～５７位からなるペプチド）
・GRKKRRQRRRAHQ（配列番号８；配列番号６に示すＴａｔタンパク質の４８～６０位からなるペプチド）
・GRKKRRQRRRA（配列番号９；配列番号６に示すＴａｔタンパク質の４８～５８位からなるペプチド）
・YGRKKRRQRRR（配列番号１０；配列番号６に示すＴａｔタンパク質の４７～５７位からなるペプチド）
・GRKKRRQRRRPPQ（配列番号１１）
・GRKKRRQRRRP（配列番号１２） Specific examples of the TAT peptide used in the present invention include the following.
RKKRRQRRR (SEQ ID NO: 7; peptide consisting of positions 49-57 of the Tat protein shown in SEQ ID NO: 6)
GRKKRRQRRRAHQ (SEQ ID NO: 8; peptide consisting of positions 48-60 of the Tat protein shown in SEQ ID NO: 6)
GRKKRRQRRRA (SEQ ID NO: 9; peptide consisting of positions 48-58 of the Tat protein shown in SEQ ID NO: 6)
YGRKKRRQRRR (SEQ ID NO: 10; peptide consisting of positions 47-57 of the Tat protein shown in SEQ ID NO: 6)
-GRKKRRQRRRPPQ (SEQ ID NO: 11)
・ GRKKRRQRRRP (SEQ ID NO: 12)
これらのタグのうち、サイトカインの細胞膜透過能の向上作用がより優れているのはＴＡＴペプチドである。一方、オリゴヒスチジンは、サイトグロビンタンパク質の細胞膜透過能の向上作用自体はＴＡＴペプチドに劣るが、オリゴヒスチジンを有するサイトグロビンタンパク質は、ＴＡＴペプチドを有するサイトグロビンタンパク質よりも低用量で抗線維化能を発揮することができる点で好ましい。このように、細胞膜透過能とは無関係にオリゴヒスチジンタグを有するサイトグロビンタンパク質の方がＴＡＴペプチドタグを有するサイトグロビンタンパク質よりも効能が高い理由としては定かではないが、少なくとも、オリゴヒスチジンタグを有するサイトグロビンタンパク質とＴＡＴペプチドタグを有するサイトグロビンタンパク質とに関して細胞膜を透過した後の細胞内における存在場所が互いに大きく異なることが挙げられる。
Among these tags, the TAT peptide has a more excellent effect of improving the cell membrane permeability of cytokines. On the other hand, oligohistidine is inferior to TAT peptide in improving the cell membrane permeability of cytoglobin protein, but cytoglobin protein having oligohistidine has antifibrinolytic ability at a lower dose than cytoglobin protein having TAT peptide. It is preferable in that it can be exerted. Thus, the reason why the cytoglobin protein having an oligohistidine tag is more effective than the cytoglobin protein having a TAT peptide tag regardless of the cell membrane permeability is not clear, but at least it has an oligohistidine tag. The location of the cytoglobin protein and the cytoglobin protein having a TAT peptide tag in the cell after permeating the cell membrane is significantly different from each other.
本発明において、サイトグロビンタンパク質におけるタグの結合場所としては特に限定されないが、サイトグロビンタンパク質の好ましい細胞膜透過能を得る観点、及び／又は、好ましい抗線維化能を得る観点から、Ｎ末端が挙げられる。また、タグは、サイトグロビンタンパク質に直接結合していてもよいし、数個のアミノ酸残基、例えば１～５個のアミノ酸残基を介して間接的に結合していてもよい。更に、本発明において、タグで修飾されたサイトグロビンタンパク質は、上記のサイトグロビンタンパク質配列、上記のタグ配列、並びに、上記のサイトグロビンタンパク質配列及びタグ配列の間に介在してよいアミノ酸残基の他に、タグ配列のサイトグロビンタンパク質側とは反対側、及び／又はサイトグロビンタンパク質のタグ配列とは反対側に、１～５個のアミノ酸残基を更に有していてもよい。
In the present invention, the binding site of the tag in the cytoglobin protein is not particularly limited, but the N-terminal can be mentioned from the viewpoint of obtaining a preferable cell membrane penetrating ability of the cytoglobin protein and / or a preferable antifibrotic ability. .. In addition, the tag may be directly bound to the cytoglobin protein, or may be indirectly bound via several amino acid residues, for example, 1 to 5 amino acid residues. Furthermore, in the present invention, the tag-modified cytoglobin protein is an amino acid residue that may intervene between the cytoglobin protein sequence, the tag sequence, and the cytoglobin protein sequence and the tag sequence. Alternatively, it may further have 1 to 5 amino acid residues on the opposite side of the tag sequence from the cytoglobin protein side and / or on the opposite side of the tag sequence of the cytoglobin protein.
本発明の抗線維化剤は、上記（１）～（３）のポリペプチドと上記のタグとが結合したポリペプチドであり、その製造方法としては特に限定されない。上記（１）～（３）のポリペプチドと上記のタグとを別々に調製し、その後、両者を結合させてもよいし、上記（１）～（３）のポリペプチドと上記のタグとの融合ポリペプチドとして調製してもよい。上記（１）～（３）のポリペプチド、タグ、及び融合ポリペプチドは、当業者に利用可能な各種の遺伝子組み換え技術によって宿主細胞を用いて生物学的に調製することができる。また、上記（１）～（３）のポリペプチド、タグ、及び融合ポリペプチドは、当業者に利用可能な固相合成法などのペプチド合成反応を利用した有機化学的方法で調製することもでき、また、ペプチドシンセサイザーを用いた自動合成により調製することもできる。
The antifibrotic agent of the present invention is a polypeptide in which the above-mentioned polypeptides (1) to (3) and the above-mentioned tag are bound, and the production method thereof is not particularly limited. The above-mentioned polypeptides (1) to (3) and the above-mentioned tag may be prepared separately, and then both may be bound to each other, or the above-mentioned polypeptides (1) to (3) and the above-mentioned tag may be combined. It may be prepared as a fusion polypeptide. The polypeptides, tags, and fusion polypeptides of (1) to (3) above can be biologically prepared using host cells by various gene recombination techniques available to those skilled in the art. In addition, the above-mentioned polypeptides (1) to (3), tags, and fusion polypeptides can also be prepared by an organic chemical method utilizing a peptide synthesis reaction such as a solid phase synthesis method available to those skilled in the art. It can also be prepared by automatic synthesis using a peptide synthesizer.
他の成分
本発明の抗線維化剤は、前述の有効成分の他に、治療対象となる疾患の種類に応じて、他の薬理活性成分を含んでいてもよい。 Other Ingredients The antifibrotic agent of the present invention may contain other pharmacologically active ingredients in addition to the above-mentioned active ingredients, depending on the type of disease to be treated.
本発明の抗線維化剤は、前述の有効成分の他に、治療対象となる疾患の種類に応じて、他の薬理活性成分を含んでいてもよい。 Other Ingredients The antifibrotic agent of the present invention may contain other pharmacologically active ingredients in addition to the above-mentioned active ingredients, depending on the type of disease to be treated.
また、本発明の抗線維化剤は、前記有効成分の他に、所望の投与形態及び製剤形態に調製するために、必要に応じて、薬学的に許容される担体や添加剤を含んでいてもよい。このような担体や添加剤としては、希釈剤、賦形剤、結合剤、崩壊剤、滑沢剤、懸濁化剤、溶解補助剤、安定化剤、甘味剤、着色剤、矯味剤、矯臭剤、界面活性剤、保湿剤、保存剤、ｐＨ調整剤、緩衝剤、粘稠化剤等が挙げられる。
In addition to the active ingredient, the antifibrotic agent of the present invention contains, if necessary, a pharmaceutically acceptable carrier or additive in order to prepare a desired dosage form and formulation form. May be good. Such carriers and additives include diluents, excipients, binders, disintegrants, lubricants, suspending agents, solubilizers, stabilizers, sweeteners, colorants, flavoring agents, and odorants. Examples thereof include agents, surfactants, moisturizers, preservatives, pH adjusters, buffers, thickeners and the like.
剤型
本発明の抗線維化剤の剤型については、特に制限されず、その投与形態等に応じて適宜設定すればよい。本発明の抗線維化剤の剤型として、具体的には、注射剤、シロップ剤、細胞懸濁液、リポソーム製剤等の液状製剤；錠剤、硬カプセル剤、軟カプセル剤、顆粒剤、散剤、丸剤等の固形状製剤等が挙げられる。また、注射剤にする場合には、使用前に生理食塩水等で溶解する用時調製用粉末（例えば凍結乾燥粉末）の形態であってもよい。 The dosage form of the anti-fibrotic agent dosage forms the present invention is not particularly limited, it may be appropriately set depending on the dosage forms and the like. Specific examples of the dosage form of the antifibrinolytic agent of the present invention include liquid preparations such as injections, syrups, cell suspensions, and liposome preparations; tablets, hard capsules, soft capsules, granules, powders, etc. Examples include solid preparations such as pills. Further, when it is used as an injection, it may be in the form of a powder for preparation at the time of use (for example, lyophilized powder) which is dissolved in physiological saline or the like before use.
本発明の抗線維化剤の剤型については、特に制限されず、その投与形態等に応じて適宜設定すればよい。本発明の抗線維化剤の剤型として、具体的には、注射剤、シロップ剤、細胞懸濁液、リポソーム製剤等の液状製剤；錠剤、硬カプセル剤、軟カプセル剤、顆粒剤、散剤、丸剤等の固形状製剤等が挙げられる。また、注射剤にする場合には、使用前に生理食塩水等で溶解する用時調製用粉末（例えば凍結乾燥粉末）の形態であってもよい。 The dosage form of the anti-fibrotic agent dosage forms the present invention is not particularly limited, it may be appropriately set depending on the dosage forms and the like. Specific examples of the dosage form of the antifibrinolytic agent of the present invention include liquid preparations such as injections, syrups, cell suspensions, and liposome preparations; tablets, hard capsules, soft capsules, granules, powders, etc. Examples include solid preparations such as pills. Further, when it is used as an injection, it may be in the form of a powder for preparation at the time of use (for example, lyophilized powder) which is dissolved in physiological saline or the like before use.
用途
本発明の抗線維化剤は、生体内で組織の線維化を抑制することによって予防又は治療効果が期待される疾患に適用して使用される。 Uses The antifibrotic agent of the present invention is used by applying it to a disease that is expected to have a preventive or therapeutic effect by suppressing tissue fibrosis in vivo.
本発明の抗線維化剤は、生体内で組織の線維化を抑制することによって予防又は治療効果が期待される疾患に適用して使用される。 Uses The antifibrotic agent of the present invention is used by applying it to a disease that is expected to have a preventive or therapeutic effect by suppressing tissue fibrosis in vivo.
本発明の抗線維化剤は、線維化の予防又は治療に用いることができ、また、線維化組織を母地とする癌の予防にも用いることができる。本発明の抗線維化剤は、様々な組織の線維化の予防又は治療に用いることができ、組織の具体例としては、肝臓（肝星細胞）、膵臓（膵星細胞）、腎臓（尿細管上皮近傍の線維芽細胞）、脳、胸腺、肺、乳房、心臓、胃、腸、皮膚、骨髄等が挙げられ、好ましくは、肝臓、膵臓、腎臓が挙げられ、より好ましくは肝臓が挙げられる。
The antifibrotic agent of the present invention can be used for the prevention or treatment of fibrosis, and can also be used for the prevention of cancer based on fibrotic tissue. The antifibrotic agent of the present invention can be used for the prevention or treatment of fibrosis of various tissues, and specific examples of the tissues include liver (hepatic stellate cell), pancreas (pancreatic stellate cell), kidney (urinary tubule). Fibrotic cells in the vicinity of the epithelium), brain, thoracic gland, lung, breast, heart, stomach, intestine, skin, bone marrow and the like, preferably liver, pancreas, kidney, and more preferably liver.
より具体的には、本発明の抗線維化剤は、様々な線維化疾患の予防又は治療に用いることができ、線維化疾患の具体例としては、肝線維症、肝硬変、膵嚢胞性線維症、腎線維症、グリオーシス、びまん性胸腺線維症、肺線維症、間質性肺炎、心筋線維症、心筋梗塞、胃繊維症、腸線維症、皮膚線維症、骨髄繊維症が挙げられ、好ましくは、肝線維症、肝硬変、膵嚢胞性線維症、腎線維症が挙げられ、より好ましくは肝線維症、肝硬変が挙げられ、さらに好ましくは肝硬変が挙げられる。
More specifically, the antifibrotic agent of the present invention can be used for the prevention or treatment of various fibrotic diseases, and specific examples of fibrotic diseases include liver fibrosis, liver cirrhosis, and pancreatic cystic fibrosis. , Renal fibrosis, gliosis, diffuse thoracic fibrosis, pulmonary fibrosis, interstitial pneumonia, myocardial fibrosis, myocardial infarction, gastric fibrosis, intestinal fibrosis, cutaneous fibrosis, myeloid fibrosis. , Liver fibrosis, liver cirrhosis, pancreatic cystic fibrosis, renal fibrosis, more preferably liver fibrosis, liver cirrhosis, and further preferably liver cirrhosis.
また、本発明の抗線維化剤は、様々な癌の予防にも用いることができ、癌の具体例としては、肝臓癌、膵臓癌、腎臓癌、脳腫瘍、胸腺癌、肺癌、乳癌、胃癌、大腸癌、結腸癌、皮膚癌、骨肉腫、軟骨肉腫が挙げられ、好ましくは、肝臓癌、膵臓癌、腎臓癌が挙げられ、より好ましくは肝臓癌が挙げられる。
The antifibrotic agent of the present invention can also be used for prevention of various cancers, and specific examples of cancers include liver cancer, pancreatic cancer, kidney cancer, brain cancer, thoracic adenocarcinoma, lung cancer, breast cancer, gastric cancer, and the like. Colorectal cancer, colon cancer, skin cancer, osteosarcoma, chondrosarcoma can be mentioned, preferably liver cancer, pancreatic cancer, kidney cancer, and more preferably liver cancer.
本発明の抗線維化剤において、投与対象となる生物は、線維化抑制が求められる生物であればよく、ヒト、ラット、ハムスター、モルモット、マウス、ウシ、ヒツジ、ブタ、ヤギ、サル、ウサギ等の哺乳動物等が挙げられ、好ましくはヒトが挙げられる。
In the antifibrotic agent of the present invention, the organism to be administered may be any organism that is required to suppress fibrosis, such as humans, rats, hamsters, guinea pigs, mice, cows, sheep, pigs, goats, monkeys, and rabbits. Mammals and the like, preferably humans.
投与方法
本発明の抗線維化剤の投与形態としては、例えば、局所投与、皮下投与、腹腔内投与、筋肉内投与、静脈内投与、経直腸的、皮内投与等の非経口投与；経口投与が挙げられ、適用する疾患の種類等に応じて適宜設定すればよい。 Administration method Examples of the administration form of the antifibrotic agent of the present invention include parenteral administration such as local administration, subcutaneous administration, intraperitoneal administration, intramuscular administration, intravenous administration, transrectal administration, and intradermal administration; oral administration. However, it may be appropriately set according to the type of disease to be applied.
本発明の抗線維化剤の投与形態としては、例えば、局所投与、皮下投与、腹腔内投与、筋肉内投与、静脈内投与、経直腸的、皮内投与等の非経口投与；経口投与が挙げられ、適用する疾患の種類等に応じて適宜設定すればよい。 Administration method Examples of the administration form of the antifibrotic agent of the present invention include parenteral administration such as local administration, subcutaneous administration, intraperitoneal administration, intramuscular administration, intravenous administration, transrectal administration, and intradermal administration; oral administration. However, it may be appropriately set according to the type of disease to be applied.
本発明の抗線維化剤の投与量については、適用する疾患の種類、投与対象者の年齢、性別、体重、症状の程度、投与形態等に応じて適宜設定すればよいが、例えば、１日当たり、８～１０ｍｇ／６０ｋｇ、好ましくは９～１０ｍｇ／６０ｋｇ、より好ましくは９．５～９．７ｍｇ／６０ｋｇ程度となる量を１回又は数回に分けて投与することができる。
The dose of the antifibrotic agent of the present invention may be appropriately set according to the type of disease to be applied, the age, sex, body weight, degree of symptom, administration form, etc. of the subject to be administered. For example, per day. , 8 to 10 mg / 60 kg, preferably 9 to 10 mg / 60 kg, more preferably about 9.5 to 9.7 mg / 60 kg, can be administered once or in several divided doses.
以下、本発明を実施例により更に詳細に説明するが、本発明はこれらの実施例によって何ら限定されるものではない。なお、図面において、「*」はP＜0.05、「**」はP＜0.01、「***」はP＜0.001であることを示している。
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. In the drawings, "*" indicates P <0.05, "**" indicates P <0.01, and "***" indicates P <0.001.
試験例１：ＨＨＳｔｅＣ細胞及び初代培養ヒト星細胞に及ぼすＨｉｓ－ｒｈＣＹＧＢの効果（ｉｎ ｖｉｔｒｏ）
本試験例では、６個のヒスチジン残基からなるオリゴヒスチジンタグが配列番号５に示すサイトグロビンのＮ末端に結合した融合ポリペプチドＨｉｓ－ｒｈＣＹＧＢを調製し、調製されたＨｉｓ－ｒｈＣＹＧＢについて、ＨＨＳｔｅＣ細胞及び初代培養ヒト星細胞に及ぼす影響を検証した。 Test Example 1: Effect of His-rhCYGB on HHSteC cells and primary cultured human stellate cells (in vitro)
In this test example, a fusion polypeptide His-rhCYGB in which an oligohistidine tag consisting of 6 histidine residues was bound to the N-terminal of cytoglobin shown in SEQ ID NO: 5 was prepared, and for the prepared His-rhCYGB, HHSteC cells were prepared. And the effect on primary cultured human stellate cells was examined.
本試験例では、６個のヒスチジン残基からなるオリゴヒスチジンタグが配列番号５に示すサイトグロビンのＮ末端に結合した融合ポリペプチドＨｉｓ－ｒｈＣＹＧＢを調製し、調製されたＨｉｓ－ｒｈＣＹＧＢについて、ＨＨＳｔｅＣ細胞及び初代培養ヒト星細胞に及ぼす影響を検証した。 Test Example 1: Effect of His-rhCYGB on HHSteC cells and primary cultured human stellate cells (in vitro)
In this test example, a fusion polypeptide His-rhCYGB in which an oligohistidine tag consisting of 6 histidine residues was bound to the N-terminal of cytoglobin shown in SEQ ID NO: 5 was prepared, and for the prepared His-rhCYGB, HHSteC cells were prepared. And the effect on primary cultured human stellate cells was examined.
（Ｈｉｓ－ｒｈＣＹＧＢの調製）
ヒトＣｙｇｂ ｃＤＮＡをｐＲＳＥＴＡベクター（ヒスチジンタグサイトを含む）に挿入してシークエンスを確認した。ベクターをＥ．ｃｏｌｉ ＢＬ２１ＡＩに感染させ、感染クローンを取得後、５０μｇ／ｍｌアンピシリン、１０μｇ／ｍｌ テトラサイクリンを含む３．２ＬのLuria-Bertani溶液で３７℃振盪培養した。０．１ｗ／ｗ％アラビノースによって、融合ポリペプチドＨｉｓ－ｒｈＣＹＧＢを誘導した。Ｈｉｓ－ｒｈＣＹＧＢはfast protein liquid chromatography（ＦＰＬＣ）を用いてクリーンアップし、引き続いて、Ｓｅｐｈａｄｅｘ Ｇ２５カラムを用いてさらなるクリーンアップと脱塩とを行った。精製したＨｉｓ－ｒｈＣＹＧＢにエンドトキシンのコンタミネーションはなく、細胞への毒性も否定された。さらに、ピリジンヘモクロモーゲンアッセイにてヘムの存在も確認された。なお、調製されたＨｉｓ－ｒｈＣＹＧＢは、配列番号１３に示す配列を有している。 (Preparation of His-rhCYGB)
The human Cygb cDNA was inserted into the pRSETA vector (including histidine tag site) to confirm the sequence. Vector to E. After infecting with E. coli BL21AI and obtaining an infected clone, the cells were cultured with shaking at 37 ° C. in 3.2 L of Luria-Bertani solution containing 50 μg / ml ampicillin and 10 μg / ml tetracycline. The fusion polypeptide His-rhCYGB was induced with 0.1 w / w% arabinose. His-rhCYGB was cleaned up using fast protein liquid chromatography (FPLC), followed by further cleanup and desalting using a Sephadex G25 column. The purified His-rhCYGB had no endotoxin contamination, and its toxicity to cells was ruled out. In addition, the presence of heme was confirmed by the pyridine hemochromogen assay. The prepared His-rhCYGB has the sequence shown in SEQ ID NO: 13.
ヒトＣｙｇｂ ｃＤＮＡをｐＲＳＥＴＡベクター（ヒスチジンタグサイトを含む）に挿入してシークエンスを確認した。ベクターをＥ．ｃｏｌｉ ＢＬ２１ＡＩに感染させ、感染クローンを取得後、５０μｇ／ｍｌアンピシリン、１０μｇ／ｍｌ テトラサイクリンを含む３．２ＬのLuria-Bertani溶液で３７℃振盪培養した。０．１ｗ／ｗ％アラビノースによって、融合ポリペプチドＨｉｓ－ｒｈＣＹＧＢを誘導した。Ｈｉｓ－ｒｈＣＹＧＢはfast protein liquid chromatography（ＦＰＬＣ）を用いてクリーンアップし、引き続いて、Ｓｅｐｈａｄｅｘ Ｇ２５カラムを用いてさらなるクリーンアップと脱塩とを行った。精製したＨｉｓ－ｒｈＣＹＧＢにエンドトキシンのコンタミネーションはなく、細胞への毒性も否定された。さらに、ピリジンヘモクロモーゲンアッセイにてヘムの存在も確認された。なお、調製されたＨｉｓ－ｒｈＣＹＧＢは、配列番号１３に示す配列を有している。 (Preparation of His-rhCYGB)
The human Cygb cDNA was inserted into the pRSETA vector (including histidine tag site) to confirm the sequence. Vector to E. After infecting with E. coli BL21AI and obtaining an infected clone, the cells were cultured with shaking at 37 ° C. in 3.2 L of Luria-Bertani solution containing 50 μg / ml ampicillin and 10 μg / ml tetracycline. The fusion polypeptide His-rhCYGB was induced with 0.1 w / w% arabinose. His-rhCYGB was cleaned up using fast protein liquid chromatography (FPLC), followed by further cleanup and desalting using a Sephadex G25 column. The purified His-rhCYGB had no endotoxin contamination, and its toxicity to cells was ruled out. In addition, the presence of heme was confirmed by the pyridine hemochromogen assay. The prepared His-rhCYGB has the sequence shown in SEQ ID NO: 13.
（線維症関連タンパク質の発現に及ぼすＨｉｓ－ｒｈＣＹＧＢの効果）
細胞株のＨＨＳｔｅＣ細胞（ScienCell Research Laboratoriesより購入）を実験に用いた。４０μｇ／ｍＬのＨｉｓ－ｒｈＣＹＧＢをリン酸緩衝溶液（５０ｍＭ ＮａＨ2ＰＯ4，３００ｍＭ ＮａＣｌ）に含む溶液を調製し、０～４８時間、ＨＨＳｔｅＣ細胞に暴露し、暴露前（０分）、並びに暴露開始後１時間、４時間、８時間、２４時間及び４８時間の時点における細胞内ＣＹＧＢ及び線維症関連タンパク質をウエスタンブロットにて確認した。ウエスタンブロット像を図１に示す。図１に示されるように、Ｈｉｓ－ｒｈＣＹＧＢに暴露された細胞内のＣＹＧＢ濃度は時間依存的に増加した。また、発現していたαＳＭＡは時間依存的に減少した。発現していたＩ型コラーゲン（ＣＯＬ１Ａ１）も時間依存的に減少し、４８時間後には消失した。なお、ＧＡＰＤＨはハウスキーピングタンパク質である。なお、ウエスタンブロットは３回独立して実験し、再現性を確認した。 (Effect of His-rhCYGB on expression of fibrosis-related proteins)
HHSteC cells from the cell line (purchased from ScienCell Research Laboratories) were used in the experiment. A solution containing 40 μg / mL His-rhCYGB in a phosphate buffer solution (50 mM NaH 2 PO 4 , 300 mM NaCl) was prepared and exposed to HHSteC cells for 0 to 48 hours before exposure (0 minutes) and onset of exposure. Intracellular CYGB and fibrosis-related proteins were confirmed by Western blotting at 1 hour, 4 hours, 8 hours, 24 hours and 48 hours. A Western blot image is shown in FIG. As shown in FIG. 1, the intracellular CYGB concentration exposed to His-rhCYGB increased in a time-dependent manner. In addition, the expressed αSMA decreased in a time-dependent manner. The expressed type I collagen (COL1A1) also decreased in a time-dependent manner and disappeared after 48 hours. GAPDH is a housekeeping protein. Western blotting was independently tested three times to confirm reproducibility.
細胞株のＨＨＳｔｅＣ細胞（ScienCell Research Laboratoriesより購入）を実験に用いた。４０μｇ／ｍＬのＨｉｓ－ｒｈＣＹＧＢをリン酸緩衝溶液（５０ｍＭ ＮａＨ2ＰＯ4，３００ｍＭ ＮａＣｌ）に含む溶液を調製し、０～４８時間、ＨＨＳｔｅＣ細胞に暴露し、暴露前（０分）、並びに暴露開始後１時間、４時間、８時間、２４時間及び４８時間の時点における細胞内ＣＹＧＢ及び線維症関連タンパク質をウエスタンブロットにて確認した。ウエスタンブロット像を図１に示す。図１に示されるように、Ｈｉｓ－ｒｈＣＹＧＢに暴露された細胞内のＣＹＧＢ濃度は時間依存的に増加した。また、発現していたαＳＭＡは時間依存的に減少した。発現していたＩ型コラーゲン（ＣＯＬ１Ａ１）も時間依存的に減少し、４８時間後には消失した。なお、ＧＡＰＤＨはハウスキーピングタンパク質である。なお、ウエスタンブロットは３回独立して実験し、再現性を確認した。 (Effect of His-rhCYGB on expression of fibrosis-related proteins)
HHSteC cells from the cell line (purchased from ScienCell Research Laboratories) were used in the experiment. A solution containing 40 μg / mL His-rhCYGB in a phosphate buffer solution (50 mM NaH 2 PO 4 , 300 mM NaCl) was prepared and exposed to HHSteC cells for 0 to 48 hours before exposure (0 minutes) and onset of exposure. Intracellular CYGB and fibrosis-related proteins were confirmed by Western blotting at 1 hour, 4 hours, 8 hours, 24 hours and 48 hours. A Western blot image is shown in FIG. As shown in FIG. 1, the intracellular CYGB concentration exposed to His-rhCYGB increased in a time-dependent manner. In addition, the expressed αSMA decreased in a time-dependent manner. The expressed type I collagen (COL1A1) also decreased in a time-dependent manner and disappeared after 48 hours. GAPDH is a housekeeping protein. Western blotting was independently tested three times to confirm reproducibility.
また、Ｈｉｓ－ｒｈＣＹＧＢを含まない（０μｇ／ｍＬ）リン酸緩衝溶液、並びに、Ｈｉｓ－ｒｈＣＹＧＢを５μｇ／ｍＬ、１０μｇ／ｍＬ、２０μｇ／ｍＬ、４０μｇ／ｍＬ、又は８０μｇ／ｍＬの濃度でリン酸緩衝溶液中に含む溶液を調製し、４８時間、ＨＨＳｔｅＣ細胞に暴露し、細胞内ＣＹＧＢをウエスタンブロットにて確認した。ウエスタンブロット像を図２に示す。図２に示されるように、Ｈｉｓ－ｒｈＣＹＧＢに暴露された細胞内のＣＹＧＢ濃度は用量依存的に増加し、４０μｇ／ｍＬ以上でプラトーに達した。また、発現していたαＳＭＡ、及びＩ型コラーゲン（ＣＯＬ１Ａ１）は用量依存的に減少した。なお、ウエスタンブロットは３回独立して実験し、再現性を確認した。
In addition, a phosphate buffer solution containing no His-rhCYGB (0 μg / mL) and His-rhCYGB at a concentration of 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, or 80 μg / mL. The solution contained in the solution was prepared, exposed to HHSteC cells for 48 hours, and intracellular CYGB was confirmed by Western blot. A Western blot image is shown in FIG. As shown in FIG. 2, the intracellular CYGB concentration exposed to His-rhCYGB increased in a dose-dependent manner and reached a plateau at 40 μg / mL and above. In addition, the expressed αSMA and type I collagen (COL1A1) decreased in a dose-dependent manner. Western blotting was independently tested three times to confirm reproducibility.
（線維症関連遺伝子の転写に及ぼすＨｉｓ－ｒｈＣＹＧＢの効果）
４０μｇ／ｍＬのＨｉｓ－ｒｈＣＹＧＢをリン酸緩衝溶液中に含む溶液を調製し、０～４８時間、ＨＨＳｔｅＣ細胞に暴露し、暴露前（０分）、並びに暴露開始後１時間、４時間、８時間、２４時間及び４８時間の時点におけるαＳｍａ、Ｃｏｌ３ａ１、及びＴｇｆβ－１（Transforming growth factor β-1）のｍＲＮＡ量を測定した。その結果、Ｈｉｓ－ｒｈＣＹＧＢに暴露された細胞におけるαＳｍａ、Ｃｏｌ３ａ１、及びＴｇｆβ－１のｍＲＮＡ発現量が時間依存的に低下したことを確認した。 (Effect of His-rhCYGB on transcription of fibrosis-related genes)
A solution containing 40 μg / mL His-rhCYGB in a phosphate buffer solution was prepared and exposed to HHSteC cells for 0 to 48 hours before exposure (0 minutes) and 1 hour, 4 hours, and 8 hours after the start of exposure. , The amount of αSma, Col3a1 and Tgfβ-1 (Transforming growth factor β-1) mRNA at 24 hours and 48 hours was measured. As a result, it was confirmed that the mRNA expression levels of αSma, Col3a1 and Tgfβ-1 in the cells exposed to His-rhCYGB decreased in a time-dependent manner.
４０μｇ／ｍＬのＨｉｓ－ｒｈＣＹＧＢをリン酸緩衝溶液中に含む溶液を調製し、０～４８時間、ＨＨＳｔｅＣ細胞に暴露し、暴露前（０分）、並びに暴露開始後１時間、４時間、８時間、２４時間及び４８時間の時点におけるαＳｍａ、Ｃｏｌ３ａ１、及びＴｇｆβ－１（Transforming growth factor β-1）のｍＲＮＡ量を測定した。その結果、Ｈｉｓ－ｒｈＣＹＧＢに暴露された細胞におけるαＳｍａ、Ｃｏｌ３ａ１、及びＴｇｆβ－１のｍＲＮＡ発現量が時間依存的に低下したことを確認した。 (Effect of His-rhCYGB on transcription of fibrosis-related genes)
A solution containing 40 μg / mL His-rhCYGB in a phosphate buffer solution was prepared and exposed to HHSteC cells for 0 to 48 hours before exposure (0 minutes) and 1 hour, 4 hours, and 8 hours after the start of exposure. , The amount of αSma, Col3a1 and Tgfβ-1 (Transforming growth factor β-1) mRNA at 24 hours and 48 hours was measured. As a result, it was confirmed that the mRNA expression levels of αSma, Col3a1 and Tgfβ-1 in the cells exposed to His-rhCYGB decreased in a time-dependent manner.
また、Ｈｉｓ－ｒｈＣＹＧＢを含まない（０μｇ／ｍＬ）リン酸緩衝溶液、並びに、Ｈｉｓ－ｒｈＣＹＧＢを５μｇ／ｍＬ、１０μｇ／ｍＬ、２０μｇ／ｍＬ、又は４０μｇ／ｍＬの濃度でリン酸緩衝溶液に含む溶液を調製し、４８時間、ＨＨＳｔｅＣ細胞に暴露し、αＳｍａ、Ｃｏｌ３ａ１、及びＴｇｆβ－１のｍＲＮＡ量を測定した。結果を図３に示す。図３に示されるように、Ｈｉｓ－ｒｈＣＹＧＢに暴露された細胞において、αＳｍａ、Ｃｏｌ３ａ１、及びＴｇｆβ－１のｍＲＮＡ発現は用量依存的に低下した。また、いずれの線維症関連遺伝子についても、わずか１０μｇ／ｍＬの用量で転写量の有意な低下が認められ、αＳｍａについては、わずか５μｇ／ｍＬの用量でも転写量の有意な低下が認められた。
In addition, a phosphate buffer solution containing no His-rhCYGB (0 μg / mL) and a solution containing His-rhCYGB at a concentration of 5 μg / mL, 10 μg / mL, 20 μg / mL, or 40 μg / mL in the phosphate buffer solution. Was prepared, exposed to HHSteC cells for 48 hours, and the mRNA levels of αSma, Col3a1 and Tgfβ-1 were measured. The results are shown in FIG. As shown in FIG. 3, mRNA expression of αSma, Col3a1, and Tgfβ-1 was dose-dependently reduced in cells exposed to His-rhCYGB. In addition, for all fibrosis-related genes, a significant decrease in transcription amount was observed at a dose of only 10 μg / mL, and for αSma, a significant decrease in transcription amount was observed even at a dose of only 5 μg / mL.
（初代培養ヒト星細胞に対する効果）
なお、ＨＨＳｔｅＣ細胞の代わりに、ユニバーシティーカレッジロンドンから提供された初代培養ヒト星細胞を用い、上述と同様の試験を行った。その結果、線維症関連タンパク質の発現に及ぼすＨｉｓ－ｒｈＣＹＧＢの効果、及び線維症関連遺伝子の転写に及ぼすＨｉｓ－ｒｈＣＹＧＢの効果のいずれについても、図１～図３と同様の結果が得られた。 (Effect on primary cultured human stellate cells)
In addition, instead of HHSteC cells, primary cultured human stellate cells provided by University College London were used, and the same test as described above was performed. As a result, the same results as those in FIGS. 1 to 3 were obtained for both the effect of His-rhCYGB on the expression of fibrosis-related proteins and the effect of His-rhCYGB on the transcription of fibrosis-related genes.
なお、ＨＨＳｔｅＣ細胞の代わりに、ユニバーシティーカレッジロンドンから提供された初代培養ヒト星細胞を用い、上述と同様の試験を行った。その結果、線維症関連タンパク質の発現に及ぼすＨｉｓ－ｒｈＣＹＧＢの効果、及び線維症関連遺伝子の転写に及ぼすＨｉｓ－ｒｈＣＹＧＢの効果のいずれについても、図１～図３と同様の結果が得られた。 (Effect on primary cultured human stellate cells)
In addition, instead of HHSteC cells, primary cultured human stellate cells provided by University College London were used, and the same test as described above was performed. As a result, the same results as those in FIGS. 1 to 3 were obtained for both the effect of His-rhCYGB on the expression of fibrosis-related proteins and the effect of His-rhCYGB on the transcription of fibrosis-related genes.
試験例２：肝線維化モデルに及ぼすＨｉｓ－ｒｈＣＹＧＢの効果（ｉｎ ｖｉｖｏ）
本試験例では、試験例１で調製されたＨｉｓ－ｒｈＣＹＧＢについて、チオアセトアミド（ＴＡＡ）で誘発される肝炎・肝線維化マウスモデルに対する効果を検証した。 Test Example 2: Effect of His-rhCYGB on liver fibrosis model (in vivo)
In this test example, the effect of His-rhCYGB prepared in Test Example 1 on a thioacetamide (TAA) -induced hepatitis / liver fibrosis mouse model was verified.
本試験例では、試験例１で調製されたＨｉｓ－ｒｈＣＹＧＢについて、チオアセトアミド（ＴＡＡ）で誘発される肝炎・肝線維化マウスモデルに対する効果を検証した。 Test Example 2: Effect of His-rhCYGB on liver fibrosis model (in vivo)
In this test example, the effect of His-rhCYGB prepared in Test Example 1 on a thioacetamide (TAA) -induced hepatitis / liver fibrosis mouse model was verified.
次の３群の肝炎・肝線維化マウスモデルを作製した。
（ｉ：ＴＡＡモデル）ＴＡＡを１０週間、週２回、腹腔内投与した。投与期間中に、ＴＡＡ投与量を、５０ｍｇ／ｋｇから４００ｍｇ／ｋｇに漸増した。また、６週間目～１０週目の５週間は、ＴＡＡに加えて生理食塩水を、週２回、尾静脈から静脈投与した（コントロール）。
（ii：ＴＡＡ－ＣＹ２モデル）ＴＡＡを上記（ｉ）と同様に１０週間腹腔内投与し、９週目～１０週目の２週間は、ＴＡＡに加えて、Ｈｉｓ－ｒｈＣＹＧＢを、２ｍｇ／ｋｇの用量で週２回、尾静脈から静脈投与した。
（iiiＴＡＡ－ＣＹ５モデル）ＴＡＡを上記（ｉ）と同様に１０週間腹腔内投与し、６週目～１０週目の５週間は、ＴＡＡに加えて、Ｈｉｓ－ｒｈＣＹＧＢを、２ｍｇ／ｋｇの用量で週２回、尾静脈から静脈投与した。
投与量はパイロットスタディーの結果に基づいて決定した。 The following three groups of hepatitis / liver fibrosis mouse models were prepared.
(I: TAA model) TAA was intraperitoneally administered twice a week for 10 weeks. During the dosing period, the TAA dose was gradually increased from 50 mg / kg to 400 mg / kg. In addition to TAA, physiological saline was intravenously administered from the tail vein twice a week for 5 weeks from the 6th week to the 10th week (control).
(Ii: TAA-CY2 model) TAA was intraperitoneally administered for 10 weeks in the same manner as in (i) above, and during the 2 weeks from 9th to 10th week, in addition to TAA, His-rhCYGB was 2 mg / kg. The dose was intravenously administered from the tail vein twice a week.
(IiiTAA-CY5 model) TAA was intraperitoneally administered for 10 weeks in the same manner as in (i) above, and for 5 weeks from the 6th week to the 10th week, His-rhCYGB was administered at a dose of 2 mg / kg in addition to TAA. It was administered intravenously from the tail vein twice a week.
The dose was determined based on the results of the pilot study.
（ｉ：ＴＡＡモデル）ＴＡＡを１０週間、週２回、腹腔内投与した。投与期間中に、ＴＡＡ投与量を、５０ｍｇ／ｋｇから４００ｍｇ／ｋｇに漸増した。また、６週間目～１０週目の５週間は、ＴＡＡに加えて生理食塩水を、週２回、尾静脈から静脈投与した（コントロール）。
（ii：ＴＡＡ－ＣＹ２モデル）ＴＡＡを上記（ｉ）と同様に１０週間腹腔内投与し、９週目～１０週目の２週間は、ＴＡＡに加えて、Ｈｉｓ－ｒｈＣＹＧＢを、２ｍｇ／ｋｇの用量で週２回、尾静脈から静脈投与した。
（iiiＴＡＡ－ＣＹ５モデル）ＴＡＡを上記（ｉ）と同様に１０週間腹腔内投与し、６週目～１０週目の５週間は、ＴＡＡに加えて、Ｈｉｓ－ｒｈＣＹＧＢを、２ｍｇ／ｋｇの用量で週２回、尾静脈から静脈投与した。
投与量はパイロットスタディーの結果に基づいて決定した。 The following three groups of hepatitis / liver fibrosis mouse models were prepared.
(I: TAA model) TAA was intraperitoneally administered twice a week for 10 weeks. During the dosing period, the TAA dose was gradually increased from 50 mg / kg to 400 mg / kg. In addition to TAA, physiological saline was intravenously administered from the tail vein twice a week for 5 weeks from the 6th week to the 10th week (control).
(Ii: TAA-CY2 model) TAA was intraperitoneally administered for 10 weeks in the same manner as in (i) above, and during the 2 weeks from 9th to 10th week, in addition to TAA, His-rhCYGB was 2 mg / kg. The dose was intravenously administered from the tail vein twice a week.
(IiiTAA-CY5 model) TAA was intraperitoneally administered for 10 weeks in the same manner as in (i) above, and for 5 weeks from the 6th week to the 10th week, His-rhCYGB was administered at a dose of 2 mg / kg in addition to TAA. It was administered intravenously from the tail vein twice a week.
The dose was determined based on the results of the pilot study.
上記３群における血清中のＡＳＴ及びＡＬＴレベルの測定結果を図４に示す。図４に示すように、ＴＡＡ投与により血清中ＡＳＴ、ＡＬＴはそれぞれ１５７．９、１３０．１ＩＵ／Ｌへと上昇した一方、Ｈｉｓ－ｒｈＣＹＧＢを２週間投与した群（ＴＡＡ－ＣＹ２）ではそれらの値がそれぞれ８８．４、５０．１ＩＵ／Ｌへと有意に低下し（ｐ＜０．０１）、Ｈｉｓ－ｒｈＣＹＧＢを５週間投与した群（ＴＡＡ－ＣＹ５）ではさらに６５．９、５１．５ＩＵ／Ｌへと有意に低下した（ｐ＜０．００１）。つまり、Ｈｉｓ－ｒｈＣＹＧＢ投与によって血清中ＡＳＴ・ＡＬＴレベルが有意に低下したことが認められた。
FIG. 4 shows the measurement results of AST and ALT levels in serum in the above three groups. As shown in FIG. 4, serum AST and ALT increased to 157.9 and 130.1 IU / L, respectively, by TAA administration, while those values were obtained in the group (TAA-CY2) in which His-rhCYGB was administered for 2 weeks. Significantly decreased to 88.4 and 50.1 IU / L, respectively (p <0.01), and further 65.9, 51.5 IU / L in the group (TAA-CY5) administered with His-rhCYGB for 5 weeks. Significantly decreased to (p <0.001). That is, it was confirmed that the serum AST / ALT level was significantly reduced by the administration of His-rhCYGB.
上記３群における線維症関連遺伝子αＳｍａ、Ｃｏｌ１ａ１、Ｔｇｆβ－１、Ｔｇｆβ－３、Ｔｉｍｐ－１ と 炎症性因子 Ｔｎｆ－α、Ｃｃｌ－２、Ｃｘｃｌ－２それぞれのｍＲＮＡを測定した結果を図５に示す。図５に示すように、いずれのｍＲＮＡへの転写も、Ｈｉｓ－ｒｈＣＹＧＢの投与によって有意に低減されることが認められた。
The results of measuring the mRNAs of the fibrosis-related genes αSma, Col1a1, Tgfβ-1, Tgfβ-3, Timp-1 and the inflammatory factors Tnf-α, Ccl-2, and Cxcl-2 in the above three groups are shown in FIG. .. As shown in FIG. 5, it was found that transcription to any mRNA was significantly reduced by administration of His-rhCYGB.
上記３群について、肝臓組織を、Ｈ＆Ｅ（ヘマトキシリン・エオジン）染色；コラーゲン繊維を染色するためのＳｉＲ（シリウスレッド）染色；並びに星細胞活性化マーカーαＳＭＡ、好中球マーカーＮｅｕ、及びマクロファージマーカーＣＤ６８を核対比染色するためのＤＡＰＩ（４，６－ジアミジノ－２－フェニルインドール）染色に供した。結果を図６に示す。図６のＨ＆Ｅ染色及びシリウスレッド染色の結果に示されるとおり、組織学的にも、Ｈｉｓ－ｒｈＣＹＧＢの投与によって線維化反応が抑制されたことを確認した。また、図６のαＳＭＡ－ＤＡＰＩ、Ｎｅｕ－ＤＡＰＩ、及びＣＤ６８－ＤＡＰＩの染色結果に示されるとおり、星細胞活性化マーカーであるαＳＭＡ、好中球マーカーであるＮｅｕ、及びマクロファージマーカーであるＣＤ６８の全ての発現がＨｉｓ－ｒｈＣＹＧＢの投与によって低減したことを確認した。
For the above three groups, H & E (hematoxylin and eosin) staining; SiR (sirius red) staining for staining collagen fibers; and astrocyte activation marker αSMA, neutrophil marker Neu, and macrophage marker CD68 were used to stain liver tissue. It was subjected to DAPI (4,6-diamidino-2-phenylindole) staining for nuclear counterstaining. The results are shown in FIG. As shown in the results of H & E staining and Sirius red staining in FIG. 6, it was histologically confirmed that the fibrotic reaction was suppressed by the administration of His-rhCYGB. In addition, as shown in the staining results of αSMA-DAPI, Neu-DAPI, and CD68-DAPI in FIG. 6, all of αSMA, which is a stellate cell activation marker, Neu, which is a neutrophil marker, and CD68, which is a macrophage marker. It was confirmed that the expression of His-rhCYGB was reduced by administration of His-rhCYGB.
試験例３：ＨＨＳｔｅＣ細胞に及ぼすＴＡＴ－ｒｈＣＹＧＢの効果（ｉｎ ｖｉｔｒｏ）
本試験例では、配列番号１０に示すＴＡＴペプチドタグが配列番号５に示すサイトグロビンのＮ末端に結合した融合ポリペプチドＴＡＴ－ｒｈＣＹＧＢを調製し、調製されたＴＡＴ－ｒｈＣＹＧＢについて、星細胞に及ぼす影響を検証した。 Test Example 3: Effect of TAT-rhCYGB on HHSteC cells (in vitro)
In this test example, a fusion polypeptide TAT-rhCYGB in which the TAT peptide tag shown in SEQ ID NO: 10 was bound to the N-terminal of cytoglobin shown in SEQ ID NO: 5 was prepared, and the prepared TAT-rhCYGB had an effect on stellate cells. Was verified.
本試験例では、配列番号１０に示すＴＡＴペプチドタグが配列番号５に示すサイトグロビンのＮ末端に結合した融合ポリペプチドＴＡＴ－ｒｈＣＹＧＢを調製し、調製されたＴＡＴ－ｒｈＣＹＧＢについて、星細胞に及ぼす影響を検証した。 Test Example 3: Effect of TAT-rhCYGB on HHSteC cells (in vitro)
In this test example, a fusion polypeptide TAT-rhCYGB in which the TAT peptide tag shown in SEQ ID NO: 10 was bound to the N-terminal of cytoglobin shown in SEQ ID NO: 5 was prepared, and the prepared TAT-rhCYGB had an effect on stellate cells. Was verified.
（ＴＡＴ－ｒｈＣＹＧＢの調製）
試験例１で用いたベクターに、ＴＥＶプロテアーゼ認識シークエンスとＴＡＴサイトとを導入し、試験例１と同様にして融合タンパク質を誘導した。誘導された融合タンパク質は、ＴＡＴタグだけでなくＨｉｓタグも有しているため、ＴＥＶプロテアーゼで融合タンパク質を処理することでＨｉｓタグを除去した。これによって、ＴＡＴペプチドタグがサイトグロビンに結合したＴＡＴ－ｒｈＣＹＧＢを得た。なお、調製されたＴＡＴ－ｒｈＣＹＧＢは、配列番号１４に示す配列を有している。 (Preparation of TAT-rhCYGB)
A TEV protease recognition sequence and a TAT site were introduced into the vector used in Test Example 1 to induce a fusion protein in the same manner as in Test Example 1. Since the derived fusion protein has not only a TAT tag but also a His tag, the His tag was removed by treating the fusion protein with TEV protease. As a result, TAT-rhCYGB in which the TAT peptide tag was bound to cytoglobin was obtained. The prepared TAT-rhCYGB has the sequence shown in SEQ ID NO: 14.
試験例１で用いたベクターに、ＴＥＶプロテアーゼ認識シークエンスとＴＡＴサイトとを導入し、試験例１と同様にして融合タンパク質を誘導した。誘導された融合タンパク質は、ＴＡＴタグだけでなくＨｉｓタグも有しているため、ＴＥＶプロテアーゼで融合タンパク質を処理することでＨｉｓタグを除去した。これによって、ＴＡＴペプチドタグがサイトグロビンに結合したＴＡＴ－ｒｈＣＹＧＢを得た。なお、調製されたＴＡＴ－ｒｈＣＹＧＢは、配列番号１４に示す配列を有している。 (Preparation of TAT-rhCYGB)
A TEV protease recognition sequence and a TAT site were introduced into the vector used in Test Example 1 to induce a fusion protein in the same manner as in Test Example 1. Since the derived fusion protein has not only a TAT tag but also a His tag, the His tag was removed by treating the fusion protein with TEV protease. As a result, TAT-rhCYGB in which the TAT peptide tag was bound to cytoglobin was obtained. The prepared TAT-rhCYGB has the sequence shown in SEQ ID NO: 14.
（線維症関連タンパク質の発現に及ぼすＴＡＴ－ｒｈＣＹＧＢの効果）
細胞株のＨＨＳｔｅＣ細胞を実験に用いた。４０μｇ／ｍＬのＴＡＴ－ｒｈＣＹＧＢをリン酸緩衝溶液に含む溶液を調製し、０～４８時間、ＨＨＳｔｅＣ細胞に暴露し、暴露前（－）、並びに暴露開始後０．２５時間、０．５時間、１時間、４時間、８時間、２４時間及び４８時間の時点における細胞内ＣＹＧＢ及び線維症関連タンパク質を測定した。結果を図７に示す。図７に示されるように、ＴＡＴ－ｒｈＣＹＧＢに暴露された細胞内のＣＹＧＢ濃度は時間依存的に増加し、発現していたαＳＭＡ及びＩ型コラーゲン（ＣＯＬ１Ａ１）は時間依存的に減少した。なお、ＧＡＰＤＨはハウスキーピングタンパク質である。 (Effect of TAT-rhCYGB on expression of fibrosis-related proteins)
HHSteC cells from the cell line were used in the experiment. A solution containing 40 μg / mL TAT-rhCYGB in a phosphate buffer solution was prepared and exposed to HHSteC cells for 0 to 48 hours before exposure (-) and 0.25 hours and 0.5 hours after the start of exposure. Intracellular CYGB and fibrosis-related proteins were measured at 1 hour, 4 hours, 8 hours, 24 hours and 48 hours. The results are shown in FIG. As shown in FIG. 7, the intracellular CYGB concentration exposed to TAT-rhCYGB increased in a time-dependent manner, and the expressed αSMA and type I collagen (COL1A1) decreased in a time-dependent manner. GAPDH is a housekeeping protein.
細胞株のＨＨＳｔｅＣ細胞を実験に用いた。４０μｇ／ｍＬのＴＡＴ－ｒｈＣＹＧＢをリン酸緩衝溶液に含む溶液を調製し、０～４８時間、ＨＨＳｔｅＣ細胞に暴露し、暴露前（－）、並びに暴露開始後０．２５時間、０．５時間、１時間、４時間、８時間、２４時間及び４８時間の時点における細胞内ＣＹＧＢ及び線維症関連タンパク質を測定した。結果を図７に示す。図７に示されるように、ＴＡＴ－ｒｈＣＹＧＢに暴露された細胞内のＣＹＧＢ濃度は時間依存的に増加し、発現していたαＳＭＡ及びＩ型コラーゲン（ＣＯＬ１Ａ１）は時間依存的に減少した。なお、ＧＡＰＤＨはハウスキーピングタンパク質である。 (Effect of TAT-rhCYGB on expression of fibrosis-related proteins)
HHSteC cells from the cell line were used in the experiment. A solution containing 40 μg / mL TAT-rhCYGB in a phosphate buffer solution was prepared and exposed to HHSteC cells for 0 to 48 hours before exposure (-) and 0.25 hours and 0.5 hours after the start of exposure. Intracellular CYGB and fibrosis-related proteins were measured at 1 hour, 4 hours, 8 hours, 24 hours and 48 hours. The results are shown in FIG. As shown in FIG. 7, the intracellular CYGB concentration exposed to TAT-rhCYGB increased in a time-dependent manner, and the expressed αSMA and type I collagen (COL1A1) decreased in a time-dependent manner. GAPDH is a housekeeping protein.
また、ＴＡＴ－ｒｈＣＹＧＢを含まない（－）リン酸緩衝溶液、並びに、ＴＡＴ－ｒｈＣＹＧＢを０．８μｇ／ｍＬ、３．１μｇ／ｍＬ、１２．５μｇ／ｍＬ、又は５０μｇ／ｍＬの濃度で生理食塩水中に含むリン酸緩衝溶液を調製し、４８時間、ＨＨＳｔｅＣ細胞に暴露し、細胞内ＣＹＧＢを測定した。結果を図８に示す。図８に示されるように、ＴＡＴ－ｒｈＣＹＧＢに暴露された細胞内のＣＹＧＢ濃度は用量依存的に増加した。また、発現していたαＳＭＡ、及びＩ型コラーゲン（ＣＯＬ１Ａ１）は用量依存的に減少した。
In addition, a (-) phosphate buffer solution containing no TAT-rhCYGB and TAT-rhCYGB in physiological saline at a concentration of 0.8 μg / mL, 3.1 μg / mL, 12.5 μg / mL, or 50 μg / mL. A phosphate buffer solution contained in was prepared, exposed to HHSteC cells for 48 hours, and intracellular CYGB was measured. The results are shown in FIG. As shown in FIG. 8, the intracellular CYGB concentration exposed to TAT-rhCYGB increased in a dose-dependent manner. In addition, the expressed αSMA and type I collagen (COL1A1) decreased in a dose-dependent manner.
（線維症関連遺伝子の転写に及ぼすＨｉｓ－ｒｈＣＹＧＢの効果）
４０μｇ／ｍＬのＴＡＴ－ｒｈＣＹＧＢをリン酸緩衝溶液に含む溶液を調製し、０～４８時間、ＨＨＳｔｅＣ細胞に暴露し、暴露前（Ｓ－）、並びに暴露開始後１時間、４時間、８時間、２４時間及び４８時間の時点におけるαＳｍａ、Ｃｏｌ３ａ１、及びＴｇｆβ－１（Transforming growth factor β-1）のｍＲＮＡ量を測定した。その結果、ＴＡＴ－ｒｈＣＹＧＢに暴露された細胞におけるαＳｍａ、Ｃｏｌ３ａ１、及びＴｇｆβ－１のｍＲＮＡ発現量が時間依存的に低下したことを確認した。 (Effect of His-rhCYGB on transcription of fibrosis-related genes)
A solution containing 40 μg / mL TAT-rhCYGB in a phosphate buffer solution was prepared and exposed to HHSteC cells for 0 to 48 hours before exposure (S-) and 1 hour, 4 hours, 8 hours after the start of exposure. The amount of αSma, Col3a1, and Tgfβ-1 (Transforming growth factor β-1) mRNA at 24 hours and 48 hours was measured. As a result, it was confirmed that the mRNA expression levels of αSma, Col3a1 and Tgfβ-1 in the cells exposed to TAT-rhCYGB decreased in a time-dependent manner.
４０μｇ／ｍＬのＴＡＴ－ｒｈＣＹＧＢをリン酸緩衝溶液に含む溶液を調製し、０～４８時間、ＨＨＳｔｅＣ細胞に暴露し、暴露前（Ｓ－）、並びに暴露開始後１時間、４時間、８時間、２４時間及び４８時間の時点におけるαＳｍａ、Ｃｏｌ３ａ１、及びＴｇｆβ－１（Transforming growth factor β-1）のｍＲＮＡ量を測定した。その結果、ＴＡＴ－ｒｈＣＹＧＢに暴露された細胞におけるαＳｍａ、Ｃｏｌ３ａ１、及びＴｇｆβ－１のｍＲＮＡ発現量が時間依存的に低下したことを確認した。 (Effect of His-rhCYGB on transcription of fibrosis-related genes)
A solution containing 40 μg / mL TAT-rhCYGB in a phosphate buffer solution was prepared and exposed to HHSteC cells for 0 to 48 hours before exposure (S-) and 1 hour, 4 hours, 8 hours after the start of exposure. The amount of αSma, Col3a1, and Tgfβ-1 (Transforming growth factor β-1) mRNA at 24 hours and 48 hours was measured. As a result, it was confirmed that the mRNA expression levels of αSma, Col3a1 and Tgfβ-1 in the cells exposed to TAT-rhCYGB decreased in a time-dependent manner.
また、ＴＡＴ－ｒｈＣＹＧＢを含まない（０μｇ／ｍＬ）リン酸緩衝溶液、並びに、ＴＡＴ－ｒｈＣＹＧＢを５μｇ／ｍＬ、１０μｇ／ｍＬ、２０μｇ／ｍＬ、４０μｇ／ｍＬ、又は８０μｇ／ｍＬの濃度でリン酸緩衝溶液に含む溶液を調製し、４８時間、ＨＨＳｔｅＣ細胞に暴露し、αＳｍａ、Ｃｏｌ３ａ１、及びＴｇｆβ－１（Transforming growth factor β-1）のｍＲＮＡ量を測定した。結果を図９に示す。図９に示されるように、TAT－ｒｈＣＹＧＢに暴露された細胞におけるαＳｍａ、Ｃｏｌ３ａ１、及びＴｇｆβ－１（Transforming growth factor β-1）のｍＲＮＡ発現量は用量依存的に低下した。
In addition, a phosphate buffer solution containing no TAT-rhCYGB (0 μg / mL) and TAT-rhCYGB at a concentration of 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, or 80 μg / mL. The solution to be included in the solution was prepared, exposed to HHSteC cells for 48 hours, and the mRNA levels of αSma, Col3a1 and Tgfβ-1 (Transforming growth factor β-1) were measured. The results are shown in FIG. As shown in FIG. 9, the mRNA expression levels of αSma, Col3a1 and Tgfβ-1 (Transforming growth factor β-1) in the cells exposed to TAT-rhCYGB decreased in a dose-dependent manner.
試験例４：細胞透過性の比較（ｉｎ ｖｉｔｒｏ）
本試験例では、タグを有しない野生型サイトグロビンＣＹＧＢ（配列番号５のアミノ酸配列を有するもの；Ｎａｔｉｖｅ－ＣＹＧＢ）、試験例１で調製されたオリゴヒスチジンタグを有するサイトグロビン（Ｈｉｓ－ｒｈＣＹＧＢ）及び試験例３で調製されたＴＡＴペプチドタグを有するサイトグロビン（ＴＡＴ－ｒｈＣＹＧＢ）について、細胞膜透過性を検証した。 Test Example 4: Comparison of cell permeability (in vitro)
In this test example, the wild-type cytoglobin CYGB having no tag (having the amino acid sequence of SEQ ID NO: 5; Native-CYGB), the cytoglobin having the oligohistidine tag prepared in Test Example 1 (His-rhCYGB) and The cell membrane permeability of cytoglobin (TAT-rhCYGB) having a TAT peptide tag prepared in Test Example 3 was verified.
本試験例では、タグを有しない野生型サイトグロビンＣＹＧＢ（配列番号５のアミノ酸配列を有するもの；Ｎａｔｉｖｅ－ＣＹＧＢ）、試験例１で調製されたオリゴヒスチジンタグを有するサイトグロビン（Ｈｉｓ－ｒｈＣＹＧＢ）及び試験例３で調製されたＴＡＴペプチドタグを有するサイトグロビン（ＴＡＴ－ｒｈＣＹＧＢ）について、細胞膜透過性を検証した。 Test Example 4: Comparison of cell permeability (in vitro)
In this test example, the wild-type cytoglobin CYGB having no tag (having the amino acid sequence of SEQ ID NO: 5; Native-CYGB), the cytoglobin having the oligohistidine tag prepared in Test Example 1 (His-rhCYGB) and The cell membrane permeability of cytoglobin (TAT-rhCYGB) having a TAT peptide tag prepared in Test Example 3 was verified.
まず、野生型サイトグロビン（Ｎａｔｉｖｅ－ＣＹＧＢ）、及びオリゴヒスチジンタグを有するサイトグロビン（Ｈｉｓ－ｒｈＣＹＧＢ）を用意し、いずれもそれぞれ４０μｇ／ｍＬを生理食塩水中に含む溶液に調製し、それぞれの溶液を、ＨＨＳｔｅＣ細胞に４８時間暴露した。暴露後４８時間時点における細胞内のＣＹＧＢ発現をウエスタンブロットにて解析した。その結果を図１０に示す。図１０に示されるように、Ｎａｔｉｖｅ－ＣＹＧＢ自体に細胞膜透過性が認められた。さらにサイトグロビンにタグを付すことによって、細胞膜透過性が向上することが認められた。なお、Ｎａｔｉｖｅ－ＣＹＧＢの細胞膜透過性を基準として、Ｈｉｓ－ｒｈＣＹＧＢの細胞膜透過性は１７０％向上した。
First, wild-type cytoglobin (Native-CYGB) and cytoglobin having an oligohistidine tag (His-rhCYGB) are prepared, and each of them is prepared into a solution containing 40 μg / mL in physiological saline, and each solution is prepared. , HHSteC cells were exposed for 48 hours. Intracellular CYGB expression at 48 hours after exposure was analyzed by Western blot. The result is shown in FIG. As shown in FIG. 10, cell membrane permeability was observed in Native-CYGB itself. Furthermore, it was found that the cell membrane permeability was improved by tagging cytoglobin. The cell membrane permeability of His-rhCYGB was improved by 170% based on the cell membrane permeability of Native-CYGB.
次に、オリゴヒスチジンタグを有するサイトグロビン（Ｈｉｓ－ｒｈＣＹＧＢ）、及びＴＡＴペプチドタグの両方を有するサイトグロビン（ＴＡＴ－ｒｈＣＹＧＢを、いずれもそれぞれ４０μｇ／ｍＬを生理食塩水中に含む溶液を調製し、それぞれの溶液を、ＨＨＳｔｅＣ細胞に４８時間暴露した。暴露前（－）、並びに暴露後０．２５時間、０．５時間、１時間、４時間、８時間、２４時間及び４８時間の時点における細胞内のＣＹＧＢ発現をウエスタンブロットにて解析した。その結果を図１１に示す。図１１に示すように、オリゴヒスチジンタグを有するＨｉｓ－ｒｈＣＹＧＢと、ＴＡＴペプチドタグを有するＴＡＴ－ｒｈＣＹＧＢとでは、ＴＡＴ－ｒｈＣＹＧＢの方がより迅速に（具体的には０．２５時間の時点で）細胞内濃度がプラトーに達したことから、ＴＡＴ－ｒｈＣＹＧＢの方が細胞膜透過性により優れることを確認した。しかしながら、試験例１（具体的には、図３）と試験例３（具体的には、図９）との対比で示されるとおり、細胞膜透過性により劣るＨｉｓ－ｒｈＣＹＧＢの方が、より低用量で線維化関連遺伝子の転写を抑制することができる。
Next, a solution containing 40 μg / mL of cytoglobin (His-rhCYGB) having an oligohistidine tag and cytoglobin (TAT-rhCYGB) having both a TAT peptide tag in physiological saline was prepared. Solution was exposed to HHSteC cells for 48 hours. Intracellular before exposure (-) and at 0.25 hours, 0.5 hours, 1 hour, 4 hours, 8 hours, 24 hours and 48 hours after exposure. The CYGB expression of CYGB was analyzed by Western blot. The results are shown in FIG. 11. As shown in FIG. 11, the His-rhCYGB having an oligohistidine tag and the TAT-rhCYGB having a TAT peptide tag are TAT-rhCYGB. However, it was confirmed that TAT-rhCYGB was superior in cell membrane permeability because the intracellular concentration reached the plateau more rapidly (specifically, at 0.25 hours). As shown in comparison between 1 (specifically, FIG. 3) and Test Example 3 (specifically, FIG. 9), His-rhCYGB, which is inferior in cell membrane permeability, is more fibrotic-related at a lower dose. It can suppress gene transcription.
試験例５：ＨＨＳｔｅＣ細胞中におけるタグ－ｒｈＣＹＧＢ（ｉｎ ｖｉｔｒｏ）
本試験例では、試験例１で調製されたオリゴヒスチジンタグを有するサイトグロビン（Ｈｉｓ－ｒｈＣＹＧＢ）及び試験例３で調製されたＴＡＴペプチドタグを有するサイトグロビン（ＴＡＴ－ｒｈＣＹＧＢ）について、ＨＨＳｔｅＣ細胞中における存在態様を検証した。 Test Example 5: Tag-rhCYGB (in vitro) in HHSteC cells
In this test example, cytoglobin (His-rhCYGB) having an oligohistidine tag prepared in Test Example 1 and cytoglobin (TAT-rhCYGB) having a TAT peptide tag prepared in Test Example 3 are used in HHSteC cells. The mode of existence was verified.
本試験例では、試験例１で調製されたオリゴヒスチジンタグを有するサイトグロビン（Ｈｉｓ－ｒｈＣＹＧＢ）及び試験例３で調製されたＴＡＴペプチドタグを有するサイトグロビン（ＴＡＴ－ｒｈＣＹＧＢ）について、ＨＨＳｔｅＣ細胞中における存在態様を検証した。 Test Example 5: Tag-rhCYGB (in vitro) in HHSteC cells
In this test example, cytoglobin (His-rhCYGB) having an oligohistidine tag prepared in Test Example 1 and cytoglobin (TAT-rhCYGB) having a TAT peptide tag prepared in Test Example 3 are used in HHSteC cells. The mode of existence was verified.
（Ｈｉｓ－ｒｈＣＹＧＢのＨＨＳｔｅＣ細胞中における存在態様）
（１）Ｈｉｓ－ｒｈＣＹＧＢをＡｌｅｘａ Ｆｌｕｏｒでラベル化し、生理食塩水中１０μｇ／ｍＬに調製し、ＨＨＳｔｅＣ細胞に４８時間暴露した。暴露後の細胞を、リソソーム、及び小胞体についてマーカー多重染色を行った。また、ＤＡＰＩで核酸を染色した。結果を図１２（ａ）に示す。図１２（ａ）に示すように、ＨＨＳｔｅＣ細胞に取り込まれたＨｉｓ－ｒｈＣＹＧＢは、リソソームに局在するが、小胞体には存在しないことが分かった。 (Existence mode of His-rhCYGB in HHSteC cells)
(1) His-rhCYGB was labeled with Alexa Fluor, prepared at 10 μg / mL in physiological saline, and exposed to HHSteC cells for 48 hours. The exposed cells were subjected to marker multiple staining for lysosomes and endoplasmic reticulum. In addition, nucleic acids were stained with DAPI. The results are shown in FIG. 12 (a). As shown in FIG. 12 (a), His-rhCYGB incorporated into HHSteC cells was found to be localized in lysosomes but not in the endoplasmic reticulum.
（１）Ｈｉｓ－ｒｈＣＹＧＢをＡｌｅｘａ Ｆｌｕｏｒでラベル化し、生理食塩水中１０μｇ／ｍＬに調製し、ＨＨＳｔｅＣ細胞に４８時間暴露した。暴露後の細胞を、リソソーム、及び小胞体についてマーカー多重染色を行った。また、ＤＡＰＩで核酸を染色した。結果を図１２（ａ）に示す。図１２（ａ）に示すように、ＨＨＳｔｅＣ細胞に取り込まれたＨｉｓ－ｒｈＣＹＧＢは、リソソームに局在するが、小胞体には存在しないことが分かった。 (Existence mode of His-rhCYGB in HHSteC cells)
(1) His-rhCYGB was labeled with Alexa Fluor, prepared at 10 μg / mL in physiological saline, and exposed to HHSteC cells for 48 hours. The exposed cells were subjected to marker multiple staining for lysosomes and endoplasmic reticulum. In addition, nucleic acids were stained with DAPI. The results are shown in FIG. 12 (a). As shown in FIG. 12 (a), His-rhCYGB incorporated into HHSteC cells was found to be localized in lysosomes but not in the endoplasmic reticulum.
（２）Ｈｉｓ－ｒｈＣＹＧＢをＡｌｅｘａ Ｆｌｕｏｒ ４８８でラベル化し、生理食塩水中１０μｇ／ｍＬに調製し、ＨＨＳｔｅＣ細胞に２４時間暴露した。暴露後の細胞に対して、リソソームのオルガネラマーカー（LysoTracker、Molecular Probes、USA；１００ｎＭ；１５分、３７℃、５％ＣＯ2）、ミトコンドリアのオルガネラマーカー（MitoTracker、Molecular Probes；１００ｎＭ；１５分、３７℃、５％ＣＯ2）、小胞体のオルガネラマーカー（ER-Tracker、Molecular Probes；０．５μＭ；１５分、３７℃、５％ＣＯ2）、初期エンドソームのオルガネラマーカー（Early Endosomes-RFP、Thermo Fisher Scientific；３７℃、５％ＣＯ2）後期エンドソームのオルガネラマーカー（Late Endosomes-RFP、Thermo Fisher Scientific；３７℃、５％ＣＯ2）による二重免疫蛍光染色を行った。なお、核の対比染色にHoechst 33258（Thermo Fisher Scientific）を用いた。各オルガネラマーカーでの処理後、細胞をＰＢＳで３回洗浄し、Zeiss LSM 800共焦点顕微鏡（Carl Zeiss Microscopy、Germany）で細胞内局在を観察し、Pearson値により共局在を決定した。結果を図１２（ｂ）に示す。
(2) His-rhCYGB was labeled with Alexa Fluor 488, prepared at 10 μg / mL in physiological saline, and exposed to HHSteC cells for 24 hours. For the exposed cells, endosome organelle markers (LysoTracker, Molecular Probes, USA; 100 nM; 15 minutes, 37 ° C, 5% CO 2 ), mitochondrial organelle markers (MitoTracker, Molecular Probes; 100 nM; 15 minutes, 37). ℃, 5% CO 2 ), endoplasmic reticulum organelle markers (ER-Tracker, Molecular Probes; 0.5 μM; 15 minutes, 37 ℃, 5% CO 2 ), early endosome organelle markers (Early Endosomes-RFP, Thermo Fisher) Scientific; 37 ° C, 5% CO 2 ) Double immunofluorescent staining of late endosomes with organelle markers (Late Endosomes-RFP, Thermo Fisher Scientific; 37 ° C, 5% CO 2 ) was performed. Hoechst 33258 (Thermo Fisher Scientific) was used for the counterstain of the nucleus. After treatment with each organelle marker, cells were washed 3 times with PBS, intracellular localization was observed with a Zeiss LSM 800 confocal microscope (Carl Zeiss Microscopy, Germany), and colocalization was determined by Pearson values. The results are shown in FIG. 12 (b).
図１２（ｂ）から明らかなように、ＨＨＳｔｅＣ細胞に取り込まれたＨｉｓ－ｒｈＣＹＧＢは、ミトコンドリア、初期エンドソーム、後期エンドソーム、及びリソソームに局在するが、小胞体には存在しないことが分かった。
As is clear from FIG. 12 (b), His-rhCYGB incorporated into HHSteC cells is localized in mitochondria, early endosomes, anaphase endosomes, and lysosomes, but is not present in the endoplasmic reticulum.
（ＴＡＴ－ｒｈＣＹＧＢのＨＨＳｔｅＣ細胞中における存在態様）
ＨＨＳｔｅＣ細胞を２群用意し、一方をコントロール（No treatment）群、他方をＴＡＴ－ｒｈＣＹＧＢ投与群とした。ＴＡＴ－ｒｈＣＹＧＢ投与群においては、１０μｇ／ｍＬのＴＡＴ－ｒｈＣＹＧＢを４８時間暴露した。コントロール群とＴＡＴ－ｒｈＣＹＧＢ投与群それぞれについて、ＣＹＧＢ、ＳＭＡ、及びＤＡＰＩ染色を行った。ＣＹＧＢは赤、αＳＭＡは緑、ＤＡＰＩ（ＤＡＰＩによる核対比染色）は青で染色される。その結果を図１３に示す。図１３に示すように、ＴＡＴ－ｒｈＣＹＧＢ投与群ではＣＹＧＢの染色が細胞内で均一に存在することが認められたため、ＨＨＳｔｅＣ細胞に取り込まれたＴＡＴ－ｒｈＣＹＧＢは細胞内に均一に存在することが分かった。なお、ＴＡＴ－ｒｈＣＹＧＢ投与群ではαＳＭＡの染色が減弱したことが認められたため、ＴＡＴ－ｒｈＣＹＧＢ投与によりαＳＭＡの発現が低下したことも確認できた。 (Existence mode of TAT-rhCYGB in HHSteC cells)
Two groups of HHSteC cells were prepared, one was a control (No treatment) group and the other was a TAT-rhCYGB administration group. In the TAT-rhCYGB-administered group, 10 μg / mL TAT-rhCYGB was exposed for 48 hours. CYGB, SMA, and DAPI staining were performed on each of the control group and the TAT-rhCYGB administration group. CYGB is stained red, αSMA is stained green, and DAPI (DAPI nuclear counterstain) is stained blue. The result is shown in FIG. As shown in FIG. 13, in the TAT-rhCYGB-administered group, the staining of CYGB was found to be uniformly present in the cells. Therefore, it was found that the TAT-rhCYGB incorporated into the HHSteC cells was uniformly present in the cells. It was. In addition, since it was confirmed that the staining of αSMA was attenuated in the TAT-rhCYGB administration group, it was also confirmed that the expression of αSMA was decreased by the administration of TAT-rhCYGB.
ＨＨＳｔｅＣ細胞を２群用意し、一方をコントロール（No treatment）群、他方をＴＡＴ－ｒｈＣＹＧＢ投与群とした。ＴＡＴ－ｒｈＣＹＧＢ投与群においては、１０μｇ／ｍＬのＴＡＴ－ｒｈＣＹＧＢを４８時間暴露した。コントロール群とＴＡＴ－ｒｈＣＹＧＢ投与群それぞれについて、ＣＹＧＢ、ＳＭＡ、及びＤＡＰＩ染色を行った。ＣＹＧＢは赤、αＳＭＡは緑、ＤＡＰＩ（ＤＡＰＩによる核対比染色）は青で染色される。その結果を図１３に示す。図１３に示すように、ＴＡＴ－ｒｈＣＹＧＢ投与群ではＣＹＧＢの染色が細胞内で均一に存在することが認められたため、ＨＨＳｔｅＣ細胞に取り込まれたＴＡＴ－ｒｈＣＹＧＢは細胞内に均一に存在することが分かった。なお、ＴＡＴ－ｒｈＣＹＧＢ投与群ではαＳＭＡの染色が減弱したことが認められたため、ＴＡＴ－ｒｈＣＹＧＢ投与によりαＳＭＡの発現が低下したことも確認できた。 (Existence mode of TAT-rhCYGB in HHSteC cells)
Two groups of HHSteC cells were prepared, one was a control (No treatment) group and the other was a TAT-rhCYGB administration group. In the TAT-rhCYGB-administered group, 10 μg / mL TAT-rhCYGB was exposed for 48 hours. CYGB, SMA, and DAPI staining were performed on each of the control group and the TAT-rhCYGB administration group. CYGB is stained red, αSMA is stained green, and DAPI (DAPI nuclear counterstain) is stained blue. The result is shown in FIG. As shown in FIG. 13, in the TAT-rhCYGB-administered group, the staining of CYGB was found to be uniformly present in the cells. Therefore, it was found that the TAT-rhCYGB incorporated into the HHSteC cells was uniformly present in the cells. It was. In addition, since it was confirmed that the staining of αSMA was attenuated in the TAT-rhCYGB administration group, it was also confirmed that the expression of αSMA was decreased by the administration of TAT-rhCYGB.
試験例６：ＨＨＳｔｅＣ細胞に及ぼす６Ｒ－ＣＹＧＢの効果（ｉｎ ｖｉｔｒｏ）（比較例）
本試験例では、比較用の６Ｒペプチドタグ（GRRRRRRRAS：配列番号１５）が配列番号５に示すサイトグロビンのＮ末端に結合した融合ポリペプチド６Ｒ－ｒｈＣＹＧＢを調製し、調製された６Ｒ－ｒｈＣＹＧＢについて、星細胞に及ぼす影響を検証した。 Test Example 6: Effect of 6R-CYGB on HHSteC cells (in vitro) (Comparative example)
In this test example, afusion polypeptide 6R-rhCYGB in which a comparative 6R peptide tag (GRRRRRRRAS: SEQ ID NO: 15) was bound to the N-terminal of the cytoglobin shown in SEQ ID NO: 5 was prepared, and the prepared 6R-rhCYGB was prepared. The effect on stellate cells was examined.
本試験例では、比較用の６Ｒペプチドタグ（GRRRRRRRAS：配列番号１５）が配列番号５に示すサイトグロビンのＮ末端に結合した融合ポリペプチド６Ｒ－ｒｈＣＹＧＢを調製し、調製された６Ｒ－ｒｈＣＹＧＢについて、星細胞に及ぼす影響を検証した。 Test Example 6: Effect of 6R-CYGB on HHSteC cells (in vitro) (Comparative example)
In this test example, a
（６Ｒ－ｒｈＣＹＧＢの調製）
試験例１で用いたベクターに、ＴＥＶプロテアーゼ認識シークエンスと６Ｒサイトとを導入し、試験例１と同様にして融合タンパク質を誘導した。誘導された融合タンパク質は、６ＲタグだけでなくＨｉｓタグも有しているため、ＴＥＶプロテアーゼで融合タンパク質を処理することでＨｉｓタグを除去した。これによって、６Ｒペプチドタグがサイトグロビンに結合した６Ｒ－ｒｈＣＹＧＢを得た。なお、調製された６Ｒ－ｒｈＣＹＧＢは、配列番号１６に示す配列を有している。 (Preparation of 6R-rhCYGB)
A TEV protease recognition sequence and a 6R site were introduced into the vector used in Test Example 1 to induce a fusion protein in the same manner as in Test Example 1. Since the induced fusion protein has not only a 6R tag but also a His tag, the His tag was removed by treating the fusion protein with TEV protease. As a result, 6R-rhCYGB in which the 6R peptide tag was bound to cytoglobin was obtained. The prepared 6R-rhCYGB has the sequence shown in SEQ ID NO: 16.
試験例１で用いたベクターに、ＴＥＶプロテアーゼ認識シークエンスと６Ｒサイトとを導入し、試験例１と同様にして融合タンパク質を誘導した。誘導された融合タンパク質は、６ＲタグだけでなくＨｉｓタグも有しているため、ＴＥＶプロテアーゼで融合タンパク質を処理することでＨｉｓタグを除去した。これによって、６Ｒペプチドタグがサイトグロビンに結合した６Ｒ－ｒｈＣＹＧＢを得た。なお、調製された６Ｒ－ｒｈＣＹＧＢは、配列番号１６に示す配列を有している。 (Preparation of 6R-rhCYGB)
A TEV protease recognition sequence and a 6R site were introduced into the vector used in Test Example 1 to induce a fusion protein in the same manner as in Test Example 1. Since the induced fusion protein has not only a 6R tag but also a His tag, the His tag was removed by treating the fusion protein with TEV protease. As a result, 6R-rhCYGB in which the 6R peptide tag was bound to cytoglobin was obtained. The prepared 6R-rhCYGB has the sequence shown in SEQ ID NO: 16.
（線維症関連タンパク質の発現に及ぼす６Ｒ－ｒｈＣＹＧＢの効果）
細胞株のＨＨＳｔｅＣ細胞を実験に用いた。４０μｇ／ｍＬの６Ｒ－ｒｈＣＹＧＢをリン酸緩衝溶液に含む溶液を調製し、０～４８時間、ＨＨＳｔｅＣ細胞に暴露し、暴露前（－）、並びに暴露開始後１時間、４時間、８時間、２４時間及び４８時間の時点における細胞内ＣＹＧＢ及び線維症関連タンパク質を測定した。結果を図１４に示す。図１４では、試験例１の６Ｒ－ＣＹＧＢの４８時間の時点における結果を併せて示している。図１４に示されるように、６Ｒ－ｒｈＣＹＧＢに暴露された細胞内のＣＹＧＢ濃度は時間依存的に増加したが、発現していたαＳＭＡ及びＩ型コラーゲン（ＣＯＬ１Ａ１）の量は変わらなかった。なお、ＧＡＰＤＨはハウスキーピングタンパク質である。 (Effect of 6R-rhCYGB on expression of fibrosis-related proteins)
HHSteC cells from the cell line were used in the experiment. A solution containing 40 μg / mL of 6R-rhCYGB in a phosphate buffer solution was prepared and exposed to HHSteC cells for 0 to 48 hours before exposure (-) and 1 hour, 4 hours, 8 hours, 24 hours after the start of exposure. Intracellular CYGB and fibrosis-related proteins were measured at time and 48 hours. The results are shown in FIG. FIG. 14 also shows the results of 6R-CYGB of Test Example 1 at 48 hours. As shown in FIG. 14, the intracellular CYGB concentration exposed to 6R-rhCYGB increased in a time-dependent manner, but the amount of αSMA and type I collagen (COL1A1) expressed did not change. GAPDH is a housekeeping protein.
細胞株のＨＨＳｔｅＣ細胞を実験に用いた。４０μｇ／ｍＬの６Ｒ－ｒｈＣＹＧＢをリン酸緩衝溶液に含む溶液を調製し、０～４８時間、ＨＨＳｔｅＣ細胞に暴露し、暴露前（－）、並びに暴露開始後１時間、４時間、８時間、２４時間及び４８時間の時点における細胞内ＣＹＧＢ及び線維症関連タンパク質を測定した。結果を図１４に示す。図１４では、試験例１の６Ｒ－ＣＹＧＢの４８時間の時点における結果を併せて示している。図１４に示されるように、６Ｒ－ｒｈＣＹＧＢに暴露された細胞内のＣＹＧＢ濃度は時間依存的に増加したが、発現していたαＳＭＡ及びＩ型コラーゲン（ＣＯＬ１Ａ１）の量は変わらなかった。なお、ＧＡＰＤＨはハウスキーピングタンパク質である。 (Effect of 6R-rhCYGB on expression of fibrosis-related proteins)
HHSteC cells from the cell line were used in the experiment. A solution containing 40 μg / mL of 6R-rhCYGB in a phosphate buffer solution was prepared and exposed to HHSteC cells for 0 to 48 hours before exposure (-) and 1 hour, 4 hours, 8 hours, 24 hours after the start of exposure. Intracellular CYGB and fibrosis-related proteins were measured at time and 48 hours. The results are shown in FIG. FIG. 14 also shows the results of 6R-CYGB of Test Example 1 at 48 hours. As shown in FIG. 14, the intracellular CYGB concentration exposed to 6R-rhCYGB increased in a time-dependent manner, but the amount of αSMA and type I collagen (COL1A1) expressed did not change. GAPDH is a housekeeping protein.
また、６Ｒ－ｒｈＣＹＧＢを含まない（－）リン酸緩衝溶液、並びに、６Ｒ－ｒｈＣＹＧＢを５μｇ／ｍＬ、１０μｇ／ｍＬ、２０μｇ／ｍＬ、４０μｇ／ｍＬ、又は８０μｇ／ｍＬの濃度で生理食塩水中に含むリン酸緩衝溶液を調製し、４８時間、ＨＨＳｔｅＣ細胞に暴露し、細胞内ＣＹＧＢを測定した。結果を図１５に示す。図１５に示されるように、６Ｒ－ｒｈＣＹＧＢに暴露された細胞内のＣＹＧＢ濃度は用量依存的に増加したが、発現していたαＳＭＡ、及びＩ型コラーゲン（ＣＯＬ１Ａ１）の量は用量に関わらず変化しなかった。
In addition, 6R-rhCYGB-free (-) phosphate buffer solution and 6R-rhCYGB are contained in physiological saline at a concentration of 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, or 80 μg / mL. A phosphate buffer solution was prepared, exposed to HHSteC cells for 48 hours, and intracellular CYGB was measured. The results are shown in FIG. As shown in FIG. 15, the intracellular CYGB concentration exposed to 6R-rhCYGB increased in a dose-dependent manner, but the amount of αSMA expressed and type I collagen (COL1A1) changed regardless of the dose. I didn't.
（線維症関連遺伝子の転写に及ぼす６Ｒ－ｒｈＣＹＧＢの効果）
６Ｒ－ｒｈＣＹＧＢを含まない（０μｇ／ｍＬ）リン酸緩衝溶液、並びに、６Ｒ－ｒｈＣＹＧＢを５μｇ／ｍＬ、１０μｇ／ｍＬ、２０μｇ／ｍＬ、４０μｇ／ｍＬ、又は８０μｇ／ｍＬの濃度でリン酸緩衝溶液に含む溶液を調製し、４８時間、ＨＨＳｔｅＣ細胞に暴露し、αＳｍａのｍＲＮＡ転写量を測定した。結果を図１６に示す。図１６に示されるように、６Ｒ－ｒｈＣＹＧＢに暴露された細胞におけるαＳｍａのｍＲＮＡ転写量は用量に関わらず変化しなかった。 (Effect of 6R-rhCYGB on transcription of fibrosis-related genes)
6R-rhCYGB-free (0 μg / mL) phosphate buffer solution and 6R-rhCYGB in phosphate buffer solution at concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, or 80 μg / mL. A solution containing the mixture was prepared, exposed to HHSteC cells for 48 hours, and the amount of αSma mRNA transcribed was measured. The results are shown in FIG. As shown in FIG. 16, the mRNA transcription level of αSma in cells exposed to 6R-rhCYGB did not change regardless of dose.
６Ｒ－ｒｈＣＹＧＢを含まない（０μｇ／ｍＬ）リン酸緩衝溶液、並びに、６Ｒ－ｒｈＣＹＧＢを５μｇ／ｍＬ、１０μｇ／ｍＬ、２０μｇ／ｍＬ、４０μｇ／ｍＬ、又は８０μｇ／ｍＬの濃度でリン酸緩衝溶液に含む溶液を調製し、４８時間、ＨＨＳｔｅＣ細胞に暴露し、αＳｍａのｍＲＮＡ転写量を測定した。結果を図１６に示す。図１６に示されるように、６Ｒ－ｒｈＣＹＧＢに暴露された細胞におけるαＳｍａのｍＲＮＡ転写量は用量に関わらず変化しなかった。 (Effect of 6R-rhCYGB on transcription of fibrosis-related genes)
6R-rhCYGB-free (0 μg / mL) phosphate buffer solution and 6R-rhCYGB in phosphate buffer solution at concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, or 80 μg / mL. A solution containing the mixture was prepared, exposed to HHSteC cells for 48 hours, and the amount of αSma mRNA transcribed was measured. The results are shown in FIG. As shown in FIG. 16, the mRNA transcription level of αSma in cells exposed to 6R-rhCYGB did not change regardless of dose.
試験例７：サイトグロビンの鉄ヘム結合と抗線維化効果（参考例）
本試験例では、野生型のサイトグロビンのヘムに含まれる鉄イオンをコバルトイオンに置き換えたＣｏ－ＣＹＧＢの、ＨＨＳｔｅＣ細胞の抗線維化に及ぼす効果を検証した。 Test Example 7: Iron heme binding and antifibrotic effect of cytoglobin (reference example)
In this test example, the effect of Co-CYGB, which replaces iron ions contained in the heme of wild-type cytoglobin with cobalt ions, on the antifibrosis of HHSteC cells was examined.
本試験例では、野生型のサイトグロビンのヘムに含まれる鉄イオンをコバルトイオンに置き換えたＣｏ－ＣＹＧＢの、ＨＨＳｔｅＣ細胞の抗線維化に及ぼす効果を検証した。 Test Example 7: Iron heme binding and antifibrotic effect of cytoglobin (reference example)
In this test example, the effect of Co-CYGB, which replaces iron ions contained in the heme of wild-type cytoglobin with cobalt ions, on the antifibrosis of HHSteC cells was examined.
（サイトグロビンのコバルト置換）
以下のようにして、サイトグロビンに結合しているヘムに含まれる鉄イオンをコバルトイオンに置き換えた。まず、野生型サイトグロビンの補助分子族を除去することでアポタンパク質（Ａｐｏ－ＣＹＧＢ）を調製し、コバルト－プロトポルフィリン（Ｃｏ－ＰＰＩＸ）をＡｐｏ－ＣＹＧＢに挿入することでＣｏ－ＣＹＧＢを作製した。Ｃｏ－ＣＹＧＢをＰＤ－１０カラムで精製し、リン酸緩衝液で平衡化した。Ｃｏ－ＣＹＧＢの紫外線吸収スペクトルは、波長４２６ｎｍのコバルトピークで確認した。 (Cobalt substitution of cytoglobin)
The iron ion contained in the heme bound to cytoglobin was replaced with cobalt ion as follows. First, an apoprotein (Apo-CYGB) was prepared by removing the auxiliary molecular group of wild-type cytoglobin, and Co-CYGB was prepared by inserting cobalt-protoporphyrin (Co-PPIX) into Apo-CYGB. .. Co-CYGB was purified on a PD-10 column and equilibrated with phosphate buffer. The ultraviolet absorption spectrum of Co-CYGB was confirmed by a cobalt peak having a wavelength of 426 nm.
以下のようにして、サイトグロビンに結合しているヘムに含まれる鉄イオンをコバルトイオンに置き換えた。まず、野生型サイトグロビンの補助分子族を除去することでアポタンパク質（Ａｐｏ－ＣＹＧＢ）を調製し、コバルト－プロトポルフィリン（Ｃｏ－ＰＰＩＸ）をＡｐｏ－ＣＹＧＢに挿入することでＣｏ－ＣＹＧＢを作製した。Ｃｏ－ＣＹＧＢをＰＤ－１０カラムで精製し、リン酸緩衝液で平衡化した。Ｃｏ－ＣＹＧＢの紫外線吸収スペクトルは、波長４２６ｎｍのコバルトピークで確認した。 (Cobalt substitution of cytoglobin)
The iron ion contained in the heme bound to cytoglobin was replaced with cobalt ion as follows. First, an apoprotein (Apo-CYGB) was prepared by removing the auxiliary molecular group of wild-type cytoglobin, and Co-CYGB was prepared by inserting cobalt-protoporphyrin (Co-PPIX) into Apo-CYGB. .. Co-CYGB was purified on a PD-10 column and equilibrated with phosphate buffer. The ultraviolet absorption spectrum of Co-CYGB was confirmed by a cobalt peak having a wavelength of 426 nm.
（線維症関連タンパク質の発現に及ぼすＣｏ－ＣＹＧＢの効果）
Ｃｏ－ＣＹＧＢを４０μｇ／ｍＬの濃度でＨＨＳｔｅＣ細胞に４０時間暴露し、細胞内ＣＹＧＢ及び線維症関連タンパク質を測定した（Ｃｏ－ＣＹ－１，Ｃｏ－ＣＹ－２）。結果を図１７に示す。図１７では、ＨＨＳｔｅＣ細胞にＣｏ－ＣＹＧＢを暴露しなかったことを除いて同様の操作を行った場合（Ｃｏｎｔ－１，Ｃｏｎｔ－２）、及びＣｏ－ＣＹＧＢの代わりに野生型サイトグロビンを同様の条件で暴露した場合（ＰＣ１，ＰＣ２）の結果を併せて示している。 (Effect of Co-CYGB on expression of fibrosis-related proteins)
Co-CYGB was exposed to HHSteC cells at a concentration of 40 μg / mL for 40 hours, and intracellular CYGB and fibrosis-related proteins were measured (Co-CY-1, Co-CY-2). The results are shown in FIG. In FIG. 17, the same procedure was performed except that the HHSteC cells were not exposed to Co-CYGB (Cont-1, Cont-2), and wild-type cytoglobin was used instead of Co-CYGB. The results of exposure under the conditions (PC1, PC2) are also shown.
Ｃｏ－ＣＹＧＢを４０μｇ／ｍＬの濃度でＨＨＳｔｅＣ細胞に４０時間暴露し、細胞内ＣＹＧＢ及び線維症関連タンパク質を測定した（Ｃｏ－ＣＹ－１，Ｃｏ－ＣＹ－２）。結果を図１７に示す。図１７では、ＨＨＳｔｅＣ細胞にＣｏ－ＣＹＧＢを暴露しなかったことを除いて同様の操作を行った場合（Ｃｏｎｔ－１，Ｃｏｎｔ－２）、及びＣｏ－ＣＹＧＢの代わりに野生型サイトグロビンを同様の条件で暴露した場合（ＰＣ１，ＰＣ２）の結果を併せて示している。 (Effect of Co-CYGB on expression of fibrosis-related proteins)
Co-CYGB was exposed to HHSteC cells at a concentration of 40 μg / mL for 40 hours, and intracellular CYGB and fibrosis-related proteins were measured (Co-CY-1, Co-CY-2). The results are shown in FIG. In FIG. 17, the same procedure was performed except that the HHSteC cells were not exposed to Co-CYGB (Cont-1, Cont-2), and wild-type cytoglobin was used instead of Co-CYGB. The results of exposure under the conditions (PC1, PC2) are also shown.
図１７に示されるように、サイトグロビンのヘム鉄をコバルトに置き換えたＣｏ－ＣＹ－１，Ｃｏ－ＣＹ－２では、野生型サイトグロビンを暴露したＰＣ１，ＰＣ２とは異なり、サイトグロビンを暴露しなかったＣｏ－ＣＹ－１，Ｃｏ－ＣＹ－２と同様に、発現していたαＳＭＡ及びＩ型コラーゲン（ＣＯＬ１Ａ１）の量は変化しなかった。なお、ＧＡＰＤＨはハウスキーピングタンパク質である。この結果から、ヘム鉄のコバルトへの置換がサイトグロビンの抗線維化効果を阻害すること、つまり、サイトグロビンの鉄ヘム活性が抗線維化効果に関連していることが示された。
As shown in FIG. 17, Co-CY-1 and Co-CY-2 in which heme iron of cytoglobin was replaced with cobalt exposed cytoglobin, unlike PC1 and PC2 exposed to wild-type cytoglobin. Similar to the absent Co-CY-1 and Co-CY-2, the amounts of αSMA and type I collagen (COL1A1) expressed did not change. GAPDH is a housekeeping protein. From this result, it was shown that the substitution of heme iron with cobalt inhibits the antifibrotic effect of cytoglobin, that is, the iron heme activity of cytoglobin is related to the antifibrotic effect.
（線維症関連遺伝子の転写に及ぼすＣｏ－ＣＹＧＢｓの効果）
Ｃｏ－ＣＹＧＢを含まない（Ｓ－）リン酸緩衝溶液、並びに、Ｃｏ－ＣＹＧＢを４０μｇ／ｍＬの濃度（Ｃｏ－ｃｙｇｂ４０）でリン酸緩衝溶液に含む溶液を調製し、４８時間、ＨＨＳｔｅＣ細胞に暴露し、αＳｍａのｍＲＮＡ転写量を測定した。結果を図１８に示す。図１８に示されるように、Ｃｏ－ｒｈＣＹＧＢに暴露された細胞におけるαＳｍａのｍＲＮＡ転写量の量に変化は無かった。この結果からも、ヘム鉄のコバルトへの置換がサイトグロビンの抗線維化効果を阻害すること、つまり、サイトグロビンの鉄ヘム結合が抗線維化効果に関連していることが示された。 (Effect of Co-CYGBs on transcription of fibrosis-related genes)
A Co-CYGB-free (S-) phosphate buffer solution and a solution containing Co-CYGB at a concentration of 40 μg / mL (Co-cygb40) in the phosphate buffer solution were prepared and exposed to HHSteC cells for 48 hours. Then, the amount of αSma mRNA transcribed was measured. The results are shown in FIG. As shown in FIG. 18, there was no change in the amount of αSma mRNA transcription in cells exposed to Co-rhCYGB. From this result, it was shown that the substitution of heme iron with cobalt inhibits the antifibrotic effect of cytoglobin, that is, the iron heme binding of cytoglobin is related to the antifibrotic effect.
Ｃｏ－ＣＹＧＢを含まない（Ｓ－）リン酸緩衝溶液、並びに、Ｃｏ－ＣＹＧＢを４０μｇ／ｍＬの濃度（Ｃｏ－ｃｙｇｂ４０）でリン酸緩衝溶液に含む溶液を調製し、４８時間、ＨＨＳｔｅＣ細胞に暴露し、αＳｍａのｍＲＮＡ転写量を測定した。結果を図１８に示す。図１８に示されるように、Ｃｏ－ｒｈＣＹＧＢに暴露された細胞におけるαＳｍａのｍＲＮＡ転写量の量に変化は無かった。この結果からも、ヘム鉄のコバルトへの置換がサイトグロビンの抗線維化効果を阻害すること、つまり、サイトグロビンの鉄ヘム結合が抗線維化効果に関連していることが示された。 (Effect of Co-CYGBs on transcription of fibrosis-related genes)
A Co-CYGB-free (S-) phosphate buffer solution and a solution containing Co-CYGB at a concentration of 40 μg / mL (Co-cygb40) in the phosphate buffer solution were prepared and exposed to HHSteC cells for 48 hours. Then, the amount of αSma mRNA transcribed was measured. The results are shown in FIG. As shown in FIG. 18, there was no change in the amount of αSma mRNA transcription in cells exposed to Co-rhCYGB. From this result, it was shown that the substitution of heme iron with cobalt inhibits the antifibrotic effect of cytoglobin, that is, the iron heme binding of cytoglobin is related to the antifibrotic effect.
試験例８：各種サイトグロビンタンパク質と抗線維化効果
本試験例では、全長サイトグロビンの部分タンパク質のＨｉｓタグ修飾体について、抗線維化効果を検証した。 Test Example 8: Various cytoglobin proteins and anti-fibrotic effect In this test example, the anti-fibrotic effect was verified for the His-tag modified product of a partial protein of full-length cytoglobin.
本試験例では、全長サイトグロビンの部分タンパク質のＨｉｓタグ修飾体について、抗線維化効果を検証した。 Test Example 8: Various cytoglobin proteins and anti-fibrotic effect In this test example, the anti-fibrotic effect was verified for the His-tag modified product of a partial protein of full-length cytoglobin.
（サイトグロビンタンパク質）
検証したサイトグロビンタンパク質は以下の通りである。
・ｐｐ１：全長サイトグロビン（配列番号５）の１～２０番目（配列番号１７）（比較用）
・ｐｐ２：全長サイトグロビン（配列番号５）の７５～９５番目（配列番号１８）（比較用）
・ｐｐ３：全長サイトグロビン（配列番号５）の７５～１２０番目（配列番号１９）（比較用）
・ｐｐ４：全長サイトグロビン（配列番号５）の１８～１７０番目（配列番号１）
・ｐｐ５：全長サイトグロビン（配列番号５）の４～１７６番目（配列番号２）
・ｐｐ６：全長サイトグロビン（配列番号５）の１８～１９０番目（配列番号３）
・ｐｐ７：全長サイトグロビン（配列番号５）の４～１９０番目（配列番号４）
・ｐｐ８（ＣＹＧＢ）：全長サイトグロビン（配列番号５） (Cytoglobin protein)
The verified cytoglobin proteins are as follows.
-Pp1: 1st to 20th (SEQ ID NO: 17) of full-length cytoglobin (SEQ ID NO: 5) (for comparison)
-Pp2: 75th to 95th (SEQ ID NO: 18) of full-length cytoglobin (SEQ ID NO: 5) (for comparison)
-Pp3: 75th to 120th (SEQ ID NO: 19) of full-length cytoglobin (SEQ ID NO: 5) (for comparison)
-Pp4: 18th to 170th (SEQ ID NO: 1) of full-length cytoglobin (SEQ ID NO: 5)
-Pp5: 4th to 176th (SEQ ID NO: 2) of full-length cytoglobin (SEQ ID NO: 5)
-Pp6: 18th to 190th (SEQ ID NO: 3) of full-length cytoglobin (SEQ ID NO: 5)
-Pp7: 4th to 190th (SEQ ID NO: 4) of full-length cytoglobin (SEQ ID NO: 5)
-Pp8 (CYGB): Full-length cytoglobin (SEQ ID NO: 5)
検証したサイトグロビンタンパク質は以下の通りである。
・ｐｐ１：全長サイトグロビン（配列番号５）の１～２０番目（配列番号１７）（比較用）
・ｐｐ２：全長サイトグロビン（配列番号５）の７５～９５番目（配列番号１８）（比較用）
・ｐｐ３：全長サイトグロビン（配列番号５）の７５～１２０番目（配列番号１９）（比較用）
・ｐｐ４：全長サイトグロビン（配列番号５）の１８～１７０番目（配列番号１）
・ｐｐ５：全長サイトグロビン（配列番号５）の４～１７６番目（配列番号２）
・ｐｐ６：全長サイトグロビン（配列番号５）の１８～１９０番目（配列番号３）
・ｐｐ７：全長サイトグロビン（配列番号５）の４～１９０番目（配列番号４）
・ｐｐ８（ＣＹＧＢ）：全長サイトグロビン（配列番号５） (Cytoglobin protein)
The verified cytoglobin proteins are as follows.
-Pp1: 1st to 20th (SEQ ID NO: 17) of full-length cytoglobin (SEQ ID NO: 5) (for comparison)
-Pp2: 75th to 95th (SEQ ID NO: 18) of full-length cytoglobin (SEQ ID NO: 5) (for comparison)
-Pp3: 75th to 120th (SEQ ID NO: 19) of full-length cytoglobin (SEQ ID NO: 5) (for comparison)
-Pp4: 18th to 170th (SEQ ID NO: 1) of full-length cytoglobin (SEQ ID NO: 5)
-Pp5: 4th to 176th (SEQ ID NO: 2) of full-length cytoglobin (SEQ ID NO: 5)
-Pp6: 18th to 190th (SEQ ID NO: 3) of full-length cytoglobin (SEQ ID NO: 5)
-Pp7: 4th to 190th (SEQ ID NO: 4) of full-length cytoglobin (SEQ ID NO: 5)
-Pp8 (CYGB): Full-length cytoglobin (SEQ ID NO: 5)
これらｐｐ１～ｐｐ８について、亜ジチオン酸塩による還元状態の鉄ヘム活性を、可視吸収スペクトルを用いて調べた。結果を図１９に示す。なお、鉄ヘム活性が認められる場合、通常、５００～６００ｎｍ領域に２つのピークが約１：３の比率で得られる。図１９に示されるように、ｐｐ４、ｐｐ５、ｐｐ６、ｐｐ７、ｐｐ８について、５００～６００ｎｍ領域に２つのピークが約１：３の比率で認められ、鉄ヘム活性があることが分かった。
For these pp1 to pp8, the iron heme activity in the reduced state by dithionous acid was investigated using a visible absorption spectrum. The results are shown in FIG. When iron heme activity is observed, two peaks are usually obtained in the region of 500 to 600 nm at a ratio of about 1: 3. As shown in FIG. 19, for pp4, pp5, pp6, pp7, and pp8, two peaks were observed in the 500 to 600 nm region at a ratio of about 1: 3, indicating that there is iron heme activity.
（線維症関連タンパク質の発現に及ぼすＨｉｓ－ｐｐ４の効果）
細胞株のＨＨＳｔｅＣ細胞を実験に用いた。Ｈｉｓ－ｐｐ４を含まない（０μｇ／ｍＬ）リン酸緩衝溶液、並びに、Ｈｉｓ－ｐｐ４を５μｇ／ｍＬ、１０μｇ／ｍＬ、２０μｇ／ｍＬ、４０μｇ／ｍＬ、又は８０μｇ／ｍＬの濃度で生理食塩水中に含むリン酸緩衝溶液を調製し、４８時間、ＨＨＳｔｅＣ細胞に暴露し、細胞内ＣＹＧＢを測定した。結果を図２０に示す。図２０に示されるように、Ｈｉｓ－ｐｐ４に暴露された細胞内に発現していたαＳＭＡ、及びＩ型コラーゲン（ＣＯＬ１Ａ１）は用量依存的に減少した。なお、ＧＡＰＤＨはハウスキーピングタンパク質である。 (Effect of His-pp4 on expression of fibrosis-related proteins)
HHSteC cells from the cell line were used in the experiment. A phosphate buffer solution containing no His-pp4 (0 μg / mL) and His-pp4 at a concentration of 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, or 80 μg / mL in physiological saline. A phosphate buffer solution was prepared, exposed to HHSteC cells for 48 hours, and intracellular CYGB was measured. The results are shown in FIG. As shown in FIG. 20, αSMA expressed in cells exposed to His-pp4 and type I collagen (COL1A1) decreased in a dose-dependent manner. GAPDH is a housekeeping protein.
細胞株のＨＨＳｔｅＣ細胞を実験に用いた。Ｈｉｓ－ｐｐ４を含まない（０μｇ／ｍＬ）リン酸緩衝溶液、並びに、Ｈｉｓ－ｐｐ４を５μｇ／ｍＬ、１０μｇ／ｍＬ、２０μｇ／ｍＬ、４０μｇ／ｍＬ、又は８０μｇ／ｍＬの濃度で生理食塩水中に含むリン酸緩衝溶液を調製し、４８時間、ＨＨＳｔｅＣ細胞に暴露し、細胞内ＣＹＧＢを測定した。結果を図２０に示す。図２０に示されるように、Ｈｉｓ－ｐｐ４に暴露された細胞内に発現していたαＳＭＡ、及びＩ型コラーゲン（ＣＯＬ１Ａ１）は用量依存的に減少した。なお、ＧＡＰＤＨはハウスキーピングタンパク質である。 (Effect of His-pp4 on expression of fibrosis-related proteins)
HHSteC cells from the cell line were used in the experiment. A phosphate buffer solution containing no His-pp4 (0 μg / mL) and His-pp4 at a concentration of 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, or 80 μg / mL in physiological saline. A phosphate buffer solution was prepared, exposed to HHSteC cells for 48 hours, and intracellular CYGB was measured. The results are shown in FIG. As shown in FIG. 20, αSMA expressed in cells exposed to His-pp4 and type I collagen (COL1A1) decreased in a dose-dependent manner. GAPDH is a housekeeping protein.
（線維症関連遺伝子の転写に及ぼすＨｉｓ－ｐｐ４の効果）
Ｈｉｓ－ｐｐ４を含まない（０μｇ／ｍＬ）リン酸緩衝溶液、並びに、Ｈｉｓ－ｐｐ４を１０μｇ／ｍＬ、２０μｇ／ｍＬ、４０μｇ／ｍＬ、又は８０μｇ／ｍＬの濃度でリン酸緩衝溶液に含む溶液を調製し、４８時間、ＨＨＳｔｅＣ細胞に暴露し、αＳｍａのｍＲＮＡ転写量を測定した。結果を図２１に示す。図２１に示されるように、Ｈｉｓ－ｐｐ４に暴露された細胞におけるαＳｍａのｍＲＮＡ転写量は用量依存的に低下した。 (Effect of His-pp4 on transcription of fibrosis-related genes)
Prepare a phosphate buffer solution free of His-pp4 (0 μg / mL) and a solution containing His-pp4 in the phosphate buffer solution at a concentration of 10 μg / mL, 20 μg / mL, 40 μg / mL, or 80 μg / mL. Then, the cells were exposed to HHSteC cells for 48 hours, and the amount of αSma mRNA transcribed was measured. The results are shown in FIG. As shown in FIG. 21, the amount of αSma mRNA transcription in cells exposed to His-pp4 decreased in a dose-dependent manner.
Ｈｉｓ－ｐｐ４を含まない（０μｇ／ｍＬ）リン酸緩衝溶液、並びに、Ｈｉｓ－ｐｐ４を１０μｇ／ｍＬ、２０μｇ／ｍＬ、４０μｇ／ｍＬ、又は８０μｇ／ｍＬの濃度でリン酸緩衝溶液に含む溶液を調製し、４８時間、ＨＨＳｔｅＣ細胞に暴露し、αＳｍａのｍＲＮＡ転写量を測定した。結果を図２１に示す。図２１に示されるように、Ｈｉｓ－ｐｐ４に暴露された細胞におけるαＳｍａのｍＲＮＡ転写量は用量依存的に低下した。 (Effect of His-pp4 on transcription of fibrosis-related genes)
Prepare a phosphate buffer solution free of His-pp4 (0 μg / mL) and a solution containing His-pp4 in the phosphate buffer solution at a concentration of 10 μg / mL, 20 μg / mL, 40 μg / mL, or 80 μg / mL. Then, the cells were exposed to HHSteC cells for 48 hours, and the amount of αSma mRNA transcribed was measured. The results are shown in FIG. As shown in FIG. 21, the amount of αSma mRNA transcription in cells exposed to His-pp4 decreased in a dose-dependent manner.
（線維症関連タンパク質の発現、線維症関連遺伝子の転写に及ぼす他のサイトグロビンタンパク質の効果）
Ｈｉｓ－ｐｐ５～Ｈｉｓ－ｐｐ７及びＨｉｓ－ｐｐ１～Ｈｉｓ－ｐｐ３についても上記と同様に線維症関連タンパク質の発現に及ぼす影響を検証した。Ｈｉｓ－ｐｐ１については、線維症関連遺伝子の転写に及ぼす影響も検証した。Ｈｉｓ－ｐｐ５～Ｈｉｓ－ｐｐ７の結果を図２２～図２４に示す。また、Ｈｉｓ－ｐｐ１の結果を図２５（線維症関連タンパク質の発現結果）及び図２６（線維症関連遺伝子の転写結果）を示し、Ｈｉｓ－ｐｐ２及びＨｉｓ－ｐｐ３の結果を図２７及び図２８に示す。なお、図中のＰＣは、Ｈｉｓ－ｐｐ８（Ｈｉｓ－ＣＹＧＢ）の結果を示す。 (Expression of fibrosis-related proteins, effects of other cytoglobin proteins on transcription of fibrosis-related genes)
The effects of His-pp5-His-pp7 and His-pp1-His-pp3 on the expression of fibrosis-related proteins were examined in the same manner as described above. For His-pp1, we also examined the effect of fibrosis-related genes on transcription. The results of His-pp5 to His-pp7 are shown in FIGS. 22 to 24. The results of His-pp1 are shown in FIGS. 25 (fibrosis-related protein expression results) and FIG. 26 (fibrosis-related gene transcription results), and the results of His-pp2 and His-pp3 are shown in FIGS. 27 and 28. Shown. The PC in the figure shows the result of His-pp8 (His-CYGB).
Ｈｉｓ－ｐｐ５～Ｈｉｓ－ｐｐ７及びＨｉｓ－ｐｐ１～Ｈｉｓ－ｐｐ３についても上記と同様に線維症関連タンパク質の発現に及ぼす影響を検証した。Ｈｉｓ－ｐｐ１については、線維症関連遺伝子の転写に及ぼす影響も検証した。Ｈｉｓ－ｐｐ５～Ｈｉｓ－ｐｐ７の結果を図２２～図２４に示す。また、Ｈｉｓ－ｐｐ１の結果を図２５（線維症関連タンパク質の発現結果）及び図２６（線維症関連遺伝子の転写結果）を示し、Ｈｉｓ－ｐｐ２及びＨｉｓ－ｐｐ３の結果を図２７及び図２８に示す。なお、図中のＰＣは、Ｈｉｓ－ｐｐ８（Ｈｉｓ－ＣＹＧＢ）の結果を示す。 (Expression of fibrosis-related proteins, effects of other cytoglobin proteins on transcription of fibrosis-related genes)
The effects of His-pp5-His-pp7 and His-pp1-His-pp3 on the expression of fibrosis-related proteins were examined in the same manner as described above. For His-pp1, we also examined the effect of fibrosis-related genes on transcription. The results of His-pp5 to His-pp7 are shown in FIGS. 22 to 24. The results of His-pp1 are shown in FIGS. 25 (fibrosis-related protein expression results) and FIG. 26 (fibrosis-related gene transcription results), and the results of His-pp2 and His-pp3 are shown in FIGS. 27 and 28. Shown. The PC in the figure shows the result of His-pp8 (His-CYGB).
図２２～図２４から明らかな通り、Ｈｉｓ－ｐｐ５～Ｈｉｓ－ｐｐ７に暴露された細胞内に発現していたＩ型コラーゲン（ＣＯＬ１Ａ１）は用量依存的に減少した。図２０、図２２～図２４の結果を対比すると、特に、Ｈｉｓ－ｐｐ５、Ｈｉｓ－ｐｐ７、Ｈｉｓ－ｐｐ８（ＰＣ；Ｈｉｓ－ＣＹＧＢ）においてＣＯＬ１Ａ１低減効果が高かった。
As is clear from FIGS. 22 to 24, type I collagen (COL1A1) expressed in cells exposed to His-pp5 to His-pp7 decreased in a dose-dependent manner. Comparing the results of FIGS. 20 and 22 to 24, the COL1A1 reducing effect was particularly high in His-pp5, His-pp7, and His-pp8 (PC; His-CYGB).
一方で、図２５，２７，２８から明かな通り、Ｈｉｓ－ｐｐ１～Ｈｉｓ－ｐｐ３に暴露された細胞内に発現していたＩ型コラーゲン（ＣＯＬ１Ａ１）は用量に関わらず変化しなかった。なお、図２６に示すようにＨｉｓ－ｐｐ１に暴露された細胞におけるαＳｍａの転写量にも変化はなかった。
On the other hand, as is clear from FIGS. 25, 27, and 28, type I collagen (COL1A1) expressed in cells exposed to His-pp1 to His-pp3 did not change regardless of the dose. As shown in FIG. 26, there was no change in the transcription amount of αSma in the cells exposed to His-pp1.
以上の結果より、抗線維化効果の発現に、少なくともｐｐ４が有するアミノ酸配列（配列番号１）が必要であることが分かった。
From the above results, it was found that at least the amino acid sequence of pp4 (SEQ ID NO: 1) is required for the expression of the antifibrotic effect.
試験例９：肝炎・肝線維化モデルに及ぼすＨｉｓ－ＣＹＧＢ（ｉｎ ｖｉｖｏ）
Ｃ５７ＢＬ／６Ｊ野生型マウスをグループ１（Ｃｏｎｔ），グループ２（ＤＤＣ４ｗ），グループ３（ＤＤＣ４ｗ－ｒｈＣＹ２ｗ）（各群ｎ＝９）に分けた。グループ１（Ｃｏｎｔ）には通常食を、グループ２（ＤＤＣ４ｗ）には０．１重量％３、５－ジエトキシカルボニル－１、４－ジヒドロコリジン添加食（ＤＤＣ食）を、いずれも４週間給餌し、３週目と４週目では２回／週の頻度で生理食塩水を尾静脈注射した。グループ３（ＤＤＣ４ｗ－ｒｈＣＹ２ｗ）にはＤＤＣ食を４週間給餌し、３週目と４週目では１日１回２回／週の頻度、２ｍｇ／ｋｇ体重の容量で尾静脈注射した。グループ２及びグループ３は、ＤＤＣ誘発マウス胆汁うっ滞モデル（肝炎・肝線維化モデル）である。マウスは最後の腹腔内注射の２日後に屠殺し、血清及び肝臓組織を採取した。 Test Example 9: His-CYGB (in vivo) on hepatitis / liver fibrosis model
C57BL / 6J wild-type mice were divided into group 1 (Cont), group 2 (DDC4w), and group 3 (DDC4w-rhCY2w) (each group n = 9). Group 1 (Cont) was given a normal diet, and Group 2 (DDC4w) was given a 0.1 wt% 3,5-diethoxycarbonyl-1,4-dihydrocoridine-added diet (DDC diet) for 4 weeks. Feeding was performed, and saline was injected into the tail vein twice a week at the 3rd and 4th weeks. Group 3 (DDC4w-rhCY2w) was fed a DDC diet for 4 weeks, and in the 3rd and 4th weeks, it was injected into the tail vein twice a day at a frequency of 2 mg / kg body weight.Group 2 and Group 3 are DDC-induced mouse cholestasis models (hepatitis / liver fibrosis model). Mice were sacrificed 2 days after the last intraperitoneal injection and serum and liver tissue were collected.
Ｃ５７ＢＬ／６Ｊ野生型マウスをグループ１（Ｃｏｎｔ），グループ２（ＤＤＣ４ｗ），グループ３（ＤＤＣ４ｗ－ｒｈＣＹ２ｗ）（各群ｎ＝９）に分けた。グループ１（Ｃｏｎｔ）には通常食を、グループ２（ＤＤＣ４ｗ）には０．１重量％３、５－ジエトキシカルボニル－１、４－ジヒドロコリジン添加食（ＤＤＣ食）を、いずれも４週間給餌し、３週目と４週目では２回／週の頻度で生理食塩水を尾静脈注射した。グループ３（ＤＤＣ４ｗ－ｒｈＣＹ２ｗ）にはＤＤＣ食を４週間給餌し、３週目と４週目では１日１回２回／週の頻度、２ｍｇ／ｋｇ体重の容量で尾静脈注射した。グループ２及びグループ３は、ＤＤＣ誘発マウス胆汁うっ滞モデル（肝炎・肝線維化モデル）である。マウスは最後の腹腔内注射の２日後に屠殺し、血清及び肝臓組織を採取した。 Test Example 9: His-CYGB (in vivo) on hepatitis / liver fibrosis model
C57BL / 6J wild-type mice were divided into group 1 (Cont), group 2 (DDC4w), and group 3 (DDC4w-rhCY2w) (each group n = 9). Group 1 (Cont) was given a normal diet, and Group 2 (DDC4w) was given a 0.1 wt% 3,5-diethoxycarbonyl-1,4-dihydrocoridine-added diet (DDC diet) for 4 weeks. Feeding was performed, and saline was injected into the tail vein twice a week at the 3rd and 4th weeks. Group 3 (DDC4w-rhCY2w) was fed a DDC diet for 4 weeks, and in the 3rd and 4th weeks, it was injected into the tail vein twice a day at a frequency of 2 mg / kg body weight.
血清中のＡＳＴ値及びＡＬＴ値の測定を図２９に示す。図２９に示すように、グループ２（ＤＤＣ４ｗ）ではグループ１（Ｃｏｎｔ）より血清中ＡＳＴ、ＡＬＴはそれぞれ４２３．３ＩＵ／Ｌ、２０５．１ＩＵ／Ｌへと上昇しており、Ｈｉｓ－ｒｈＣＹＧＢを２週間投与したグループ３（ＤＤＣ４ｗ－ｒｈＣｙ２ｗ）ではそれらの値がそれぞれ２６６．９ＩＵ／Ｌ、１２９．５ＩＵ／Ｌへと有意に低下した（ｐ＜０．０５）。つまり、Ｈｉｓ－ｒｈＣＹＧＢ投与によって肝炎・肝線維化モデルの血清中ＡＳＴ・ＡＬＴレベルが有意に低下したことが認められた。
The measurement of AST value and ALT value in serum is shown in FIG. As shown in FIG. 29, in group 2 (DDC4w), serum AST and ALT increased from group 1 (Cont) to 423.3 IU / L and 205.1 IU / L, respectively, and His-rhCYGB was increased for 2 weeks. In group 3 (DDC4w-rhCy2w) administered, their values were significantly reduced to 266.9 IU / L and 129.5 IU / L, respectively (p <0.05). That is, it was confirmed that the serum AST / ALT level of the hepatitis / liver fibrosis model was significantly reduced by the administration of His-rhCYGB.
また、線維症関連遺伝子αＳｍａ、Ｃｏｌ１ａ１、Ｔｇｆβ－１、Ｔｇｆβ－３及び炎症性因子Ｔｎｆ－α、Ｃｃｌ－２それぞれのｍＲＮＡ転写量の測定結果を図３０に示す。図３０に示すように、いずれのｍＲＮＡ転写量も、Ｈｉｓ－ｒｈＣＹＧＢの投与によって有意に低減された。
In addition, FIG. 30 shows the measurement results of the mRNA transcription levels of the fibrosis-related genes αSma, Col1a1, Tgfβ-1, Tgfβ-3 and the inflammatory factors Tnf-α and Ccl-2. As shown in FIG. 30, the amount of transcription of each mRNA was significantly reduced by administration of His-rhCYGB.
更に、グループ１（Ｃｏｎｔ），グループ２（ＤＤＣ４ｗ），グループ３（ＤＤＣ４ｗ－ｒｈＣＹ２ｗ）の肝組織について、Ｈ＆Ｅ（ヘマトキシリン・エオジン）染色；コラーゲン線維を染色するためのＳｉＲ（シリウスレッド）染色；並びに星細胞活性化マーカーαＳＭＡ、好中球マーカーＮｅｕ、及びマクロファージマーカーＣＤ６８，胆管細胞マーカーＣＫ１９を核対比染色するためのＤＡＰＩ（４，６－ジアミジノ－２－フェニルインドール）染色に供した。結果を図３１に示す。図３１のＨ＆Ｅ染色及びシリウスレッド染色の結果に示されるとおり、組織学的にも、Ｈｉｓ－ｒｈＣＹＧＢの投与によって線維化反応が抑制されたことが示された。また、図３１のαＳＭＡ－ＤＡＰＩ、Ｎｅｕ－ＤＡＰＩ、及びＣＤ６８－ＤＡＰＩの染色結果に示されるとおり、星細胞活性化マーカーであるαＳＭＡ、好中球マーカーであるＮｅｕ、及びマクロファージマーカーであるＣＤ６８、胆管細胞マーカーＣＫ１９の全ての発現がＨｉｓ－ｒｈＣＹＧＢの投与によって低減したことが示された。
Furthermore, H & E (hematoxylin and eosin) staining; SiR (sirius red) staining for staining collagen fibers; and stars were used for the liver tissues of Group 1 (Cont), Group 2 (DDC4w), and Group 3 (DDC4w-rhCY2w). The cell activation marker αSMA, neutrophil marker Neu, macrophage marker CD68, and bile duct cell marker CK19 were subjected to DAPI (4,6-diamidino-2-phenylindole) staining for nuclear counterstaining. The results are shown in FIG. As shown in the results of H & E staining and Sirius red staining in FIG. 31, it was histologically shown that the fibrotic reaction was suppressed by the administration of His-rhCYGB. In addition, as shown in the staining results of αSMA-DAPI, Neu-DAPI, and CD68-DAPI in FIG. 31, αSMA, which is a stellate cell activation marker, Neu, which is a neutrophil marker, and CD68, which is a macrophage marker, and bile duct. It was shown that all expression of the cellular marker CK19 was reduced by administration of His-rhCYGB.
配列番号１３は、ヒスチジンタグ付きサイトグロビンである。
配列番号１４は、ＴＡＴタグ付きサイトグロビンである。
配列番号１５は、６Ｒペプチドタグである。
配列番号１６は、６Ｒペプチドタグ付きサイトグロビンである。 SEQ ID NO: 13 is a histidine-tagged cytoglobin.
SEQ ID NO: 14 is a TAT-tagged site globin.
SEQ ID NO: 15 is a 6R peptide tag.
SEQ ID NO: 16 is a 6R peptide-tagged cytoglobin.
配列番号１４は、ＴＡＴタグ付きサイトグロビンである。
配列番号１５は、６Ｒペプチドタグである。
配列番号１６は、６Ｒペプチドタグ付きサイトグロビンである。 SEQ ID NO: 13 is a histidine-tagged cytoglobin.
SEQ ID NO: 14 is a TAT-tagged site globin.
SEQ ID NO: 15 is a 6R peptide tag.
SEQ ID NO: 16 is a 6R peptide-tagged cytoglobin.
Claims (13)
- （１）配列番号１で示されるアミノ酸配列を含むポリペプチド、
（２）配列番号１で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されたアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド、及び
（３）配列番号１で示されるアミノ酸配列に対する配列同一性が８０％以上のアミノ酸配列を含み、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種のポリペプチドからなるサイトグロビンタンパク質と、前記サイトグロビンタンパク質に結合したオリゴヒスチジン及びＴＡＴペプチドの少なくともいずれかのタグと、を含む修飾サイトグロビンタンパク質を有効成分として含む、抗線維化剤。 (1) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 1.
(2) A polypeptide containing an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and having an ability to suppress fibrosis, and (3) A cytoglobin protein comprising at least one polypeptide having an amino acid sequence having an amino acid sequence identity of 80% or more with respect to the amino acid sequence represented by SEQ ID NO: 1 and having an ability to suppress fibrosis, and the above-mentioned An antifibrinolytic agent comprising a modified cytoglobin protein as an active ingredient, which comprises at least one tag of oligohistidine and TAT peptide bound to the cytoglobin protein. - 前記オリゴヒスチジンの長さが、３～１０アミノ酸残基である、請求項１に記載の抗線維化剤。 The antifibrotic agent according to claim 1, wherein the oligohistidine has a length of 3 to 10 amino acid residues.
- 前記タグがオリゴヒスチジンである、請求項１又は２に記載の抗線維化剤。 The antifibrotic agent according to claim 1 or 2, wherein the tag is oligohistidine.
- 前記タグが、前記サイトグロビンタンパク質のＮ末端に結合している、請求項１～３のいずれかに記載の抗線維化剤。 The antifibrotic agent according to any one of claims 1 to 3, wherein the tag is bound to the N-terminal of the cytoglobin protein.
- 前記ポリペプチドの長さが、１５２～１９０アミノ酸残基である、請求項１～４のいずれかに記載の抗線維化剤。 The antifibrotic agent according to any one of claims 1 to 4, wherein the polypeptide has a length of 152 to 190 amino acid residues.
- 前記ポリペプチドが、
（１１）配列番号１で示されるアミノ酸配列からなるポリペプチド、
（１２）配列番号１で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（１３）配列番号１で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能をするポリペプチド
の少なくとも１種である、請求項１～５のいずれかに記載の抗線維化剤。 The polypeptide
(11) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
(12) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 and has an inhibitory ability to suppress fibrosis, and (13). The antifibrosis according to any one of claims 1 to 5, wherein the sequence identity to the amino acid sequence shown in SEQ ID NO: 1 is 80% or more, and at least one of the polypeptides capable of suppressing fibrosis. Agent. - 前記ポリペプチドが、
（２１）配列番号２で示されるアミノ酸配列からなるポリペプチド、
（２２）配列番号１及び配列番号２で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（２３）配列番号１及び配列番号２で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種である、請求項１～５のいずれかに記載の抗線維化剤。 The polypeptide
(21) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
(22) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 and which has the ability to suppress fibrosis. (23) At least one of the polypeptides having a sequence identity of 80% or more with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 and having an ability to suppress fibrosis, according to claims 1 to 5. The antifibrinolytic agent according to any one. - 前記ポリペプチドが、
（３１）配列番号３で示されるアミノ酸配列からなるポリペプチド、
（３２）配列番号１及び配列番号３で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（３３）配列番号１及び配列番号３で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種である、請求項１～５のいずれかに記載の抗線維化剤。 The polypeptide
(31) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3.
(32) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 and which has the ability to suppress fibrosis. (33) At least one of the polypeptides having a sequence identity of 80% or more with respect to the amino acid sequences represented by SEQ ID NOs: 1 and SEQ ID NO: 3 and having an ability to suppress fibrosis, according to claims 1 to 5. The antifibrinolytic agent according to any one. - 前記ポリペプチドが、
（４１）配列番号４で示されるアミノ酸配列からなるポリペプチド、
（４２）配列番号１及び配列番号４で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（４３）配列番号１及び配列番号４で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種である、請求項１～５のいずれかに記載の抗線維化剤。 The polypeptide
(41) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4.
(42) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4 and which has the ability to suppress fibrosis. (43) At least one of the polypeptides having 80% or more sequence identity with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4 and having an ability to suppress fibrosis, according to claims 1 to 5. The antifibrinolytic agent according to any one. - 前記ポリペプチドが、
（５１）配列番号５で示されるアミノ酸配列からなるポリペプチド、
（５２）配列番号１及び配列番号５で示されるアミノ酸配列において、１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、線維化抑制能を有するポリペプチド、及び
（５３）配列番号１及び配列番号５で示されるアミノ酸配列に対する配列同一性が８０％以上であり、且つ、線維化抑制能を有するポリペプチド
の少なくとも１種である、請求項１～５のいずれかに記載の抗線維化剤。 The polypeptide
(51) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5.
(52) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5 and which has the ability to suppress fibrosis. (53) Claims 1 to 5, wherein the sequence identity with respect to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5 is 80% or more, and at least one of the polypeptides having the ability to suppress fibrosis. The antifibrinolytic agent according to any one. - 肝線維化抑制剤である、請求項１～１０のいずれかに記載の抗線維化剤。 The antifibrosis agent according to any one of claims 1 to 10, which is a liver fibrosis inhibitor.
- 肝硬変治療薬である、請求項１～１１のいずれかに記載の抗線維化剤。 The antifibrotic agent according to any one of claims 1 to 11, which is a therapeutic agent for liver cirrhosis.
- 肝癌予防薬である、請求項１～１２のいずれかに記載の抗線維化剤。 The antifibrotic agent according to any one of claims 1 to 12, which is a liver cancer preventive agent.
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Citations (1)
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| CN101863985A (en) * | 2010-06-18 | 2010-10-20 | 许瑞安 | Recombinant human fusion Tat cytoglobulin and application thereof in treating liver cancer |
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| CN101863985A (en) * | 2010-06-18 | 2010-10-20 | 许瑞安 | Recombinant human fusion Tat cytoglobulin and application thereof in treating liver cancer |
Non-Patent Citations (5)
| Title |
|---|
| ASAHINA, K. ET AL.: "Characterization of human stellate cell activation-associated protein and its expression in human liver", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1577, 2002, pages 471 - 475, XP004383779, DOI: 10.1016/S0167-4781(02)00477-3 * |
| LE, THI THANH THUY ET AL.: "Cytoglobin suppresses liver injury and fibrosis in bile duct ligated mice by regulating nitrosative and oxidative stress", HEPATOLOGY, vol. 68, no. 1, 2018, pages 128A - 129A * |
| NGUYEN, THI THANH HAI ET AL., CYTOGLOBIN OVEREXPRESSION PROTECTS MICE AGAINST TAA-INDUCED LIVER FIBROSIS DEVELOPMENT, vol. 58, no. 3, 2017, pages A782 * |
| NGUYEN, THI THANH HAI ET AL.: "Protective role of hepatic stellate cell-specific overexpression of cytoglobin against liver fibrosis in in vitro and in vivo", vol. 59, no. 1, 2018, pages A391 * |
| YOSHIZATO KATSUTOSHI, THUY LE THI THANH, SHIOTA GOSHI, KAWADA NORIFUMI: "Discovery of cytoglobin and its roles in physiology and pathology of hepatic stellate cells", PROCEEDINGS OF THE JAPAN ACADEMY, vol. 92, no. 3, 2016, pages 77 - 97, XP055760392, ISSN: 0386-2208 * |
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