WO2022270071A1 - Agent for preventing liver fibrosis, and pharmaceutical composition - Google Patents

Abstract

The present invention provides: an agent for preventing liver fibrosis, which comprises at least one nucleic acid selected from the group consisting of a nucleic acid that comprises a nucleotide sequence comprising a seed sequence for miR-6848, a double-stranded nucleic acid that contains the aforementioned nucleic acid as one of the strands, a precursor of the aforementioned nucleic acid, or a nucleic acid that expressably carries the aforementioned nucleic acid or a nucleic acid encoding the precursor of the aforementioned nucleic acid; and a pharmaceutical composition. The present invention also provides: a combination medicine which comprises the above-mentioned at least one nucleic acid and a therapeutic drug for hepatitis C virus; and a kit. The present invention further provides a method for evaluating the liver fibrosis preventing activity of a substance of interest, in which an activity to induce or enhance the expression of hsa-miR-6848-5p, hsa-miR-6848-3p or a precursor of each of hsa-miR-6848-5p and hsa-miR-6848-3p is employed as a measure.

Description

Agent and pharmaceutical composition for suppressing hepatic fibrosis

The present invention expresses a nucleic acid consisting of a base sequence containing a seed sequence of miR-6848, a double-stranded nucleic acid containing said nucleic acid as one strand, a precursor of said nucleic acid, and a nucleic acid encoding said nucleic acid or its precursor. The present invention relates to agents and pharmaceutical compositions for suppressing hepatic fibrosis, containing at least one nucleic acid selected from the group consisting of nucleic acids that can be retained.

In recent years, significant progress has been made in the treatment of chronic liver disease due to hepatitis C virus (HCV) infection. A significant virological response (Sustained Virological Response; SVR) was obtained in the majority of cases, including liver cirrhosis. However, there are many cases in which liver fibrosis progresses even after achieving SVR, leading to hepatocarcinogenesis, and suppression of liver fibrosis is still an unsolved problem. Liver fibrosis is the formation of scar tissue, the excessive accumulation of connective tissue within the liver as a result of chronic hepatocyte injury, and is caused by various types of chronic liver injury, such as hepatitis virus infection and excessive alcohol consumption. It is known.

In liver fibrosis, the central event is the activation of hepatic stellate cells (HSCs) present around hepatic blood vessels. Hepatic stellate cells are activated to become myofibroblasts during liver injury, and overproduce fibrosis-inducing factors such as TGF (transforming growth factor)-β and extracellular matrix to promote fibrosis. In addition, activated hepatic stellate cells are known not only to induce fibrosis, but also to participate in the regulation of inflammation and immune response, as well as in the composition of the microenvironment of liver cancer. Controlling the function of activated hepatic stellate cells is considered to be an important and effective approach to suppress liver fibrosis and, in turn, hepatocarcinogenesis. Agonists, CCR2/CCR5 antagonists, ASK-1 (Apoptosis signal-regulating kinase 1) inhibitors, Gallection 3 inhibitors, siRNA against HSP47, etc. are being developed).

In addition, the canonical Wnt pathway (β-catenin pathway, also called canonical Wnt pathway), a Wnt signaling pathway that plays a role in regulating stem cell proliferation and differentiation and maintaining homeostasis, is overexpressed in fibrotic tissues. has been shown to be expressed in Based on this finding, it has been proposed to use the Wnt inhibitor PRI-724 as a therapeutic drug for liver fibrosis (Non-Patent Document 1).

Furthermore, hepatic stellate cell activation has been shown to be epigenetically regulated by microRNAs (miRNAs), e.g., miR-29 family, miR-34 family, miR-15 family, miR-200 family, miR-199 family, miR-378 family, etc. are reported to be involved in fibrosis (eg, Non-Patent Document 2). It has been proposed that miR-29a be used as a preventive/therapeutic agent for hepatic fibrosis (Patent Document 1).

JP 2017-145222

Tokunaga Y. et al., Scientific Reports, 7: 325. Published online 2017 Mar 23. doi: 10.1038/s41598-017-00282-w. Jiang et al., Cell Biosci (2017) 7:34. doi: 10.1186/s13578-017-0161-7.

The present invention provides a new means for suppressing liver fibrosis. The present invention also provides new means for evaluating liver fibrosis-suppressing activity.

The present inventors found miRNAs that can suppress the expression of the α-SMA (α-smooth muscle actin) gene and the activation of the canonical Wnt pathway in activated hepatic stellate cells.

The present disclosure provides the following.
Item 1. A pharmaceutical composition for suppressing liver fibrosis, comprising at least one nucleic acid selected from the group consisting of the following a) to e).
a) A nucleic acid having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
b) Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand
d) a precursor of the nucleic acid of any of the above a) to c)
e) Nucleic acid carrying any of the nucleic acids of a) to d) or a nucleic acid encoding its precursor in an expressible manner Item 2 The nucleic acid of a) above consists of or has the base sequence shown in SEQ ID NO: 1. Item 1. The pharmaceutical composition according to item 1, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in number 1.
Item 3. Item 1 or 2, wherein the nucleic acid of b) consists of the base sequence shown in SEQ ID NO: 2 or consists of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 2. pharmaceutical composition.
Item 4 Any of Items 1 to 3, wherein the precursor of d) consists of the base sequence shown in SEQ ID NO: 3, or consists of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 3. or the pharmaceutical composition according to claim 1.
Item 5 The nucleic acid of e) above can express a nucleic acid consisting of the base sequence shown in SEQ ID NO: 4 or a nucleic acid consisting of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 4. Item 5. The pharmaceutical composition according to any one of Items 1 to 4, which is a nucleic acid prepared by the above method.
Item 6. The pharmaceutical composition according to any one of Items 1 to 5, wherein the nucleic acids a) and b) are mature microRNAs, and the precursors of d) are primary microRNAs or precursor microRNAs.
Item 7. The pharmaceutical composition of any one of Items 1 to 6, for use in a subject undergoing or having received treatment with a therapeutic agent for hepatitis C virus.
Item 8. The pharmaceutical composition according to Item 7, for use in a subject who has achieved a remarkable virologic response by treatment with a therapeutic agent for hepatitis C virus.
Item 9. The pharmaceutical composition of any one of Items 1-8 for the treatment of hepatitis C, wherein said treatment involves the use of a hepatitis C virus therapeutic agent.
Item 10 A pharmaceutical for treating hepatitis C, comprising at least one nucleic acid selected from the group consisting of the following a) to e) in combination with a therapeutic drug for hepatitis C virus.
a) A nucleic acid having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
b) Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand
d) a precursor of the nucleic acid of any of the above a) to c)
e) At least one kind of nucleic acid selected from the group consisting of a) to e) below in Item 11, a nucleic acid that retains an expressible nucleic acid that encodes any of the above a) to d) or a nucleic acid that encodes a precursor thereof A kit for the treatment of hepatitis C, comprising: , a hepatitis C virus therapeutic drug.
a) A nucleic acid having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
b) Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand
d) a precursor of the nucleic acid of any of the above a) to c)
e) Nucleic acid Item 12 hsa-miR-6848-5p, hsa-miR-6848-3p, or precursors thereof, carrying a nucleic acid encoding any one of the above a) to d) or a nucleic acid encoding the precursor thereof in an expressible manner A method for evaluating the hepatic fibrosis-suppressing activity of a test substance, using as an index the activity of inducing or enhancing the expression of
Item 13: Incubating a test substance with hepatic stellate cells; Measuring the expression level of hsa-miR-6848-5p, hsa-miR-6848-3p or their precursors in hepatic stellate cells after incubation; comparing the amount to the expression level of hsa-miR-6848-5p, hsa-miR-6848-3p or their precursors in the absence of the test substance; and hsa-miR-6848-5p, hsa-miR Item 13. The method according to Item 12, comprising the step of determining that the test substance that induces expression or increases the expression level of 6848-3p or their precursors has hepatic fibrosis-suppressing activity.
Item 14: Incubating a test substance with a cell containing a nucleic acid having a marker gene under the control of a promoter region that controls the expression of the hsa-mir-6848 gene; Measuring the intensity of a signal derived from the marker gene in the cell; comparing the intensity of the marker gene-derived signal obtained in the presence of the test substance with the intensity of the marker gene-derived signal in the absence of the test substance; 13. The method of clause 12, comprising determining that.

This disclosure also provides:
Item 1A A liver fibrosis inhibitor containing at least one nucleic acid selected from the group consisting of a) to e) below.
a) A nucleic acid having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
b) Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand
d) a precursor of the nucleic acid of any of the above a) to c)
e) Nucleic acid carrying any of the nucleic acids a) to d) or a nucleic acid encoding its precursor in an expressible manner. Item 1A, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in No. 1.
Item 3A. Item 1A or 2A, wherein the nucleic acid of b) consists of the base sequence shown in SEQ ID NO: 2, or consists of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 2. agent.
Item 4A Any of Items 1A to 3A, wherein the precursor of d) consists of the base sequence shown in SEQ ID NO: 3, or consists of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 3. or the agent according to item 1.
Item 5A The nucleic acid of e) is capable of expressing a nucleic acid consisting of the base sequence shown in SEQ ID NO: 4, or a nucleic acid consisting of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 4. The agent according to any one of Items 1A to 4A, which is a nucleic acid obtained by
Item 6A The agent according to any one of Items 1A to 5A, wherein the nucleic acids a) and b) are mature microRNAs, and the precursors of d) are primary microRNAs or precursor microRNAs.
Item 7A For a subject with liver fibrosis, or a subject with a disease or condition that can cause liver fibrosis, containing at least one nucleic acid selected from the group consisting of a) to e) below Pharmaceutical composition for use.
a) A nucleic acid having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
b) Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand
d) a precursor of the nucleic acid of any of the above a) to c)
e) For subjects undergoing or who have undergone treatment with a nucleic acid Section 8A hepatitis C virus therapeutic drug that retains an expressible nucleic acid encoding any of the above a) to d) or a nucleic acid that encodes a precursor thereof A pharmaceutical composition according to Section 7A, for use in
Item 9A The pharmaceutical composition according to Item 7A or 8A, for use in a subject who has achieved a significant virologic response by treatment with a therapeutic agent for hepatitis C virus.
Item 10A A pharmaceutical for treating hepatitis C, comprising at least one nucleic acid selected from the group consisting of the following a) to e) in combination with a therapeutic drug for hepatitis C virus.
a) A nucleic acid having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
b) Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand
d) a precursor of the nucleic acid of any of the above a) to c)
e) Nucleic acid carrying any of the above a) to d) or a nucleic acid encoding its precursor in an expressible manner At least one nucleic acid selected from the group consisting of a) to e) below in item 11A A pharmaceutical composition for the treatment of hepatitis C, comprising the use of a hepatitis C virus therapeutic.
a) A nucleic acid having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
b) Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand
d) a precursor of the nucleic acid of any of the above a) to c)
e) A nucleic acid that retains the nucleic acid encoding any of the nucleic acids a) to d) above or its precursor in an expressible manner

According to the present disclosure, activation of hepatic stellate cells can be inhibited, thereby inhibiting liver fibrosis in a subject with liver fibrosis or with a disease or condition that can cause liver fibrosis. be able to.

Fig. 4 is a graph showing the αSMA gene expression level in hepatic stellate cells introduced with mock miRNA (N.C.), miR-29, miR-342, miR-6848 or miR-449. Fig. 4 is a graph showing the expression level of the collagen I gene in hepatic stellate cells introduced with mock miRNA (N.C.), miR-29, miR-342, miR-6848 or miR-449. Fig. 10 is a graph showing the activity of TCF/LEF promoter, NFAT promoter or AP-1 promoter in hepatic stellate cells introduced with mock miRNA (N.C.), miR-29, miR-342, miR-6848 or miR-449. Fig. 2 is a schematic diagram showing an outline of an administration schedule in an animal test for evaluating suppression of liver fibrosis by a lipid membrane structure encapsulating miRNA. Normal mouse (normal), carbon tetrachloride-administered mouse (CCl4), carbon tetrachloride plus negative control mimic-encapsulated lipid membrane construct (NC), and carbon tetrachloride plus miRNA-encapsulated lipid membrane construct is a graph showing Sirius-Red stained areas of liver tissue sections taken from mice (342, 6848) administered with

Although the description given below may be based on representative embodiments or specific examples, the present invention is not limited to such embodiments or specific examples. Moreover, the upper limit and lower limit of each numerical range shown in this specification can be combined arbitrarily. Further, in the present specification, a numerical range represented by "-" or "-" means a range including numerical values at both ends thereof as upper and lower limits, unless otherwise specified.

miRNAs are non-coding RNAs with a length of about 16 to 29 bases, mostly around 22 bases, encoded on the genome of eukaryotes, and play an important role in regulating gene expression. After being transcribed from genomic DNA as primary miRNA (pri-miRNA), which is a primary transcript with a length of several hundred to several thousand bases, miRNA is processed by Drosha in the nucleus to form a precursor miRNA with a hairpin structure. (pre-miRNA). Pre-miRNA moves into the cytoplasm and is further processed by Dicer to become double-stranded miRNA. Double-stranded miRNA is incorporated into Ago protein, and one RNA strand is complexed with Ago protein to form an RNA-protein complex called RISC (RNA-induced silencing complex). This RNA strand is called mature miRNA, and has a base sequence called a seed sequence at the 2nd to 8th positions from the 5' end. The seed sequence of mature miRNA exerts the function of inhibiting the translation of the target gene by binding with the complementary sequence present in the 3' untranslated region of the target gene mRNA.

Throughout the present specification, IDs and accession numbers of miRNAs are defined in miRBase (http://www.miRbase.org/), which is a database that collects information such as base sequences of miRNAs. be. The nucleotide sequences of each miR are as registered in miRBase, and shown in the sequence listing as necessary.

In the present disclosure, at least one nucleic acid selected from the group consisting of a) to e) below can be used for suppressing liver fibrosis.
a) RNA having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
b) RNA having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand
d) a precursor of the nucleic acid of any of the above a) to c)
e) A nucleic acid that retains the nucleic acid encoding any of the nucleic acids a) to d) above or its precursor in an expressible manner

The nucleic acid a) is a nucleic acid having a base sequence consisting of GGGGGCU as a seed sequence, and is a mature miRNA or a nucleic acid having functions equivalent thereto. The chain length of the nucleic acid a) may be 16 to 29 bases, preferably 18 bases or more, and preferably 25 bases or less. Preferred examples of the nucleic acid a) include RNA consisting of the nucleotide sequence shown in SEQ ID NO: 1; RNA consisting of a nucleotide sequence having identity; and having a nucleotide sequence consisting of GGGGGCU and having 1 or 2, preferably 1 base deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 1 RNA consisting of a base sequence can be mentioned. The RNA consisting of the base sequence shown in SEQ ID NO: 1 is hsa-miR-6848-5p registered with miRBase under accession number MIMAT0027596.

The RNA in b) is a nucleic acid having a nucleotide sequence consisting of UGGUCUC as a seed sequence, and is a mature miRNA or a nucleic acid having functions equivalent thereto. The chain length of the nucleic acid b) may be 16 to 29 bases, preferably 18 bases or more, and preferably 25 bases or less. Preferred examples of the nucleic acid b) include RNA consisting of the nucleotide sequence shown in SEQ ID NO: 2; RNA consisting of a base sequence having identity; and having a base sequence consisting of UGGUCUC, and having 1 or 2, preferably 1 base deleted, substituted or added in the base sequence shown in SEQ ID NO: 2 RNA consisting of a base sequence can be mentioned. The RNA consisting of the base sequence shown in SEQ ID NO: 2 is hsa-miR-6848-3p registered in miRBase under accession number MIMAT0027597.

The double-stranded nucleic acid of c) has the nucleic acid of a) or b) on one strand.

The precursor of d) is a nucleic acid capable of producing any of the nucleic acids of a) to c) in cells, for example, pri-miRNA or pre-miRNA. Preferred examples of the precursor of d) include RNA consisting of the nucleotide sequence shown in SEQ ID NO: 3; RNA consisting of a nucleotide sequence having 95% identity; can include RNA consisting of a nucleotide sequence in which 1 to 4, more preferably 1 to 3, and particularly preferably 1 or 2 nucleotides have been deleted, substituted or added. The RNA consisting of the base sequence shown in SEQ ID NO: 3 is hsa-miR-6848 registered with miRBase under accession number MI0022694.

The nucleic acid of e) holds any nucleic acid of a) to c) or a nucleic acid encoding the precursor of d) in an expressible manner, and as a result of transcription and processing, any of a) to c) or a nucleic acid capable of expressing the precursor of d). Preferred examples of the nucleic acid of e) include DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4; and a DNA consisting of a base sequence with 100% identity; A nucleic acid that retains a DNA consisting of a base sequence in which 1 to 4, more preferably 1 to 3, and particularly preferably 1 or 2 bases have been deleted, substituted, or added so as to be expressible can be mentioned. DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4 is DNA encoding hsa-miR-6848.

The nucleic acid of e) is preferably in the form of an expression vector into which the nucleic acid of any one of a) to c) or the nucleic acid encoding the precursor of d) is integrated under the control of an appropriate promoter sequence. In addition to the promoter sequence, the expression vector may further contain any functional base sequence that regulates transcriptional expression, such as operator sequences, enhancers, and the like. Any promoter sequence may be used as long as it can function in mammalian cells, and a promoter sequence having a high ability to induce expression in hepatic stellate cells is particularly preferred.

Nucleotide sequence identity refers to an optimal alignment calculated using an algorithm known in the art (preferably, the algorithm takes into account the introduction of gaps in one or both of the sequences for optimal alignment. It means the ratio (%) of identical bases to all overlapping bases in the total number of overlapping bases.

Identity can be calculated, for example, by aligning two base sequences using NCBI BLAST-2 (National Center for Biotechnology Information Basic Local Alignment Search Tool) with default conditions.

The nucleic acids a) to e) may be chimeric nucleic acids containing deoxyribonucleotides and ribonucleotides in their base sequences, or hybrid nucleic acids consisting of a DNA strand and an RNA strand.

In addition, the nucleic acids a) to e) may be chemically modified nucleic acids. Chemical modifications of nucleic acids may be performed on any of the base, phosphate, and sugar moieties, and examples of base moieties include 5-methylcytosine, 5-propynylcytosine, phenoxazine, 9-( aminoethoxy)phenoxazine and the like, and examples of modification of the phosphate moiety include phosphodiester, phosphorothioate, boranophosphate, phosphoramidate, methylphosphonate, phosphotriester, and the like. Modification examples include 2'-OMe conversion, 2'-F conversion, 2'-MOE conversion, and cross-linking such as LNA or BNA. The type and number of modified bases in the modified nucleic acid are not particularly limited as long as the effect of suppressing hepatic fibrosis is not lost.

The nucleic acids a) to e) can be prepared using genetic recombination technology or chemical synthesis technology. Various techniques such as gene recombination, chemical synthesis of nucleic acids, and synthesis of modified nucleic acids are well known to those skilled in the art.

Nucleic acids a) to e) can be introduced by various known methods capable of introducing nucleic acids into cells, for example, nucleic acid introduction methods using viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, etc., jetPEI (PolyPlus-transfection), a nucleic acid transfection method using a non-viral carrier such as lipofectamine, a hydrodynamics method, or the like, can be used for introduction into the cells of the target animal. The invention also provides, in one aspect, the nucleic acids of a)-e) in combination with a viral vector or non-viral carrier.

The nucleic acids a) to e) can suppress the expression of the αSMA gene in activated hepatic stellate cells and can also suppress the activation of the canonical Wnt pathway. The αSMA gene is a fibroblast marker gene, and activation of the canonical Wnt pathway is involved in tissue fibrosis. Therefore, the nucleic acids a) to e) can be used as active ingredients of agents and pharmaceutical compositions for suppressing activation of hepatic stellate cells and suppressing hepatic fibrosis.

The pharmaceutical composition should be used as a medicament for suppressing liver fibrosis in subjects with or at risk of liver fibrosis, particularly subjects with diseases or conditions that can cause liver fibrosis. can be done.

In the present disclosure, "subject" includes humans and non-human animals. Examples of non-human animals include rodents including mice, rats, hamsters and guinea pigs, primates including chimpanzees and rhesus monkeys, domestic animals including pigs, cattle, goats, horses and sheep, and pet animals including dogs and cats. can be mentioned. A preferred subject is a human.

Hepatic fibrosis and its suppression are detected by immunohistological staining of liver tissue; measurement of liver fibrosis markers such as blood hyaluronic acid or type IV collagen; ultrasonography, CT, MRI, ultrasonic elastography, magnetic resonance It can be assessed using techniques such as elastography or imaging studies such as acoustoradiography.

Diseases or conditions that can cause liver fibrosis include conditions that directly affect the liver, such as bacterial infections (such as brucellosis), parasitic infections (such as echinococcosis), viral infections (such as hepatitis B, hepatitis C, etc.), nonalcoholic steatohepatitis (NASH), primary biliary cirrhosis, primary sclerosing cholangitis, congenital liver fibrosis, autoimmune hepatitis, α1-antitrypsin deficiency, copper Storage disease, fructoseemia, galactosemia, glycogen storage disease, iron overload syndrome, dyslipidemia, peroxisome disease, tyrosinemia, etc.; conditions affecting hepatic blood flow, such as Budd-Chiari syndrome, heart failure, central hepatic vein Obstruction, portal vein thrombosis, etc.; intake of drugs or chemicals that can cause fibrosis, such as alcohol, amiodarone, chlorpromazine, isoniazid, methotrexate, methyldopa, oxyphenisatin, tolbutamide, etc.; Examples include scarring due to liver surgery, bile duct stricture due to incarcerated gallstones, and the like.

In the present disclosure, "suppression of liver fibrosis" refers to improvement of the fibrosis state of the liver. including regression or disappearance of "Inhibition of liver fibrosis" is used interchangeably with prevention and/or treatment of liver fibrosis.

The pharmaceutical composition is preferably used in a subject suffering from hepatitis C, a subject suffering from hepatitis C and being treated with an HCV therapeutic agent, or a subject suffering from hepatitis C and treated with an HCV therapeutic agent. It can be used to suppress liver fibrosis in subjects who have achieved SVR with treatment. Here, SVR is also called sustained virologic negative change, and means that blood HCV RNA becomes negative both in the test at the end of anti-HCV treatment and at 12 or 24 weeks after the end of treatment. .

The pharmaceutical composition contains an effective amount of at least one nucleic acid selected from the group consisting of a) to e), and pharmaceutically acceptable substances such as buffers, antioxidants, preservatives, excipients, carriers, and the like. and other pharmaceutically active ingredients. Pharmaceutically acceptable ingredients are well known to those skilled in the art, and can be appropriately selected and used within the scope of ordinary ability of those skilled in the art according to the properties of the active ingredient and the form of the formulation.

The pharmaceutical composition is preferably administered using a drug delivery system (DDS) that can selectively introduce nucleic acids into hepatic stellate cells. Examples of such DDS include DDS having tropism to the liver, especially hepatic stellate cells, lipid membrane structures such as viral vectors, liposomes or micelles, particularly cationic lipid membrane structures suitable for intracellular delivery of nucleic acids. , which utilize DDS materials such as magnetic microparticles. In one aspect, the present invention provides a pharmaceutical composition in which at least one nucleic acid selected from the group consisting of a) to e) is carried or encapsulated in such a DDS material, and a DDS material carries or encapsulates the nucleic acid. Also provided is said nucleic acid.

The pharmaceutical composition is preferably used in the form of parenteral preparations such as injections and infusions. In addition, the administration method of the pharmaceutical composition is not particularly limited. topical administration to . In one preferred embodiment, the pharmaceutical composition is applied to the subject by intravenous administration or by topical administration at or near the fibrotic site of the liver.

The pharmaceutical composition can be used as a combination drug in combination with other drugs for diseases or conditions that can cause liver fibrosis. A combination medicament means a combination of medicaments intended to be administered together or separately, simultaneously or sequentially to a subject in need thereof, i.e. a subject with or at risk of liver fibrosis . Intended modes of administration include administration of a formulation containing at least one nucleic acid selected from the group consisting of a) to e) above and other pharmaceutically active ingredients in one formulation, or administration of separate formulations. It can include administration of at least one nucleic acid selected from the group consisting of a) to e) formulated above and other pharmaceutical agents. In addition, when they are formulated and administered separately, their administration order and administration time are not particularly limited, and they may be administered at the same time, or may be administered at different times or on different days. An example of other drugs are HCV drugs, including RNA polymerase inhibitors such as ribavirin, protease inhibitors such as asunaprevir, nonstructural protein 5A (NS5A) inhibitors such as daclatasvir, and nonstructural protein 5A (NS5A) inhibitors such as sofosbuvir. RNA polymerase inhibitors of protein 5B (NS5B), interferons, and pharmaceutical combinations thereof can be mentioned.

The combination pharmaceutical can be a kit for treating hepatitis C, containing at least one nucleic acid selected from the group consisting of a) to e) above and a therapeutic agent for HCV. The at least one nucleic acid and HCV therapeutic agent contained in the kit are administered together or separately, simultaneously or sequentially to a subject in need of treatment for hepatitis C, and administration of each formulation There are no particular restrictions on the order and timing of administration.

The present invention also provides, in one aspect, a pharmaceutical composition for the treatment of hepatitis C, comprising at least one nucleic acid selected from the group consisting of a) to e) above, wherein the treatment comprises C The pharmaceutical composition is provided, comprising the use of a therapeutic agent for hepatitis virus.

In addition, in one aspect, the present invention provides a method of suppressing liver fibrosis in a subject by administering an effective amount of a pharmaceutical composition for suppressing liver fibrosis. In one aspect, the present invention provides at least one nucleic acid selected from the group consisting of a) to e) for use in suppressing liver fibrosis in a subject, and in one aspect, liver fibrosis Provided is the use of at least one nucleic acid selected from the group consisting of a) to e) above in the manufacture of a pharmaceutical composition for inhibiting catalysis. Definitions and other explanations for each term are as described above.

The term “effective amount” used herein with respect to agents and pharmaceutical compositions for suppressing liver fibrosis means an amount sufficient to suppress liver fibrosis in a subject, for example, suppressing progression of liver fibrosis It means an amount sufficient to reduce the size of the fibrotic site. In addition, the term “effective amount” used herein with respect to agents and pharmaceutical compositions for suppressing activation of hepatic stellate cells means suppressing activation of hepatic stellate cells or inactivating activated hepatic stellate cells. means an amount sufficient to Such an effective amount is appropriately adjusted depending on the subject to be administered, degree of fibrosis and other medical factors.

Further, in one aspect of the present invention, a A method for evaluating the hepatic fibrosis-suppressing activity of a test substance as an index is provided. Here, the precursor is typically the precursor microRNA (hsa-mir-6848, SEQ ID NO: 3) or primary microRNA of hsa-miR-6848-5p and hsa-miR-6848-3p.

hsa-miR-6848-5p, hsa-miR-6848-3p, or substances capable of inducing or enhancing the expression of these precursors can induce or enhance the expression of these nucleic acids in hepatic stellate cells. It is expected that the activation of hepatic stellate cells can be suppressed and an inhibitory effect on hepatic fibrosis can be exhibited.

In one exemplary embodiment, a method for evaluating the hepatic fibrosis-suppressing activity of a test substance comprises the step of incubating the test substance with hepatic stellate cells; hsa-miR-6848-5p, hsa- measuring the expression level of miR-6848-3p or their precursors; and a test substance that induced the expression of hsa-miR-6848-5p, hsa-miR-6848-3p or their precursors or increased the expression level of liver fibrosis suppression It includes the step of determining that it has activity.

Incubation of the hepatic stellate cells with the test substance is carried out in an appropriate medium in which the hepatic stellate cells can survive, typically in a physiological buffer such as PBS or a cell culture medium such as DMEM. The test can be performed by coexisting the hepatic stellate cells and the test substance under temperature and oxygen conditions, for example, a temperature of 37° C. and 5% CO ₂ . In addition, the expression of hsa-miR-6848-5p, hsa-miR-6848-3p, or their precursors in hepatic stellate cells can be determined by hybridization, quantitative It can be carried out by PCR, RNA-Seq and other common methods capable of detecting and quantifying specific gene expression.

Expression of hsa-miR-6848-5p, hsa-miR-6848-3p, or their precursors is not detected in hepatic stellate cells not incubated with test substance but is detected in hepatic stellate cells incubated with test substance or expression levels of hsa-miR-6848-5p, hsa-miR-6848-3p, or their precursors in hepatic stellate cells incubated with the test article compared to hepatic stellate cells not incubated with the test article When it is high, the test substance can be determined to have hepatic fibrosis inhibitory activity.

In another exemplary embodiment, the method for evaluating the hepatic fibrosis-suppressing activity of a test substance includes the method of evaluating the hepatic fibrosis-suppressing activity of a test substance, comprising: measuring the intensity of the marker gene-derived signal in the cells; comparing the measured intensity of the marker gene-derived signal with the intensity of the marker gene-derived signal in the absence of the test substance; and a marker A step of determining that the test substance that increased the intensity of the gene-derived signal has hepatic fibrosis-suppressing activity. This method evaluates the effect of inducing or enhancing the expression of hsa-miR-6848-5p, hsa-miR-6848-3p, or their precursors using a reporter assay.

The marker gene is not particularly limited as long as the expression induction of the gene can be detected, for example, a gene encoding a fluorescent protein such as GFP or a chemiluminescent protein such as luciferase, or a drug resistance gene or auxotrophy of cells Genes that confer a phenotypic change on cells, such as genes that can complement , can be used.

Cells containing a nucleic acid having a marker gene under the control of the hsa-mir-6848 promoter region can be obtained, for example, by combining the nucleotide sequence of the genomic region encoding hsa-mir-6848 in hepatic stellate cells with the nucleotide sequence encoding the marker gene. It can be produced by recombination with Also, based on the human genome nucleotide sequence information, construct an expression vector containing a human hsa-mir-6848 promoter region and a marker gene recombined under its control, and introduce the expression vector into cells. can also

Incubation can be performed by coexisting the cells and the test substance in a medium or buffer solution in an appropriate container. Conditions such as incubation temperature and time may be conditions that allow the cell to express the marker gene. In addition, marker gene-derived signals can be measured by a method suitable for the marker gene used.

The present invention will be described in more detail by the following examples, but the present invention is not limited to these. In the examples, operations using commercially available kits were performed according to the kit manufacturer's protocol. It should be noted that this invention is not limited to the particular methodology, protocols, cell lines, animal species and genera, constructs and reagents described herein, which may be modified accordingly. It is easily understood by a trader.

The data shown in the examples below were obtained at n = 3-5 and shown as mean ± standard deviation. For statistical processing, significant differences between groups were verified by a t-test, and p-values of less than 0.05 were considered significant differences between groups (*P<0.05, **P<0.01).

Example 1 Suppression of αSMA Expression by Introduction of miRNA 1) miRNA
The following miRNA manufactured by Bioneer (BIN) was purchased from Cosmo Bio and used. These miRNAs are double-stranded RNAs consisting of the base sequences shown below and their complementary base sequences.
・miRNA Mimic hsa-miR-29a-3p
Product number: SMM-001- MIMAT0000086
Product name: AccuTarget ^TM Human miRNA Mimic hsa-miR-29a-3p [Accession: MIMAT0000086]
Nucleotide sequence: 42-uagcaccaucuguaucgguua-63 (SEQ ID NO: 5)
・Human miRNA Mimic hsa-miR-449a
Product number: SMM-001- MIMAT0001541
Product name: AccuTarget ^TM Human miRNA Mimic hsa-miR-449a [Accession: MIMAT0001541]
Nucleotide sequence: 16-uggcaguguauguuagcuggu-37 (SEQ ID NO: 6)
・Human miRNA Mimic hsa-miR-342-3p
Product number: SMM-001- MIMAT0000753
Product name: AccuTarget ^TM Human miRNA Mimic hsa-miR-342-3p [Accession: MIMAT0000753]
Nucleotide sequence: 61-ucucacacagaaucgcacccgu-83 (SEQ ID NO: 7)
・miRNA Mimic hsa-miR-6848-5p
Product number: SMM-001- MIMAT0027596
Product name: AccuTarget ^TM Human miRNA Mimic hsa-miR-6848-5p [Accession: MIMAT0027596]
Nucleotide sequence: 6-ugggggcugggaugggccauggu-28 (SEQ ID NO: 2)
・Mimic positive control
AccuTarget ^TM miRNA Houskeeping Positive control (GAPDH, Human)
・Mimic negative control
AccuTarget ^™ miRNA mimic Negative control

2) Expression analysis of αSMA DMEM containing 2% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin was dispensed into a 6-well plate, and human hepatic stellate cells (LX- ² Hepatic stellate cells (purchased from Millipore) were seeded and cultured. Two days later, 40 μM of each miRNA was introduced into the cells using Lipofectamine (registered trademark) RNAiMAX (life Technologies). Six hours after the introduction, human TGF-β1 (PeproTech) was added to the medium to 2 ng/mL in some wells, and the cells were further cultured for 24 hours. After the culture was completed, the cells were collected and RNA was extracted using RNeasy (registered trademark) Mini Kit (QIAGEN). The extracted RNA was reverse transcribed using Prime Script RT Master Mix (TaKaRa) to synthesize cDNA, and the expression levels of αSMA and collagen-I were quantified by real-time PCR. Real-time PCR was performed using Platinum SYBER Green qPCR SuperMix-UDG with ROX (Thermo) and the following primers.
αSMA
Forward primer: 5'-CCGACCGAATGCAGAAGGA-3' (SEQ ID NO: 8)
Reverse primer: 5'-ACAGAGTATTTGCGCTCCGAA-3' (SEQ ID NO: 9)
Collagen I (Col1α1)
Forward primer: 5'-GGGATTCCCTGGACCTAAAG-3' (SEQ ID NO: 10)
Reverse primer: 5'-GGAACACCTCGCTCTCCA-3' (SEQ ID NO: 11)

When the expression level in mock miRNA-introduced hepatic stellate cells without TGF-β1 was set to 1, the relative expression level of α-SMA in each cell is shown in Fig. 1, and the relative expression level of collagen I is shown in Fig. 2. . Expression of αSMA is suppressed in the presence of miR-29, miR-342 and miR-6848, and miR-6848 has a particularly strong effect, and the tendency to do so under stimulation of the profibrotic cytokine TGF-β1 was found to be clearer. In addition, miR-6848 was also confirmed to suppress the expression of collagen I that was particularly enhanced by TGF-β1, demonstrating its ability to suppress fibrosis at the molecular level.

Example 2 Suppression of Activation of Wnt Signaling Pathway by Introduction of miRNA DMEM containing 2% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin was dispensed into a 6-well plate to give 1×10 ⁵ cells/well. Human hepatic stellate cells (LX-2 hepatic stellate cells, purchased from Millipore) were seeded and cultured. Two days later, 40 μM of miR-6848 or mock miRNA, TCF/LEF reporter vector (Promega), NFAT reporter vector (Promega) or AP-1 reporter vector (Promega), and Renilla luciferase as an internal control The expression vector pGL4.74 [hRluc/TK] (Promega) was introduced into cells using Lipofectamine (registered trademark) RNAiMAX (life Technologies). When the TCF/LEF reporter vector activates the canonical Wnt pathway and increases the expression of TCF/LEF, the NFAT reporter vector and the AP-1 reporter vector activate the non-canonical Wnt pathway and NFAT/AP-1 It is an expression vector into which a firefly luciferase gene has been introduced under the TCF/LEF, NFAT or AP-1 promoter so that luciferase expression is induced when the expression of is increased.

Six hours after the introduction, human TGF-β1 (PeproTech) was added to 2 ng/mL in some wells, and human Wnt3A (biotechne) was added to 100 ng/mL in some wells. It was added to the medium and cultured for an additional 24 hours. After culturing, the cells were harvested and whole cell lysates were prepared, and changes in each promoter activity were evaluated by dual luciferase assay. Luciferase activity was measured using a Dual Luciferase Reporter Assay System (Promega) with a luminometer (GloMax®-Multi Detection System, Promega). Corrected and taken as promoter activity.

Figure 3 shows the relative promoter activity values in each cell when the promoter activity in mock miRNA-introduced hepatic stellate cells to which TGF-β1 and Wnt3A are not added is set to 1. In miR-6848-introduced hepatic stellate cells, Wnt3A-induced suppression of TCF/LEF promoter activity was observed, but no such tendency was observed for NFAT and AP-1 promoter activities. This suggests that miR-6848 suppresses canonical Wnt pathway activation, but not non-canonical Wnt pathway activation.

Example 3 Inhibition of liver fibrosis by introduction of miRNA into liver fibrosis model mice 1) Preparation of miR-6848-encapsulated MEND It was manufactured according to the method described in JP 2016-141664. Specifically, to a tert-butanol solution containing a cationic lipid, cholesterol, and methoxyethylene glycol 2000 dimethylglycerol, an aqueous solution containing miR-6848 was added under stirring to obtain a lipid-miR-6848 mixed solution, and then quenched. MENDs encapsulating miR-6848 (miR-6848) were obtained by injecting the lipid-miR-6848 mixed solution into an acid buffer (pH 4.0) under agitation, followed by addition of PBS followed by ultrafiltration purification. 6848 encapsulated MEND) were manufactured. Similarly, miR-342-encapsulated MENDs (miR-342-encapsulated MENDs) and negative control mimics (AccuTarget ^TM miRNA mimic Negative control #1, Purification: BioRP, Bionner) were also encapsulated in MENDs (negative control mimic Encapsulated MEND) was manufactured. A 20% PBS solution of each manufactured MEND was used for administration to mice.

2) Induction of hepatic fibrosis and administration of miR-6848-encapsulated MEND C57BL/6J mice (male, 6 weeks old) were acclimatized for 1 week, then 2 μL/g body weight of 20% carbon tetrachloride solution (in olive oil). A solution of carbon tetrachloride) or vehicle (olive oil only) was administered intraperitoneally twice a week for 4 weeks. In parallel with the administration of carbon tetrachloride, 1 μL / g body weight of miR-6848-encapsulated MEND solution, miR-342-encapsulated MEND solution, negative control mimic-encapsulated MEND or vehicle (PBS) was administered once a week with carbon tetrachloride. Intravenous administration (orbital plexus administration; Retro Orbital Injection) was performed on a different day. Four weeks after the start of carbon tetrachloride administration, livers were collected from the mice. The test schedule is shown in FIG.

A tissue section was created from the collected liver and stained with Sirius Red. For sections after staining, the area of the region stained red with Sirius Red was measured using image analysis software WinROOF2018 Ver 4.15.0 (MITANI) and divided by the area of the entire observation field.

The results are shown in Figure 5. Mice administered with carbon tetrachloride alone (CCl4 in the figure) showed less Sirius-Red staining area (type I and III type collagen) increased. In addition to carbon tetrachloride, miR-6848-encapsulated MEND was administered (6848 in the figure). , NC) had a reduced Sirius-Red staining area. These results confirmed that miR-6848 exhibited an inhibitory effect on hepatic fibrosis in vivo.

SEQ ID NO: 1 hsa-miR-6848-5p nucleotide sequence SEQ ID NO: 2 hsa-miR-6848-3p nucleotide sequence SEQ ID NO: 3 hsa-miR-6848 nucleotide sequence SEQ ID NO: 4 DNA encoding hsa-miR-6848 Base sequence SEQ ID NO: 5 hsa-miR-29a-3p base sequence SEQ ID NO: 6 hsa-miR-449a base sequence SEQ ID NO: 7 hsa-miR-342-3p base sequence SEQ ID NO: 8 Base of forward primer for αSMA gene amplification SEQ ID NO: 9 Nucleotide sequence of reverse primer for amplifying αSMA gene SEQ ID NO: 10 Nucleotide sequence of forward primer for amplifying collagen I gene SEQ ID NO: 11 Nucleotide sequence of reverse primer for amplifying collagen I gene

Claims (14)

A pharmaceutical composition for suppressing hepatic fibrosis, comprising at least one nucleic acid selected from the group consisting of a) to e) below.
a) A nucleic acid having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
b) Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand
d) a precursor of the nucleic acid of any of the above a) to c)
e) A nucleic acid that retains the nucleic acid encoding any of the nucleic acids a) to d) above or its precursor in an expressible manner 2. The pharmaceutical composition according to claim 1, wherein the nucleic acid of a) consists of the base sequence shown in SEQ ID NO: 1 or consists of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 1. . 3. The medicament according to claim 1 or 2, wherein the nucleic acid b) consists of the base sequence shown in SEQ ID NO: 2, or consists of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 2. Composition. 4. Any one of claims 1 to 3, wherein the precursor of d) consists of the base sequence shown in SEQ ID NO: 3, or consists of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 3. The pharmaceutical composition according to item 1. The nucleic acid of e) holds a nucleic acid consisting of the base sequence shown in SEQ ID NO: 4, or a nucleic acid consisting of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 4 so that it can be expressed. The pharmaceutical composition according to any one of claims 1 to 4, which is The pharmaceutical composition according to any one of claims 1 to 5, wherein the nucleic acids a) and b) are mature microRNAs, and the precursors d) are primary microRNAs or precursor microRNAs. The pharmaceutical composition according to any one of claims 1 to 6, for use in subjects undergoing or having received treatment with a therapeutic drug for hepatitis C virus. The pharmaceutical composition according to claim 7, for use in a subject who has achieved a remarkable virologic response by treatment with a therapeutic drug for hepatitis C virus. A pharmaceutical composition according to any one of claims 1 to 8 for the treatment of hepatitis C, wherein said treatment involves the use of a therapeutic agent for hepatitis C virus. A pharmaceutical for treating hepatitis C, comprising at least one nucleic acid selected from the group consisting of the following a) to e) in combination with a therapeutic drug for hepatitis C virus.
a) A nucleic acid having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
b) Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand
d) a precursor of the nucleic acid of any of the above a) to c)
e) A nucleic acid that retains the nucleic acid encoding any of the nucleic acids a) to d) above or its precursor in an expressible manner A kit for treating hepatitis C, comprising at least one nucleic acid selected from the group consisting of the following a) to e) and a therapeutic drug for hepatitis C virus.
a) A nucleic acid having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
b) Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases
c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand
d) a precursor of the nucleic acid of any of the above a) to c)
e) A nucleic acid that retains the nucleic acid encoding any of the nucleic acids a) to d) above or its precursor in an expressible manner A method of evaluating the hepatic fibrosis-suppressing activity of a test substance, using the activity of inducing or enhancing the expression of hsa-miR-6848-5p, hsa-miR-6848-3p, or their precursors as an index. Incubating a test substance with hepatic stellate cells; Measuring the expression level of hsa-miR-6848-5p, hsa-miR-6848-3p or their precursors in hepatic stellate cells after incubation; , comparing the expression levels of hsa-miR-6848-5p, hsa-miR-6848-3p, or their precursors in the absence of the test substance; and hsa-miR-6848-5p, hsa-miR-6848 13. The method according to claim 12, comprising the step of determining that the test substance that induces the expression of -3p or their precursors or increases the expression level thereof has hepatic fibrosis-suppressing activity. incubating a test substance with cells containing a nucleic acid having a marker gene under the control of a promoter region that controls the expression of the hsa-mir-6848 gene; measuring the intensity of the marker gene-derived signal in the cell; A step of comparing the intensity of the marker gene-derived signal with the intensity of the marker gene-derived signal in the absence of the test substance; and determining that the test substance that increased the intensity of the marker gene-derived signal has hepatic fibrosis-suppressing activity. 13. The method of claim 12, comprising the step of:

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