Pharmaceutical composition for preventing or treating liver disease associated with fibrosis

Abstract

To provide a medicine for preventing or treating a liver disease associated with fibrosis, which contains micro RNA as an active ingredient.SOLUTION: A pharmaceutical composition for preventing or treating a liver disease associated with fibrosis contains at least one kind of micro RNA selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089 and miR 5787 as an active ingredient.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for preventing or treating liver diseases accompanied by fibrosis, extracellular vesicles, a culture, and a method for producing extracellular vesicles for suppressing liver fibrosis.

Background Art

[0002] Liver fibrosis (hepatic fibrosis) mainly occurs along with inflammation caused by hepatitis virus or alcohol, and is particularly likely to occur when the inflammation becomes chronic. It is known that hepatocellular carcinoma is likely to develop from chronic hepatitis or liver cirrhosis accompanied by liver fibrosis. So far, although promising antiviral drugs have been developed for viral cirrhosis or liver cancer, they are not curative.

[0003] In recent years, along with changes in lifestyle, liver disorders, precancerous lesions such as hepatitis or liver cirrhosis, and liver cancer caused by chronic metabolic abnormalities due to excessive alcohol consumption, overeating, and lack of exercise have been increasing. Since cirrhosis or liver cancer caused by metabolic abnormalities is related to lifestyle diseases such as obesity and diabetes, it has become a social problem. However, at present, the only treatment methods are symptomatic treatments such as diet and exercise therapy.

[0004] The present inventors have hitherto advanced the research on liver fibrosis inhibitors and disclosed a liver fibrosis inhibitor containing dantrolene or a pharmaceutically acceptable salt thereof as an active ingredient (see Patent Document 1).

[0005] In recent years, methods for diagnosing cancer by measuring microRNA have been developed. MicroRNA (hereinafter also referred to as "miRNA or miR") is an RNA approximately 18 to 25 bases in length that exists within cells and is a type of non-coding RNA thought to have the function of regulating the expression of other genes. To date, a marker for diagnosing colorectal cancer consisting of microRNA molecules has been disclosed (see Patent Document 2). Also, exosomes containing a predetermined microRNA have been disclosed for the purpose of treating various diseases (see Patent Document 3). Furthermore, regarding microRNA and liver fibrosis, for example, it has been disclosed that miR 6848-5p and miR 6848-3p are involved in liver fibrosis (see Patent Document 4). However, the relationship between microRNA and fibrosis in the liver has not been sufficiently studied and has not yet reached practical application at present.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a pharmaceutical composition containing microRNA or a precursor thereof as an active ingredient for preventing or treating liver diseases accompanied by fibrosis.

Means for Solving the Problems

[0008] The inventors of the present invention have been conducting research on liver regeneration therapy using mesenchymal stem cells, particularly liver cirrhosis regeneration therapy. In the search for a method for producing mesenchymal stem cells optimized for the regeneration of cirrhotic liver, it has been found that a predetermined microRNA is deeply involved in suppressing liver fibrosis, and the present invention has been completed.

[0009] That is, the present invention is as follows. 〔1〕A pharmaceutical composition for preventing or treating a liver disease accompanied by fibrosis, comprising at least one microRNA selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089 and miR 5787 or a precursor thereof as an active ingredient. 〔2〕The pharmaceutical composition according to the above 〔1〕, characterized by containing a medium retaining the microRNA or a precursor thereof. 〔3〕The pharmaceutical composition according to the above 〔2〕, wherein the medium retaining the microRNA or a precursor thereof is an extracellular vesicle selected from exosomes, microvesicles, liposomes (lipid bilayer) or apoptotic bodies. 〔4〕The pharmaceutical composition according to the above 〔3〕, wherein the extracellular vesicle is an extracellular vesicle derived from mesenchymal stem cells. 〔5〕The extracellular vesicle is an extracellular vesicle derived from a cell into which a polynucleotide encoding at least one microRNA selected from exogenous miR 1237-5p, miR 204-3p, miR 7977, miR 6089 and miR 5787 or a precursor thereof has been introduced, and the microRNA or a precursor thereof is expressed. The pharmaceutical composition according to the above 〔3〕 or 〔4〕. 〔6〕The pharmaceutical composition according to the above 〔1〕 or 〔2〕, which is administered to a subject in combination with mesenchymal stem cells. 〔7〕A polynucleotide encoding at least one microRNA selected from exogenous miR-1237-5p, miR-204-3p, miR-7977, miR-6089, and miR-5787 or their precursors is introduced into a cell, and the extracellular vesicles derived from the cells expressing the microRNA or their precursors, wherein the extracellular vesicles contain at least one microRNA selected from miR-1237-5p, miR-204-3p, miR-7977, miR-6089, and miR-5787 or their precursors. 〔8〕The extracellular vesicles according to the above 〔7〕, wherein the extracellular vesicles are extracellular vesicles derived from mesenchymal stem cells. 〔9〕A culture obtained by culturing cells into which a polynucleotide encoding at least one microRNA selected from exogenous miR-1237-5p, miR-204-3p, miR-7977, miR-6089, and miR-5787 or their precursors is introduced and the at least one microRNA or their precursors are expressed. 〔10〕Step A: Introducing a polynucleotide encoding at least one microRNA selected from exogenous miR-1237-5p, miR-204-3p, miR-7977, miR-6089, and miR-5787 or their precursors into mesenchymal stem cells; Step B: Culturing the mesenchymal stem cells obtained in Step A to obtain a culture; Step C: Recovering extracellular vesicles from the culture; A method for producing extracellular vesicles for suppressing liver fibrosis, comprising Steps A to C, and containing at least one microRNA selected from exogenous miR-1237-5p, miR-204-3p, miR-7977, miR-6089, and miR-5787 or their precursors.

[0010] Also, as another aspect of the present invention, (I) a method for preventing or treating a liver disease accompanied by fibrosis, comprising administering an effective amount of at least one microRNA selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787 or their precursors to a subject in need thereof, (II) the use of at least one microRNA selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787 or their precursors, and polynucleotides encoding them, for the manufacture of a pharmaceutical composition for preventing or treating a liver disease accompanied by fibrosis, and (III) at least one microRNA selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787 or their precursors, and polynucleotides encoding them, for use in preventing or treating a liver disease accompanied by fibrosis can be mentioned. Here, "effective amount" means the amount required for administration to expect a preventive or therapeutic effect in a subject. Therefore, it can be adjusted according to the severity of the disease of the administration subject, the dosage form to be administered, age, weight, sex, administration time, administration route, treatment period, and the like.

Effects of the Invention

[0011] By using the pharmaceutical composition of the present disclosure, it becomes possible to prevent or treat a liver disease accompanied by fibrosis.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 3E

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0013] As a pharmaceutical composition for preventing or treating liver diseases accompanied by fibrosis in the present specification, there is no particular limitation as long as it is a pharmaceutical composition for preventing or treating liver diseases accompanied by fibrosis, which contains at least one microRNA selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787 (hereinafter also referred to as "the present microRNA") or a precursor thereof as an active ingredient, and is hereinafter also referred to as "the present pharmaceutical composition". In addition, as the extracellular vesicles in the present specification, there is no particular limitation as long as they are extracellular vesicles derived from cells into which a polynucleotide encoding at least one microRNA selected from exogenous miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787 or a precursor thereof is introduced and the microRNA or a precursor thereof is expressed, and which contain at least one microRNA selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787 or a precursor thereof, and are hereinafter also referred to as "the present extracellular vesicles". Furthermore, as the culture in the present specification, there is no particular limitation as long as it is a culture obtained by culturing cells into which a polynucleotide encoding at least one microRNA selected from exogenous miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787 or a precursor thereof is introduced and the microRNA or a precursor thereof is expressed, and is hereinafter also referred to as "the present culture".In addition, as a method for producing extracellular vesicles for suppressing liver fibrosis in the present specification, step A of introducing a polynucleotide encoding at least one microRNA selected from exogenous miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787 or their precursors into mesenchymal stem cells; step B of culturing the mesenchymal stem cells obtained in step A to obtain a culture; step C of recovering extracellular vesicles from the culture; A method for producing extracellular vesicles for suppressing liver fibrosis, which comprises steps A to C and contains at least one microRNA selected from exogenous miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787 or their precursors, is not particularly limited, and is hereinafter also referred to as "the method for producing the extracellular vesicles of the present case".

[0014] Here, the microRNA of the present case may be a single-stranded mature microRNA or a double-stranded microRNA having a base sequence complementary to the single-stranded mature microRNA. Among the microRNAs of the present case, the polynucleotides of the single-stranded mature microRNAs have the sequences shown in Table 1 below, respectively. The numbers in parentheses are the IDs and Accession Nos. of miRBase (https: / / mirbase.org / ).

[0015]

Table 1

[0016] In addition, the microRNA of the present case also includes variants of the single-stranded mature microRNA shown in the following (a) or (b). (a) A polynucleotide in which one or several bases are deleted, added, or substituted in the base sequence shown in any of SEQ ID NOs: 1 to 5, and the transcription product has an action of suppressing liver fibrosis (b) A polynucleotide having a sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more with the nucleotide sequence shown in any of SEQ ID NOs: 1 to 5, wherein the transcription product has an inhibitory effect on liver fibrosis

[0017] In the above (a) or (b), whether it has an inhibitory effect on liver fibrosis can be determined, for example, by using a polynucleotide in which one or several bases are deleted, added, or substituted in the nucleotide sequence shown in any of SEQ ID NOs: 1 to 5, or a polynucleotide having a sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more with the nucleotide sequence shown in any of SEQ ID NOs: 1 to 5. According to the method described in the following examples, the polynucleotide is introduced into hepatic stellate cells (HHSteC) and cultured, and it can be confirmed by whether the expression of fibrosis markers is suppressed. As the above fibrosis markers, one kind selected from ACTA2, COL1A1, ELN, COL1A2, and COL3A1, preferably two kinds, more preferably three kinds, and particularly preferably four kinds can be mentioned for evaluation. Examples of the combination of two kinds include COL1A2 and COL3A1, examples of the combination of three kinds include ACTA2, COL1A2, and COL3A1, and COL1A2, COL3A1, and ELN, and examples of the combination of four kinds include ACTA2, COL1A1, COL1A2, and COL3A1.

[0018] As for the above-mentioned "one or several", 1 to 5 are preferable, 1 to 4 are more preferable, 1 to 3 are further preferable, 1 to 2 are particularly preferable, and 1 is most preferable. Also, it is preferable to retain the seed sequence consisting of 6 to 7 bases at the 2nd to 7th or 8th position counted from the 5' end in the base sequence shown in any of SEQ ID NOs: 1 to 5. That is, as (a), it is a polynucleotide in which the seed sequence is retained in the base sequence shown in SEQ ID NOs: 1 to 5, and one or several bases are deleted, added, or substituted in the base sequence shown in SEQ ID NOs: 1 to 5, and the transcription product has an inhibitory effect on liver fibrosis; as (b), it is a polynucleotide in which the seed sequence is retained in the base sequence shown in SEQ ID NOs: 1 to 5 and has 80% or more sequence identity with the base sequence shown in SEQ ID NOs: 1 to 5, and the transcription product has an inhibitory effect on liver fibrosis.

[0019] The above-mentioned precursor of microRNA is a stem-loop structure, which contains the above single-stranded mature microRNA or its variant in the precursor strand, and means an RNA that can generate mature miRNA by cleavage or cleavage of a double strand, etc. Specifically, pri-miRNA, pre-miRNA, and double-stranded miRNA consisting of mature miRNA and its antisense strand can be mentioned. The length of the precursor of microRNA can be 50 to 100 nt, preferably 65 to 85 nt.

[0020] In order to improve the stability, the above-mentioned microRNA or their precursors may have any number of bases, such as 1 to 15 bases, 1 to 10 bases, 1 to 5 bases, 1 to 3 bases added to the 5' end and / or 3' end. In order to increase the affinity with the complementary strand nucleic acid, it may be an analog modified with a sugar-modified nucleotide analog, a phosphodiester bond-modified nucleotide analog, etc., or a nucleic acid derivative. Furthermore, the present microRNA may also be a microRNA mimic, which is a double-stranded RNA in which the passenger strand is chemically modified.

[0021] As the sugar moiety-modified nucleotide analogs, any chemical substances can be added or substituted for part or all of the chemical structure of the sugar of the nucleotide. Specific examples of the sugar moiety-modified nucleotide analogs include nucleotide analogs substituted with 2'-O-methylribose, nucleotide analogs substituted with 2'-O-propylribose, nucleotide analogs substituted with 2'-methoxyethoxyribose, nucleotide analogs substituted with 2'-O-methoxyethylribose, nucleotide analogs substituted with 2'-O-[2-(guanidium)ethyl]ribose, nucleotide analogs substituted with 2'-fluororibose, bridged nucleic acid (BNA) having two cyclic structures by introducing a cross-linked structure into the sugar moiety, more specifically, locked nucleic acid (LNA) in which the oxygen atom at the 2' position and the carbon atom at the 4' position are cross-linked via a methylene group, and ethylene bridged nucleic acid (ENA) [Nucleic Acid Research, 32, e175 (2004)]. Furthermore, peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxy peptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], and peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)] and the like can be mentioned.

[0022] As the phosphodiester bond-modified nucleotide analogs, those obtained by adding or substituting any chemical substances for part or all of the chemical structure of the phosphodiester bond of the nucleotide can be mentioned. Specific examples of the phosphodiester bond-modified nucleotide analogs include nucleotide analogs substituted with phosphorothioate bonds, nucleotide analogs substituted with N3'-P5' phosphoramidate bonds, etc. [Cell Engineering, 16, 1463-1473 (1997)] [RNAi Method and Antisense Method, Kodansha (2005)].

[0023] As the above nucleic acid derivative, any molecule may be used as long as it is a molecule in which another chemical substance is added to the nucleic acid in order to improve nuclease resistance, stabilize, increase affinity for complementary strand nucleic acid, increase cell permeability, or visualize, compared to the nucleic acid. Specific examples include 5'-polyamine-added derivatives, cholesterol-added derivatives, steroid-added derivatives, bile acid-added derivatives, vitamin-added derivatives, Cy5-added derivatives, Cy3-added derivatives, 6-FAM-added derivatives, and biotin-added derivatives.

[0024] The polynucleotide encoding the present microRNA or its precursor may be incorporated into an expression vector. Examples of the expression vector include an expression vector that can be brought into contact with a cell to introduce the polynucleotide encoding the present microRNA or its precursor into the cell and express the predetermined present microRNA or its precursor encoded by the polynucleotide encoding the present microRNA or its precursor in the cell. Specifically, a viral vector or a non-viral vector described below can be mentioned.

[0025] The origin of the present microRNA is not particularly limited, and examples include human, monkey, pig, dog, cat, horse, sheep, mouse, and rabbit.

[0026] In this specification, "fibrosis" in the liver refers to the excessive formation of fibrous connective tissue in the liver caused by various causes such as non-alcoholic steatohepatitis, persistent infection with hepatitis virus, excessive alcohol intake, autoimmune mechanisms, intrahepatic cholestasis, drug-induced, metal metabolism disorders, and congestive liver. The hepatitis that causes fibrosis is not particularly limited, and examples include non-alcoholic steatohepatitis; viral hepatitis such as hepatitis B and hepatitis C; chronic hepatitis; acute hepatitis; alcoholic hepatitis; liver cirrhosis; and hepatitis associated with liver cancer.

[0027] In this specification, the liver diseases are not particularly limited as long as they are liver diseases accompanied by fibrosis, but include chronic hepatitis B, chronic hepatitis C, non-alcoholic steatohepatitis (also called NASH: MASH (metabolic dysfunction-associated steatohepatitis)), non-alcoholic fatty liver (NAFL), and other non-alcoholic fatty liver diseases (NAFLD: also called MASLD (metabolic dysfunction-associated steatotic liver disease)), fatty liver associated with metabolic dysfunction (MASLD), autoimmune diseases, liver cirrhosis, and hepatocellular carcinoma.

[0028] In this specification, "prevention" means preventing or delaying the onset of a disease by administering the pharmaceutical composition to a subject before the onset of the disease, and does not necessarily mean completely suppressing the onset. Specifically, it means suppressing or delaying the progression of fibrosis in the liver by administering the pharmaceutical composition to a subject. Note that the onset of a disease means that the symptoms of the disease appear in the body.

[0029] In this specification, "treatment" means reducing the symptoms of the disease by administering the pharmaceutical composition to a subject after the onset of the disease, and does not necessarily mean completely suppressing the symptoms of the disease. Specifically, if one or more of the pathological conditions of fibrosis or the biological signs of the pathological condition in the liver of the disease can be improved by administering the pharmaceutical composition, it can be said that the liver disease accompanied by fibrosis can be treated using the pharmaceutical composition.

[0030] The present pharmaceutical composition may contain a medium that holds microRNAs or their precursors. The medium that holds microRNAs or their precursors only needs to be able to hold microRNAs or their precursors. Here, holding microRNAs or their precursors can include a state in which all or part of the microRNAs or their precursors are encapsulated, adhered to, or embedded. Examples of the medium that holds the microRNAs or their precursors include extracellular vesicles such as exosomes, microvesicles, liposomes (lipid bilayers), or apoptotic bodies, and micelles, ionizable lipids, and non-cationic lipids, and exosomes are preferred.

[0031] The above exosomes are released from most cell types and can be found in many body fluids, and contain nucleic acids such as microRNAs or mRNAs and proteins. Further, in other preferred embodiments, the above exosomes can include exosomes that are positive for at least one of the cell exosome markers consisting of the group of CD (cluster of differentiation) 9, CD81, CD63, CD41a, CD41b, CD42b, CD61, CD62P, and syntenin.

[0032] The size of the above exosomes can be mentioned as an average particle diameter of 10 to 1000 nm, preferably 20 to 200 nm, and more preferably 30 to 140 nm. The average particle diameter can be measured by dynamic light scattering, interference light microscopy observation, or electron microscopy observation.

[0033] The extracellular vesicles such as exosomes according to the present invention are preferably extracellular vesicles derived from mesenchymal stem cells. Exosomes derived from mesenchymal stem cells are preferred in that they have an anti-inflammatory effect and a wound healing promoting effect, that exosomes can be easily recovered because mesenchymal stem cells can be cultured without serum, and that exosomes derived from mesenchymal stem cells are also highly safe because mesenchymal stem cells have almost no risk of canceration, and they contain various growth factors.

[0034] In addition, the origin of the cells used to prepare the above exosomes is not limited to humans, and other mammals (for example, mice, rats, rabbits, dogs, cats, monkeys, sheep, cows, horses) may also be used.

[0035] As used herein, the term "mesenchymal stem cell" means a population of stem cells or their progenitor cells that can differentiate into all or some of the mesenchymal cells such as osteoblasts, chondroblasts, and adipoblasts. More specifically, dental pulp stem cells, bone marrow-derived stem cells, adipose tissue-derived stem cells, etc. can be mentioned. Further, the above mesenchymal stem cells may be derived from induced pluripotent stem cells (iPS cells). By using iPS cell-derived mesenchymal stem cells, it becomes possible to maintain stable quality without depending on donors.

[0036] The extracellular vesicles of this case are extracellular vesicles derived from cells into which a polynucleotide encoding at least one microRNA selected from exogenous miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787 or their precursors has been introduced and the microRNA or their precursors are expressed. Here, the cells in which the microRNA or their precursors are expressed overexpress the at least one microRNA or their precursors compared to natural cells, for example, 2-fold, 3-fold, or 5-fold or more. The method for introducing a polynucleotide encoding at least one microRNA selected from exogenous miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787 into cells is not particularly limited, and examples include methods of introducing a polynucleotide encoding a microRNA by known genetic engineering techniques. Specifically, transfection using a viral vector, electroporation using a non-viral vector, lipofection, and microinjection can be mentioned.

[0037] Examples of the virus vector include virus vectors such as an adenovirus vector, a retrovirus vector, an adeno-associated virus vector, a poxvirus, a vaccinia virus vector, a baculovirus vector, and a herpes virus vector. Examples of the non-virus vector include a plasmid vector, a phagemid vector, a cosmid vector, and a bacteriophage.

[0038] The extracellular vesicles in this specification may be extracellular vesicles derived from cells into which a polynucleotide encoding two kinds of exogenous microRNAs or their precursors has been introduced and the microRNAs or their precursors have been expressed. Examples of the two kinds of exogenous microRNAs include miR 1237-5p and miR 204-3p; miR 1237-5p and miR 7977; miR 1237-5p and miR 6089; miR 1237-5p and miR 5787; miR 204-3p and miR 7977; miR 204-3p and miR 6089; miR 204-3p and miR 5787; miR 7977 and miR 6089; miR 7977 and miR 5787; miR 6089 and miR 5787; can be mentioned.

[0039] The extracellular vesicles in this specification may be extracellular vesicles derived from cells into which a polynucleotide encoding three kinds of exogenous microRNAs or their precursors has been introduced and the microRNAs or their precursors have been expressed. Examples of the three kinds of exogenous microRNAs include miR 1237-5p, miR 204-3p and miR 7977; miR 1237-5p, miR 204-3p and miR 6089; miR 1237-5p, miR 204-3p and miR 5787; miR 1237-5p, miR 7977, and miR 6089; miR 1237-5p, miR 7977, and miR 5787; miR 1237-5p, miR 6089, and miR 5787; miR 204-3p, miR 7977, and miR 6089; miR 204-3p, miR 7977, and miR 5787; miR 204-3p, miR 6089, and miR 5787; miR 7977, miR 6089, and miR 5787; can be mentioned.

[0040] The extracellular vesicles in this specification may be extracellular vesicles derived from cells into which a polynucleotide encoding four kinds of foreign microRNAs or their precursors has been introduced and the microRNAs or their precursors have been expressed. As the four kinds of foreign microRNAs, miR 1237-5p, miR 204-3p, miR 7977, and miR 6089; miR 1237-5p, miR 204-3p, miR 7977, and miR 5787; miR 1237-5p, miR 204-3p, miR 5787, and miR 6089; miR 1237-5p, miR 7977, miR 6089, and miR 5787; miR 204-3p, miR 7977, miR 6089, and miR 5787; can be mentioned.

[0041] The extracellular vesicles in this specification may be extracellular vesicles derived from cells into which a polynucleotide encoding five kinds of foreign microRNAs or their precursors has been introduced and the microRNAs or their precursors have been expressed. As the five kinds of foreign microRNAs, miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787; can be mentioned.

[0042] This pharmaceutical composition may be used in combination with mesenchymal stem cells or in combination with a pharmaceutical for preventing or treating other liver diseases accompanied by fibrosis. Examples of the method of "using in combination with mesenchymal stem cells" include a method of treating with mesenchymal stem cells and then using this pharmaceutical composition, a method of using this pharmaceutical composition and mesenchymal stem cells simultaneously, and a method of treating with this pharmaceutical composition and then using mesenchymal stem cells. Similarly, examples of the method of "using in combination with a pharmaceutical for preventing or treating other liver diseases accompanied by fibrosis" include a method of treating with a pharmaceutical for preventing or treating other liver diseases accompanied by fibrosis and then using this pharmaceutical composition, a method of using this pharmaceutical composition and a pharmaceutical for preventing or treating other liver diseases accompanied by fibrosis simultaneously, and a method of treating with this pharmaceutical composition and then using a pharmaceutical for preventing or treating other liver diseases accompanied by fibrosis.

[0043] In this culture, the "culture" refers to a product obtained by introducing a polynucleotide encoding at least one microRNA selected from exogenous miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787 or their precursors into cells to express the at least one microRNA or their precursors, and culturing the cells in a known liquid medium or solid medium, which means a state containing the cultured cells. It is also possible to remove the cells from the above culture to obtain a culture solution. This culture solution contains extracellular vesicles derived from the cultured cells. And among the extracellular vesicles, some contain at least one microRNA selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787 according to the introduced polynucleotide.

[0044] As the medium for the above-mentioned culturing, it can be appropriately adjusted according to the cells to be cultured. For example, in the case of mesenchymal stem cells, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), StemFit (registered trademark) For Mesenchymal Stem Cell (MSC) can be mentioned.

[0045] Extracellular vesicles can be recovered from the present culture. Such methods include ultracentrifugation in which the medium is centrifuged to remove cells and contaminants and the supernatant is recovered, polymer precipitation method, immunoprecipitation method, and methods using commercially available kits for recovering extracellular vesicles. The ultracentrifugation method can recover extracellular vesicles by centrifugation at 500 - 3500 rpm for 2 - 60 minutes in advance and then ultracentrifugation (for example, 100000 - 1000000 G, 30 minutes - 12 hours). Thereafter, it may be purified by filtration through a filter or the like.

[0046] The present pharmaceutical composition may contain appropriate diluents, buffers, suspending agents, dispersing agents, preservatives, stabilizers, and excipients commonly used in the manufacture of drugs or pharmaceutical compositions. Also, the form of the present pharmaceutical composition can be a parenteral preparation such as an injection or infusion, or an oral preparation.

[0047] The present pharmaceutical composition can be administered via various routes including subcutaneous, intramuscular, intravenous, transdermal, or oral. The preferred dosage can be appropriately determined by those skilled in the art in light of various relevant factors such as the patient's age, sex, weight, health status, and severity of the disease. Specifically, as the weight of the microRNA or their precursors as the active ingredient, 0.01 - 500 mg / 60 kg body weight per day, preferably 0.1 - 10 mg / 60 kg body weight can be mentioned. Also, as the dosage of extracellular vesicles, 10 mg - 25 g / 60 kg body weight per day, preferably 100 mg - 5 g / 60 kg body weight can be mentioned.

[0048] Examples of the administration target of the present pharmaceutical composition include patients diagnosed with liver diseases accompanied by fibrosis and patients diagnosed with suspected liver diseases accompanied by fibrosis.

[0049] All the contents described in all patent documents and non-patent documents cited in this specification are incorporated herein by reference in their entirety.

[0050] Hereinafter, the present invention will be described more specifically by way of examples. However, the technical scope of the present invention is not limited to these illustrations.

[0051] [Example 1] miRNAs highly expressed in extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) First, among 2632 types of miRNAs, miRNAs highly expressed in each extracellular vesicle were analyzed by array using commercially available extracellular vesicles derived from MSCs and extracellular vesicles derived from MSCs of liver cirrhosis cases.

[0052] Extracellular vesicles (MSC-EVs) derived from MSCs of liver cirrhosis cases were collected by the following method. · MSCs from 3 liver cirrhosis cases were cultured for 2 days in a medium partially modified based on StemFit For Mesenchymal Stem Cell (Ajinomoto Co., Inc.). · After changing the medium (DMEM+F12 medium: Thermo Fisher Scientific) and culturing for 2 days, the culture supernatant was collected. · The collected culture supernatant was centrifuged at 500G, 5 min, 4°C, and then the supernatant was collected. · Further centrifuged at 2000G, 30 min, 4°C, and then the supernatant was collected. · The collected supernatant was filtered through a PVDF 0.22 μm filter. · An equal amount of 16% PEG+NaCl was added to the filtered solution and left standing at 4°C overnight. · The solution left standing overnight was centrifuged at 3300G, 60 min, 4°C, and then the supernatant was removed. · Add 1 mL of phosphate buffered saline (PBS) thereto and perform ultracentrifugation at 100,000 G for 70 min at 4°C. · Remove the supernatant, add an appropriate amount of PBS, shake and mix for about 30 min to obtain MSC-EVs of cirrhotic cases as an extracellular vesicle suspension.

[0053] Next, array analysis was performed based on MSC-EVs of three commercially available healthy individuals (26-year-old male, 29-year-old female, 30-year-old male) and MSC-Evs of the above three cirrhotic cases. For the microarray analysis, MSC-EVs were treated with QIAzol Lysis Reagent (QIAGEN) and performed using an oligo DNA probe for microRNA detection (Toray Industries). High expression of miRNA was determined by the signal intensity (1000 or more) after normalization by global normalization.

[0054] As a result, 39 types, 52 types, and 63 types of miRNAs were confirmed to be highly expressed in MSC-EVs of three commercially available healthy individuals, respectively, and 39 types of miRNAs were obtained as those highly expressed in all three individuals. In addition, 45 types, 79 types, and 79 types of miRNAs were confirmed to be highly expressed in MSC-Evs of the above three cirrhotic cases, respectively, and 40 types of miRNAs were obtained as those highly expressed in all three individuals. Then, 36 types of miRNAs were confirmed to be highly expressed in common between the 39 types obtained by the array analysis of commercially available MSC-EVs and the 40 types obtained by the array analysis of MSC-EVs of cirrhotic cases.

[0055] [Example 2] Selection of miRNAs effective for fibrosis suppression According to Example 1, 36 types of miRNAs highly expressed in EVs derived from MSC of healthy individuals and cirrhotic cases were confirmed. Next, among these, miRNAs with low intracellular expression in hepatic stellate cells (HHSteC), which become a factor of fibrosis when activated, were examined.

[0056] The intracellular miRNA expression in hepatic stellate cells (HHSteC) was performed by microarray. The microarray analysis was carried out by treating HHSteC with the RNeasy® Mini Kit (QIAGEN) and using an oligo DNA probe for microRNA detection (Toray Industries). High miRNA expression was determined by the signal intensity (1000 or more) after normalization by global normalization.

[0057] As a result of the microarray analysis, among 36 miRNAs highly expressed in the MSC-derived EVs of healthy individuals and liver cirrhosis cases obtained in Example 1, 11 miRNAs (miR-1908-5p, miR-204-3p, miR-3960, miR-6126, miR-7977, miR-1237-5p, miR-5787, miR-6075, miR-6089, miR-6893-5p, miR-7704) were confirmed to be lowly expressed in HHSteC cells. The results of the RNA expression analysis by microarray in Example 1 and this example for the above 11 types are shown in Table 2.

[0058] [Table 2]

[0059] [Example 3] By Examples 1 and 2, 11 miRNAs highly expressed in extracellular vesicles (EVs) derived from mesenchymal stem cells (MSC) and effective in suppressing fibrosis were selected. It was confirmed whether these miRNAs were expressed in mesenchymal stem cells derived from iPS cells (hereinafter also referred to as "iMSC").

[0060] First, human iPS cells were cultured in a medium partially modified based on StemFit For Mesenchymal Stem Cell (Ajinomoto) to obtain iMSC, mesenchymal stem cells derived from iPS cells. Next, extracellular vesicles (MSC-EVs) were collected in the same manner as in Example 1, and microarray analysis was performed in the same manner as in Example 2. The results are shown in Table 3.

[0061]

Table 3

[0062] From Table 3, it was confirmed that the above 11 types of microRNAs were highly expressed also in iMSC.

[0063] [Example 4] Selection of miRNAs Effective for Suppressing Fibrosis Among the 11 types of miRNAs obtained in Example 2, it was not clear whether they suppress or enhance the fibrosis of HHSteC cells. Therefore, it was examined whether each miRNA has an effect of suppressing fibrosis. Specifically, a cocktail of 11 types of miRNAs was prepared, and if fibrosis was enhanced in a group of 10 types of miRNAs excluding only one type of miRNA among them compared to the group of 11 types of miRNAs, the removed miRNA was judged to have an effect of suppressing fibrosis and was selected. The selection was specifically performed by the following method.

[0064] (1) A miR cocktail (total 10 pmol) was prepared by mixing 11 types of miRNAs or 10 types of miRNAs excluding one type from the above 11 types in a PBS solution, and 50 μL of DMEM containing the same was adjusted. (2) 3 μL of Lipofectamin RNAiMax (Thermo Fisher Scientific) was mixed with 50 μL of DMEM. (3) The two above (1) and (2) were mixed and left at room temperature for 5 minutes or more. (4) Hepatic stellate cells (HHSteC) were collected, further activated with transforming growth factor (TGF)-β, and then seeded at 5×10 4 cells each in a 24-well plate (DMEM + 10% Exofree FBS; 500 μL + TGF-β (5 μg / mL)) (5) 50 μL each of the turbid liquid left at room temperature in (3) was administered to the seeded plates. (Final concentration; RNA: 5 pmol / well, Lipofectamine: 1.5 μL / well) (6) Incubated at 37°C for 2 days, collected the incubated samples, and evaluated the fibrosis markers (ACTA2, COL1A1, ELN) by real-time PCR.

[0065] Figure 1 shows the relative ratios of each of the 10 miRNA groups when the fold change of the 11 miRNA groups is set to 1.0. Note that ACTA2 is a gene expressed when hepatic stellate cells are activated, COL1A1 is a fibrosis-related gene, and ELN is a major component of elastic fibers. It can be evaluated that if the expression of any of them is suppressed, it will lead to the suppression of fibrosis.

[0066] Based on the results of Figure 1, B, E, F, G, I (miR-204-3p, miR-7977, miR-1237-5p, miR-5787, miR-6089) were selected as candidates for miRNAs effective in suppressing fibrosis.

[0067] [Example 5] Fibrosis suppression effect by the selected 5 miRNAs The fibrosis suppression effect in HHSteC was confirmed using the 5 miRNAs selected in Example 4. HHSteC cells were used in the same manner as in Example 4. The cells were divided into the following 4 groups, and the fibrosis suppression effect by 5 miRNA groups or 4 miRNA groups was examined. Control group: HHSteC (2×10 4 cells) TGF-β group: HHSteC (2×10 4 cells) + TGF-β (2 ng / mL) 5 miRNA group: HHSteC (2×10 4 cells) + TGF-β (2 ng / mL) + miR-1237-5p, miR-204-3p, miR-7977, miR-6089, miR-5787 (B + E + F + G + I: 0.28 μg each, Total 1.4 μg) 4 miRNA group: HHSteC (2×10 4(number) + TGF-β (2 ng / mL) + 4 out of miR-1237-5p, miR-204-3p, miR-7977, miR-6089, miR-5787 (4 combinations out of B, E, F, G, I: 0.35 μg each, Total 1.4 μg)

[0068] Figure 2 shows the relative ratios of the TGF-β group, the 5 miRNA group, and the 4 miRNA group when the Fold change of the control group is set to 1.0

[0069] From Figure 2, it was found that the expression of all genes was suppressed in the 5 miRNA group even in the presence of TGF-β. In particular, ACTA2 and ELN, which are significantly involved as factors in liver fibrosis, were suppressed to the same level as the control. Furthermore, the expression of ACTA2, COL1A1, and ELN was decreased in any 4 miRNAs compared to the case where only TGF-β was added, and in particular, the expression of ELN was decreased to the same level as the control. Therefore, it was confirmed that liver fibrosis can be suppressed by using miR-1237-5p, 204-3p, 7977, 6089, or 5787 alone or in combination

[0070] [Example 6] Fibrosis inhibitory effect of the selected 5 miRNAs alone In Example 5, among the 5 miRNAs selected in Example 4, the fibrosis inhibitory effect of the 5 miRNA group or the 4 miRNA group was examined, but the fibrosis inhibitory effect in HHSteC was confirmed using a single miRNA

[0071] HHSteC cells were used in the same manner as in Example 4. The fibrosis inhibitory effect of the single miRNA group was examined by dividing them into the following 8 groups Control group: HHSteC (2×10 4 (number) TGF-β group: HHSteC (2×10 4 (number) + TGF-β (2 ng / mL) 5 miRNA group: HHSteC (2×10 4(cells) + TGF-β (2 ng / mL) + miR-1237-5p, miR-204-3p, miR-7977, miR-6089, miR-5787 (each 0.28 μg, Total 1.4 μg) miRNA alone group: HHSteC (2×10 4 (cells) + TGF-β (2 ng / mL) + miR 1237-5p (1.4 μg) HHSteC (2×10 4 (cells) + TGF-β (2 ng / mL) + miR 204-3p (1.4 μg) HHSteC (2×10 4 (cells) + TGF-β (2 ng / mL) + miR 7977 (1.4 μg) HHSteC (2×10 4 (cells) + TGF-β (2 ng / mL) + miR 6089 (1.4 μg) HHSteC (2×10 4 (cells) + TGF-β (2 ng / mL) + miR 5787 (1.4 μg)

[0072] The antifibrotic effect of miRNA was performed in the same manner as in Example 4 except that ACTA2, COL1A1, ELN, and in addition, COL1A2 and COL3A1, which are fibrosis markers, were evaluated by real-time PCR method. Note that COL1A2 and COL3A1 are genes related to fibrosis.

[0073] The relative ratios of the TGF-β group and the miRNA alone group when the Fold change of the control group was set to 1.0 are shown in FIGS. 3A to 3E. In FIGS. 3A to 3E, the upper row shows the Control group, the TGF-β group, and the 5 miRNA groups, and the lower row shows the Control group, the TGF-β group, and the miRNA alone group.

[0074] From FIGS. 3A to 3E, not only the 5 miRNA groups but also each of miR 204-3p, miR 7977, miR 1237-5p, miR 5787 or miR 6089 in the miRNA alone group suppressed the expression of the fibrosis marker gene in the presence of TGF-β. When examined individually, miR 204-3p reduced the expression of ACTA2 to the control level, and also reduced the expression of COL1A2, COL3A1, and ELN by about 50% compared to the TGF-β group. miR 7977 reduced the expression of COL3A1 by about 60%, the expression of ACTA2 by about 50%, and the expression of ELN by about 35% compared to the TGF-β group. miR 1237-5p reduced the expression of COL1A2 by 70%, the expression of ACTA2 and COL1A3 by about 60%, and the expression of COL1A1 and ELN by about 50% compared to the TGF-β group. miR 5787 reduced the expression of COL1A2 and COL3A1 by 70%, the expression of COL1A1 by about 50%, and the expression of ACTA2 and ELN by about 40% compared to the TGF-β group. miR 6089 reduced the expression of COL3A1 and ELN by about 80% and the expression of COL1A2 by about 60% compared to the TGF-β group. From the above results, it was confirmed that not only a combination of 5 types but also the individual use of miR 1237-5p, miR 204-3p, miR 7977, miR 6089, or miR 5787 can suppress liver fibrosis.

[0075] [Example 7] Fibrosis inhibitory effect in mice by the selected 5 miRNAs In the above, the fibrosis inhibitory effect was evaluated using HHSteC cells, but here the fibrosis inhibitory effect was evaluated using mice.

[0076] A solution mixed at a ratio of corn oil: carbon tetrachloride (CCl4) = 3:1 was administered intraperitoneally to Col1-GFP mice (male) at 200 μL twice a week for 8 weeks to prepare a liver cirrhosis model mouse. At the 8th week, 10 μg (15 μg of each miRNA) of a cocktail of 5 miRNAs, miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787, was intravenously injected twice (5 miRNA group). The control was without administration of the 5 miRNA cocktail (control group).

[0077] Liver tissue was collected from mice, and the collected liver tissue was immersed in formalin and paraffin-embedded unstained specimens were prepared from the formalin-fixed tissue. For the above paraffin-embedded unstained specimens, hematoxylin-eosin (HE) staining and Sirius red staining were performed by a conventional method. The results are shown in Fig. 4. The fibrosis area was calculated based on the red area of Sirius red. From the results of HE staining, it was confirmed that none of the tissues had fat attached.

[0078] From Fig. 4, the fibrosis area of the control group was 13%, while that of the 5-miRNA administration group was 9%. It was confirmed that the 5-miRNA administration group reduced the fibrosis area by 30% compared with the control group. Therefore, it was shown that the fibrosis inhibitory effect of miRNA evaluated in HHSteC cells also showed the same effect in mice.

[0079] [Example 8] Fibrosis inhibitory effect by administration of extracellular vesicles From the results of Examples 1 and 2, it has been confirmed that five miRNAs, miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787, are highly expressed in extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs). Therefore, the fibrosis inhibitory effect by administering MSC-derived EVs was examined.

[0080] Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) of three commercially available healthy individuals were collected in the same manner as in Example 1. Also, HHSteC cells were used in the same manner as in Example 4. The fibrosis inhibitory effect by extracellular vesicle administration was examined by dividing into the following three groups. Control group: HHSteC (2×10 5 cells) TGF-β group: HHSteC (2×10 5 cells) + TGF-β (2 ng / mL) Extracellular vesicle administration group: HHSteC (2×10 5 cells) + TGF-β (2 ng / mL) + MSC-EVs (2×10 6 cells) The inhibitory effect of extracellular vesicles on fibrosis was evaluated by real-time PCR for fibrosis markers ACTA2, COL1A1, and ELN.

[0081] Figure 5 shows the relative ratios of the TGF-β group and the extracellular vesicle administration group, respectively, when the Fold change of the control group was set to 1.0.

[0082] In Figure 5, when MSC-EVs were administered, the expression of ACTA2 was decreased by about 40%, the expression of COL1A1 was decreased by about 55%, the expression of ELN was decreased by about 80%, and the expression of ELN was decreased by about 70% compared with the TGF-β group. Therefore, it was confirmed that not only when miR 1237-5p, miR 204-3p, miR 7977, miR 6089, or miR 5787 was administered, but also when extracellular vesicles containing the above microRNAs were administered, liver fibrosis could be suppressed.

Claims

1. A pharmaceutical composition for preventing or treating a liver disease accompanied by fibrosis, comprising as an active ingredient at least one microRNA selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787, or a precursor thereof.

2. The pharmaceutical composition according to claim 1, characterized in that it comprises a medium that retains the microRNA or a precursor thereof.

3. The pharmaceutical composition according to claim 2, wherein the medium retaining the microRNA or a precursor thereof is an extracellular vesicle selected from the group consisting of exosomes, microvesicles, liposomes (lipid bilayer membranes) and apoptotic vesicles.

4. The pharmaceutical composition according to claim 3 , wherein the extracellular vesicles are extracellular vesicles derived from mesenchymal stem cells.

5. The pharmaceutical composition according to claim 3 or 4, wherein the extracellular vesicles are extracellular vesicles derived from a cell into which a polynucleotide encoding at least one exogenous microRNA selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787 or a precursor thereof has been introduced and the microRNA or a precursor thereof has been expressed.

6. The pharmaceutical composition according to claim 1 or 2, which is administered to a subject in combination with mesenchymal stem cells.

7. An extracellular vesicle derived from a cell into which a polynucleotide encoding at least one type of exogenous microRNA selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787, or a precursor thereof, has been introduced and the microRNA or a precursor thereof has been expressed, the extracellular vesicle comprising at least one type of microRNA selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787, or a precursor thereof.

8. The extracellular vesicle of claim 7, wherein the extracellular vesicle is an extracellular vesicle derived from a mesenchymal stem cell.

9. A culture obtained by introducing a polynucleotide encoding at least one exogenous microRNA selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787, or a precursor thereof, and culturing cells that express the microRNA or a precursor thereof.

10. A step A of introducing a polynucleotide encoding at least one kind of exogenous microRNA selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787, or a precursor thereof, into mesenchymal stem cells; Step B: culturing the mesenchymal stem cells obtained in step A to obtain a culture; A step C of recovering extracellular vesicles from the culture; A method for producing extracellular vesicles for suppressing liver fibrosis, comprising exogenous at least one microRNA or a precursor thereof selected from miR 1237-5p, miR 204-3p, miR 7977, miR 6089, and miR 5787, the method comprising steps A to C.

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