Fibrosis Treatment Drugs

Abstract

To provide a fibrosis treatment agent.SOLUTION: A fibrosis treatment agent contains (A) a polypeptide composed of an amino sequence represented by the sequence number 1, or (B) a polypeptide that is composed of an amino acid sequence 85% or more identical to the amino sequence represented by the sequence number 1 and inhibits the expression of at least one gene selected from the group consisting of COL1A1, COL1A2, and αSMA.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to therapeutic agents for treating fibrosis, particularly liver fibrosis. [Background technology]

[0002] Liver cirrhosis is the final stage of chronic hepatitis, regardless of the underlying liver disease, and is a fatal disease that progresses to liver cancer at an annual rate of 6-8%. Liver cirrhosis is a pathological condition in which hepatic stellate cells, whose main function is vitamin A storage, become activated, take on a fibroblast-like morphology, and the liver parenchyma is replaced by extracellular matrix proteins such as type I collagen, resulting in a decrease in functional liver cells. Therefore, it is important to control the activation of hepatic stellate cells and remove the extracellular matrix in the treatment of liver cirrhosis. However, there are currently no drugs targeting hepatic stellate cells, let alone drugs for cirrhosis, and treatment is limited to symptomatic treatment such as maintaining liver function.

[0003] In addition, because activation of hepatic stellate cells occurs due to inflammation caused by hepatic cell damage, active development of drugs for treating liver cirrhosis targeting hepatocytes, inflammatory cells, or hepatic stellate cells is underway. However, most of the drugs currently undergoing clinical trials target hepatocytes or inflammatory cells, and very few target hepatic stellate cells. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2019-108291 A Summary of the Invention [Problem to be solved by the invention]

[0005] The objective of the present invention is to provide a therapeutic agent for fibrosis. [Means for solving the problem]

[0006] The present inventors discovered that HSD17B4 (17beta-hydroxysteroid dehydrogenase type 4) protein suppresses collagen production in hepatic stellate cells and have made further improvements.

[0007] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. (A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or (B) A therapeutic agent for fibrosis, comprising an amino acid sequence that is 85% or more identical to the amino acid sequence shown in SEQ ID NO:1 and a polypeptide that suppresses expression of at least one gene selected from the group consisting of COL1A1, COL1A2, and αSMA. Section 2. Item 1. A therapeutic agent for fibrosis, comprising an expression vector incorporating a polynucleotide encoding the polypeptide (A) or the polypeptide (B) according to Item 1. Section 3. Item 3. The fibrosis therapeutic agent according to Item 1 or 2, wherein the fibrosis is liver fibrosis. Effect of the Invention

[0008] Drugs for treating fibrosis are provided. [Brief description of the drawings]

[0009] [Figure 1A] The chemical formula of epoxy beads (N) and the chemical formula of epoxy beads with Lawsone immobilized thereon (L) are shown. [Figure 1B] 1 shows the results of SDS-PAGE analysis of proteins reacted with epoxy beads. [Figure 1C] The results of silver staining of proteins separated by SDS-PAGE and MALDI-MS analysis are shown. [Figure 2A] The results of analysis by quantitative PCR are shown. [Figure 2B] The results of analysis by Western blotting are shown. [Figure 3A] The results of analysis by quantitative PCR are shown. [Figure 3B] The results of analysis by Western blotting are shown. [Figure 4] The results of analysis by quantitative PCR are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Each embodiment included in the present disclosure will be described in further detail below.

[0011] A therapeutic agent for fibrosis encompassed by the present disclosure contains a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide consisting of an amino acid sequence showing 85% or more identity to the amino acid sequence shown in SEQ ID NO: 1. In this specification, the therapeutic agent for fibrosis may be referred to as the "therapeutic agent for fibrosis of the present disclosure."

[0012] The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence that constitutes the human-derived HSD17B4 protein.

[0013] The HSD17B4 protein is a multifunctional peroxisomal protein involved in fatty acid β-oxidation and steroid metabolism. HSD17B4 is also called D-bifunctional protein (DBP).

[0014] A polypeptide consisting of an amino acid sequence showing 85% or more identity to the amino acid sequence shown in SEQ ID NO: 1 is preferably a polypeptide consisting of an amino acid sequence having 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 1. More preferably, it is 95% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0015] Amino acid sequence identity can be calculated using the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: / / www.ncbi.nlm.nih.gov / BLAST / with default parameters.

[0016] Examples of polypeptides consisting of an amino acid sequence showing 85% or more identity with the amino acid sequence shown in SEQ ID NO: 1 include polypeptides consisting of an amino acid sequence in which one or two or more amino acids have been deleted, substituted, inserted or added in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. The upper limit of the number of deleted, substituted, inserted or added amino acids is, for example, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0017] Techniques for introducing mutations such as deletion, substitution, insertion, or addition of one or more amino acids into a specific amino acid sequence are known in the art and can be carried out by any method, such as restriction enzyme treatment, treatment with exonuclease or DNA ligase, site-directed mutagenesis, random mutagenesis, etc.

[0018] A polypeptide consisting of an amino acid sequence showing 85% or more identity with the amino acid sequence shown in SEQ ID NO: 1 preferably suppresses the expression of at least one gene selected from the group consisting of COL1A1, COL1A2, and αSMA. A polypeptide consisting of an amino acid sequence showing 85% or more identity with the amino acid sequence shown in SEQ ID NO: 1 may suppress the expression of only one gene, or may suppress the expression of two or more genes. For example, it may suppress the expression of the gene COL1A1, COL1A2, or αSMA, or it may suppress the expression of the gene COL1A1 and COL1A2, COL1A1 and αSMA, or it may suppress the expression of the gene COL1A1, COL1A2, and αSMA.

[0019] COL1A1 is a protein that produces a component of type I collagen called the pro-α1 chain. COL1A1 is known as a hepatic stellate cell activation-related factor. COL1A2 is a protein that produces a component of type I collagen called the pro-α2 chain. COL1A2 is known as a hepatic stellate cell activation-related factor. Pro-type I collagen is formed by the combination of two α1 chains and one α2 chain.

[0020] αSMA (α-smooth muscle actin) is an actin protein expressed in smooth muscle. αSMA is also called ACTA2. αSMA is known as a hepatic stellate cell activation-associated factor.

[0021] The expression of the COL1A1, COL1A2, and αSMA genes is confirmed by measuring the amount of mRNA of the genes encoding the respective proteins by quantitative PCR, more specifically, by the method described in the Examples. For example, if the addition of a polypeptide consisting of an amino acid sequence that shows 85% or more identity to the amino acid sequence shown in SEQ ID NO:1 reduces the amount of mRNA of the COL1A1, COL1A2, or αSMA gene compared to when the polypeptide is not added, it is determined that the expression of the gene is suppressed.

[0022] A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide consisting of an amino acid sequence showing 85% or more identity to the amino acid sequence shown in SEQ ID NO: 1, can be produced using a general protein chemical synthesis method (e.g., liquid phase method and solid phase method) based on the information of the amino acid sequence shown in SEQ ID NO: 1. In addition, the above-mentioned polypeptide can be produced by genetic engineering techniques using a polynucleotide encoding the polypeptide.

[0023] The fibrosis therapeutic agent of the present disclosure also includes a fibrosis therapeutic agent containing an expression vector incorporating a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1, or a polypeptide consisting of an amino acid sequence showing 85% or more identity to the amino acid sequence shown in SEQ ID NO:1.

[0024] The base sequence of the polynucleotide is not particularly limited, so long as it encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1, or a polypeptide consisting of an amino acid sequence that shows 85% or more identity to the amino acid sequence shown in SEQ ID NO:1.

[0025] The polynucleotide is preferably a polynucleotide that is compatible with the codon usage frequency in humans. "Compatible with the codon usage frequency in humans" means that when there are multiple codons corresponding to one amino acid, the codon most frequently used in humans is used. It is preferable that 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the open reading frame of the above-mentioned polynucleotide is a polynucleotide that is compatible with the codon usage frequency in humans.

[0026] Polynucleotides can be easily obtained by chemical polynucleotide synthesis methods (eg, the phosphoramidite method) or genetic engineering techniques.

[0027] The vector preferably contains the polynucleotide in an expressible manner. Examples of the vector used in the present disclosure include viral vectors such as retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors; plasmid vectors, etc.

[0028] In the fibrosis therapeutic agent of the present disclosure, the content of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or the polypeptide consisting of an amino acid sequence showing 85% or more identity with the amino acid sequence shown in SEQ ID NO: 1, or the expression vector incorporating a polynucleotide encoding the polypeptide is not particularly limited as long as it suppresses the expression of at least one gene selected from the group consisting of COL1A1, COL1A2, and αSMA. For example, it can be 0.001 to 99% by mass. The fibrosis treatment agent of the present disclosure preferably contains as an active ingredient a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide consisting of an amino acid sequence showing 85% or more identity to the amino acid sequence shown in SEQ ID NO: 1, or an expression vector incorporating a polynucleotide encoding the polypeptide.

[0029] The fibrosis therapeutic agent of the present disclosure may contain known components that can be contained in a pharmaceutical composition in addition to the above-mentioned components. Examples of such components include pharma- ceutically acceptable bases, carriers, and / or additives (e.g., solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, antioxidants, preservatives, coating agents, colorants, gastric mucosa protective agents, other antifibrotic agents, etc.). Such known components may be used alone or in combination of two or more.

[0030] The form of the fibrosis therapeutic agent of the present disclosure is not particularly limited, and examples thereof include tablets, pills, granules, fine granules, powders, capsules, chewable tablets, pellets, troches, jellies, syrups, liquids, suspensions, inhalants, injections, suppositories, retention enemas, and nasal drops.

[0031] Examples of fibrosis include fibrosis in organs or tissues, such as liver fibrosis, pulmonary fibrosis (interstitial pneumonia), renal fibrosis, myocardial fibrosis, and bone marrow fibrosis.

[0032] The subject to which the fibrosis therapeutic agent is administered is preferably a mammal. It may be not only a human but also a non-human mammal. Examples of non-human mammals include mammals kept as pets, livestock, laboratory animals, etc. Examples of such non-human mammals include dogs, cats, monkeys, cows, horses, sheep, goats, pigs, rabbits, mice, rats, camels, llamas, etc.

[0033] Humans to which the pharmaceutical composition may be administered include, for example, patients with fibrosis in organs or tissues, such as hepatic fibrosis, pulmonary fibrosis (interstitial pneumonia), renal fibrosis, myocardial fibrosis, and myelofibrosis, or those suspected of having these diseases; and humans in which expression of at least one gene selected from the group consisting of COL1A1, COL1A2, and αSMA is enhanced.

[0034] The administration method may be, for example, oral administration or parenteral administration (for example, intravenous, intraarterial, intramuscular, subcutaneous, peritoneal, rectal, transdermal, topical, etc.).

[0035] The administration (ingestion) amount of the fibrosis therapeutic agent of the present disclosure is not particularly limited and is determined depending on the age, sex, severity of symptoms, method of ingestion, etc. of the subject to be ingested.

[0036] According to the present disclosure, it is possible to suppress the expression of at least one gene selected from the group consisting of COL1A1, COL1A2, and αSMA, which are involved in fibrosis, and therefore it is possible to suppress fibrosis in organs, tissues, etc. Therefore, the fibrosis therapeutic agent of the present disclosure can be used for the treatment of fibrosis. In addition, according to the present disclosure, the expression of at least one gene selected from the group consisting of COL1A1, COL1A2, and αSMA, which are hepatic stellate cell activation-related factors, can be suppressed, thereby suppressing the activation of hepatic stellate cells. According to the present disclosure, the expression of at least one gene selected from the group consisting of COL1A1, COL1A2, and αSMA, which are hepatic stellate cell activation-related factors, can be suppressed, thereby suppressing liver fibrosis. Therefore, the fibrosis treatment drug of the present disclosure can be used particularly for the treatment of liver fibrosis.

[0037] In addition, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes all arbitrary combinations of the constituent features described in this specification.

[0038] In addition, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter included in the present disclosure. In other words, the present disclosure includes all subject matter consisting of all combinations of each combinable characteristic described in this specification. EXAMPLES

[0039] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, the experiments are performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "volume %".

[0040] [ka]

[0041] Lawsone (2-hydroxy-1,4-naphthoquinone) represented by formula (1), which is known to suppress COL1A1 and COL1A2 expression (Patent Document 1), was immobilized on epoxy beads (TAS8848N1110, Tamagawa Seiki) (the immobilization method was performed according to the Tamagawa Seiki protocol). The beads were diluted to 1.0x10 using RIPA buffer (Abcam). 7 Proteins were extracted from primary cultured hepatic stellate cells (HHSteCs). 200 μg of extracted protein was diluted with binding buffer (20 mM HEPES-NaOH (pH 7.9), 150 mM KCl, 1 mM MgCl2, 0.2 mM CaCl2, 0.2 mM EDTA, 10% (v / v) glycerol, 0.1% NP-40, 0.2 mM PMSF) to adjust the concentration to 1 mg / mL and reacted with Lawsone-immobilized beads overnight. The next day, the beads were fixed with a magnet and washed three times with binding buffer. Proteins bound to the beads were eluted with 0.1 M glycine-HCl (pH 2.5) and neutralized with 1 M Tris-HCl (pH 9.0). The proteins were separated by SDS-PAGE and visualized by silver staining (Fuji Film Wako). The detected proteins were analyzed by MALDI-MS (contracted to the Osaka City University Artificial Photosynthesis Research Center). The results are shown in Figure 1A-C.

[0042] 1.0x10 5Primary cultured hepatic stellate cells (HHSteC) were seeded on the wells. The cells were cultured for 1 day at 37°C and 5% CO2 in D-MEM medium (Fujifilm Wako) supplemented with 2% fetal bovine serum (Thermo Fisher Scientific) and 1% SteCGS (ScienCell Research Laboratories). pCMV6-hHSD17B4 plasmid was introduced at 0, 0.3, or 1.0 μg / well using Lipofectamine 3000 (Thermo Fisher Scientific). The amount of DNA introduced was adjusted using pCMV6-Empty plasmid. The next day, the medium was replaced with D-MEM medium supplemented with 0.2% fetal bovine serum, and the cells were cultured for another 2 days at 37°C and 5% CO2. The RNA levels of hepatic stellate cell activation-related factors COL1A1, COL1A2, and αSMA, and CYGB (cytoglobin), a hepatic stellate cell activation inhibitor, were determined using quantitative PCR. Protein levels were determined using Western blotting. The results are shown in Figure 2A and B.

[0043] The mRNA levels of COL1A1, COL1A2, and αSMA were attenuated in a dose-dependent manner by the HSD17B4 plasmid, whereas no change was observed in the mRNA level of CYGB (Fig. 2A). Similar to the mRNA levels, the protein levels of COL1A1, COL1A2, and αSMA were attenuated in an HSD17B4 plasmid dose-dependent manner, whereas the protein level of CYGB was unchanged (FIG. 2B).

[0044] 1.0x10 5Primary cultured hepatic stellate cells (HHSteC) were seeded on the cells. The cells were cultured for 1 day at 37°C and 5% CO2 in D-MEM medium (Fujifilm Wako) supplemented with 2% fetal bovine serum (Thermo Fisher Scientific) and 1% SteCGS (ScienCell Research Laboratories). hHSD17B4 siRNA (Dharmacon) was transfected at 0, 3, or 10 nM using Lipofectamine RNAi Max (Thermo Fisher Scientific). The amount of RNA transfected was adjusted with control siRNA (Thermo Fisher Scientific). The next day, the medium was replaced with D-MEM medium supplemented with 0.2% fetal bovine serum, and the cells were cultured for another 2 days at 37°C and 5% CO2. RNA levels of COL1A1, COL1A2, αSMA, and CYGB were determined using quantitative PCR. Protein levels were determined using Western blotting. The results are shown in Figures 3A and B.

[0045] As expected, the mRNA levels of COL1A1, COL1A2, and αSMA were increased in a dose-dependent manner by HSD17B4-siRNA (Fig. 3A), whereas the mRNA level of CYGB was increased contrary to expectations (Fig. 3A). Similar to mRNA, the protein levels of COL1A1, COL1A2, αSMA, and CYGB were increased in a dose-dependent manner by HSD17B4-siRNA. Furthermore, since no change in CYGB expression levels was observed when a plasmid that forcibly expressed HSD17B4 was introduced, it was presumed that the increase in CYGB expression levels due to introduction of HSD17B4 siRNA occurred through a pathway other than HSD17B4 (a side reaction of HSD17B4 siRNA).

[0046] The rhHSD17B4 protein was prepared as follows. The pET30a-hHSD17B4 plasmid was introduced into E. coli strain BL21 (Takara) to express the rhHSD17B4 protein (His-Tag and S-Tag fused to the N-terminus). The rhHSD17B4 protein was bound to a Ni-NTA agarose column using the His-Tag and eluted with imidazole. The rhHSD17B4 protein was further purified by dialysis against dialysis buffer and used in the following experiments.

[0047] 1.0x10 5 Primary cultured hepatic stellate cells (HHSteC) were seeded. The cells were cultured for 1 day at 37 °C and 5% CO2 in D-MEM medium (Fujifilm Wako) supplemented with 2% fetal bovine serum (Thermo Fisher Scientific) and 1% SteCGS (ScienCell Research Laboratories). The medium was replaced with D-MEM medium supplemented with 0.2% fetal bovine serum, and 40 μg / mL of rhHSD17B4 protein was added. As a control, the same amount of dialysis buffer (solution used in purification: 10 mM Kpi (pH 7.4), 0.1 mM DTT, 10% Glycerol, 0.025% Tween-20) as the amount of rhHSD17B4 protein solution was added. The cells were then cultured for 2 days at 37 °C and 5% CO2. The RNA amounts of COL1A1, COL1A2, and αSMA were determined using quantitative PCR. The results are shown in Figure 4. Note that Figure 4 also shows a control (CNT) that was treated in the same manner except that rhHSD17B4 protein was not added.

[0048] Addition of rhHSD17B4 protein to the medium attenuated the mRNA levels of COL1A1, COL1A2, and αSMA.

Claims

1. (A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or (B) a polypeptide having an amino acid sequence showing 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1, and suppressing the expression of at least one gene selected from the group consisting of COL1A1 and COL1A2. A therapeutic agent for fibrosis comprising the compound.

2. A therapeutic agent for fibrosis, comprising an expression vector incorporating a polynucleotide encoding the polypeptide (A) or the polypeptide (B) according to claim 1.

3. The fibrosis therapeutic agent according to claim 1 or 2, wherein the fibrosis is liver fibrosis.

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