KR20230118040A - Pharmaceutical composition for the prevention or treatment of liver fibrosis comprising a miR-4449 inhibitor as an active ingredient in patients with non-alcoholic steatohepatitis - Google Patents

Abstract

The present invention provides miR-4449 as a biomarker for diagnosing the onset or progression of liver fibrosis in patients with non-alcoholic steatohepatitis, and that the prevention or treatment of liver fibrosis is possible through the inhibition of miR-4449. As confirmed at the human level, miR-4449 is provided as a target for the prevention or treatment of liver fibrosis.

Description

Pharmaceutical composition for the prevention or treatment of liver fibrosis comprising a miR-4449 inhibitor as an active ingredient in patients with non-alcoholic steatohepatitis non-alcoholic steatohepatitis}

The present invention provides miR-4449 as a target for preventing or treating liver fibrosis in non-alcoholic steatohepatitis patients.

Nonalcoholic fatty liver disease (NAFLD) is a disease in which triglyceride is excessively accumulated in the liver regardless of alcohol intake, and the prevalence is increasing worldwide. It is reported to have

NAFLD is divided into simple steatosis and nonalcoholic steatohepatitis (NASH). Simple fatty liver, which accounts for about 75% of all NAFLD, is a state in which excessive fat is deposited and is generally benign with a good clinical prognosis. Although considered a disease, NASH, which accounts for about 25%, is accompanied by hepatocellular damage and inflammation along with fat deposition, and is highly likely to progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma.

Although there is a need for drugs to prevent the treatment and progression of non-alcoholic steatohepatitis, a drug that effectively improves fibrosis and steatohepatitis has not been developed to date.

The present inventors have proposed miR-4449 as a biomarker for the differential diagnosis of non-alcoholic steatohepatitis from simple fatty liver patients among NAFLD patients to prevent disease progression and enable early treatment (10-2020-0065623). Accordingly, the present inventors further completed the present invention by intensively studying whether the biomarker could serve as a target for treatment.

KR 10-2020-0065623

A technical problem to be achieved by the present invention is to provide miR-4449 as a biomarker for diagnosing the onset or possibility of liver fibrosis in patients with non-alcoholic fatty liver disease.

In addition, the present invention has confirmed that the progress of liver fibrosis can be inhibited through the inhibition of miR-4449, and thus provides a pharmaceutical composition for preventing or treating liver fibrosis containing an inhibitor of miR-4449 as an active ingredient. .

In addition, the present invention is a method for providing information for diagnosing the onset or possibility of liver fibrosis in patients with non-alcoholic fatty liver disease and a drug screening for the development of drugs capable of preventing or treating the onset of liver fibrosis in patients with non-alcoholic fatty liver disease. provides a way

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

In order to solve the above problems, the present invention provides miR-4449 as a target for preventing or treating liver fibrosis.

In addition, the present invention provides a pharmaceutical composition for preventing or treating liver fibrosis, including an agent that inhibits the expression of miRNA-4449.

As an embodiment of the present invention, the agent may be at least one selected from the group consisting of siRNA, aptamer, antisense oligonucleotide, and compound.

As another embodiment of the present invention, the agent may increase the level of merilin expression reduced by overexpressed miRNA-4449 in liver cells and may increase the phosphorylation level of TAZ in liver cells.

As another embodiment of the present invention, the composition may be provided for use in preventing or treating liver fibrosis in patients with non-alcoholic steatohepatitis.

In addition, the present invention provides a screening method for a drug for preventing or treating liver fibrosis, comprising the following steps:

(1) treating liver cells with a candidate substance and measuring the expression level of miRNA-4449; and

(2) selecting the candidate substance as a preventive or therapeutic agent for non-alcoholic steatohepatitis when the expression level of miRNA-4449 is decreased.

In addition, the present invention provides an information providing method for predicting the risk of liver fibrosis progression in patients with non-alcoholic fatty liver disease, including the following steps:

(1) preparing a biological sample from a non-alcoholic fatty liver patient;

(2) checking the level of miRNA-4449 in the biological sample; and

(3) classifying into a high-risk group for liver fibrosis when the expression level of the miRNA-4449 is higher than that of the control group using non-alcoholic fatty liver patients who have not progressed in fibrosis as a control group.

The present invention relates to miR-4449 as a biomarker capable of diagnosing the progress or possibility of liver fibrosis in patients with non-alcoholic steatohepatitis. As confirmed that it can inhibit, the present invention is considered to be useful for preventing the progression to liver fibrosis or for diagnosing liver fibrosis in patients with non-alcoholic steatohepatitis.

Figure 1 is a heat map comparing miRNA expression in serum in 15 patients with non-alcoholic steatohepatitis without uncomplicated fatty liver or fibrosis and 9 patients with non-alcoholic steatohepatitis with fibrosis.
Figure 2 confirms the expression level of miR-4449 in the liver tissue of non-alcoholic steatohepatitis patients without uncomplicated fatty liver or fibrosis and non-alcoholic steatohepatitis patients with fibrosis by performing RT-RCR.
3 is a diagram predicting the secondary structure of Merlin as a target of miR-4449 and its interaction.
Figure 4 confirms the expression level of Merlin in the liver tissue of non-alcoholic steatohepatitis patients with or without fibrosis by performing RT-RCR.
Figure 5 compares and confirms the expression level of Merlin when miR-4449 mimics or inhibitors are treated in liver cells.
Figure 6 compares and confirms the phosphorylation level of TAZ when liver cells are treated with miR-4449 mimics or inhibitors.
Figure 7 compares and confirms the expression of genes related to hepatic fibrosis by treating hepatic stellate cells with the supernatant obtained by treating liver cells with miR-4449 mimics or inhibitors.

The present inventors studied the effect of miR-4449, which is overexpressed in non-alcoholic fatty liver disease, and in the case of patients with steatohepatitis with advanced fibrosis among patients with non-alcoholic fatty liver disease, the expression level of miR-4449 was significantly higher than that of non-alcoholic fatty liver disease or non-fibrotic patients. It was confirmed that it was higher than that of patients with alcoholic steatohepatitis, and it was confirmed that miR-4449 affects the expression of Merlin and is involved in the activation of TAZ, which is involved in the mechanism of liver fibrosis, and the present invention was completed.

Accordingly, the present invention provides miR-4449 as a target for preventing or treating liver fibrosis.

The present inventors confirmed a high level of miR-4449 expression in the liver tissue of non-alcoholic steatohepatitis patients with advanced fibrosis compared to simple fatty liver or non-alcoholic steatohepatitis patients without advanced fibrosis. It was confirmed that treatment with the mimic reduced the expression of Merlin and treatment with the inhibitor of miR-4449 increased the expression of Merlin. In addition, the present inventors confirmed that the activity of TAZ, an important signal regulating liver fibrosis, was regulated by the expression level of miR-4449.

Accordingly, the present inventors provide a pharmaceutical composition for preventing or treating liver fibrosis comprising a miR-4449 inhibitor as an active ingredient.

In the present invention, the miR-4449 inhibitor inhibits the expression of miR-4449, and inhibiting the expression of miR-4449 in the present specification prevents the action of miR-4449 with a target or induces degradation of miR-4449. siRNA, aptamers, antisense oligonucleotides, and compounds, and the like.

In addition, the present invention provides an information providing method for diagnosing a high-risk group patient who is highly likely to progress to liver fibrosis in a patient diagnosed with non-alcoholic fatty liver disease.

In order to measure the expression level of miRNA-4449 present in a sample, various agents capable of detecting microRNA can be used.

In the present invention, “diagnosis” refers to determining the susceptibility of a subject to a specific disease or disorder, determining whether an individual currently has a specific disease or disorder, or determining a subject suffering from a specific disease or disorder determining the prognosis of, or therametrics (eg, monitoring the condition of an individual to provide information about the efficacy of a treatment).

In addition, the present invention provides a kit for distinguishing a high-risk group of steatohepatitis accompanied by liver fibrosis for patients with non-alcoholic fatty liver disease, including an agent capable of measuring the expression level of miRNA-4449.

In the present invention, the high-risk group for steatohepatitis with liver fibrosis means that liver fibrosis has already occurred or is highly likely to occur.

In the present invention, antisense oligonucleotides, primers, probes, and/or antibodies that specifically bind to miRNA-4449 can be used as miRNA-4449 expression level measurement agents, and known methods used to measure gene expression levels, such as For example, the expression level of miRNA-4449 can be measured by PCR using primers or by hybridization using a probe. In addition to the measurement agent, the kit may include various tools and reagents known in the art for facilitating detection, for example, a suitable carrier, a label material capable of generating a detectable signal, and a stabilizer.

In the present invention, the biological sample may be a non-invasive sample, and the non-invasive sample may be blood, serum, urine, or saliva, preferably blood or serum, and more preferably serum. It is not limited.

In the present invention, miRNA-4449 is a functional equivalent of the oligonucleotide constituting it, for example, some base sequences of the miRNA-4449 oligonucleotide are modified by deletion, substitution, or insertion, It is a concept that includes variants and mimics that can have the same functional action as the microRNA-4449 nucleic acid molecule.

In the present invention, "antisense oligonucleotide" is a nucleotide sequence that binds complementary to the miRNA and suppresses its expression, and includes, but is not limited to, antisenseRNA and antagonist miRNA.

In the present invention, "probe" refers to a nucleic acid fragment such as RNA or DNA composed of several to hundreds of bases that can specifically bind to miRNA, and is labeled so that the presence or absence of a specific miRNA can be confirmed. The probe may be prepared in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, and the like, and may be labeled with biotin, FITC, rhodamine, DIG, or the like, or labeled with a radioactive isotope.

In the present invention, the kit may be a kit including essential elements required to perform RT-PCR, but is not limited thereto. In the case of an RT-PCR kit as an example, in addition to each primer pair specific for a marker gene, a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors , DEPC-water, sterile water, and the like.

In addition, the present invention can provide a method for providing information for diagnosing a high-risk group for liver fibrosis in non-alcoholic fatty liver patients, including measuring the expression level of miRNA-4449 in a patient's biological sample, and the information providing method The step of comparing the measured expression level of miRNA-4449 with that of non-alcoholic fatty liver patients in which fibrosis has not progressed, and determining a high-risk group for liver fibrosis if higher than this level may further include.

The miRNA expression level measurement is RT-PCR, competitive RT-PCR, real-time RT-PCR, quantitative RT-PCR, RNase protection assay (RPA) RNase protection assay), Northern blotting, or DNA chip method, but is not limited thereto.

In the present invention, the miRNA expression inhibitor is characterized in that an inhibitor specific to miRNA-4449 is used, and the expression inhibitor reduces the level of miRNA-4449 in cells by reducing the expression level of miRNA4449 or promoting its degradation. or specifically bind to miRNA-4449 to reduce its activity, and is characterized in that it is selected from the group consisting of siRNA, aptamer, antisense oligonucleotide and compound. The compound may be used as long as it inhibits the expression of miRNA among antioxidants, substances affecting cell growth, and cell signaling substances. However, it is not limited thereto, and any substance that inhibits the expression of miRNA-4449 may be used.

In the present invention, "siRNA" refers to a small RNA fragment of 21-25 nucleotides in size produced by cleavage of double-stranded RNA by Dicer, which specifically binds to miRNA having a complementary sequence and inhibits its expression. For the purpose of the present invention, it means to inhibit the expression of miRNA by specifically binding to miRNA-4449. siRNA can be synthesized chemically or enzymatically. The method for preparing siRNA is not particularly limited, and methods known in the art may be used. For example, a method for chemically synthesizing siRNA directly, a method for synthesizing siRNA using in vitro transcription, and a method for cutting long double-stranded RNA synthesized by in vitro transcription using an enzyme , expression methods through intracellular delivery of shRNA expression plasmids or viral vectors, and expression methods through intracellular delivery of polymerase chain reaction (PCR)-induced siRNA expression cassettes, but are not limited thereto.

In the present invention, the introduction of the miRNA inhibitor into cells may be performed using a microinjection method, a calcium phosphate co-precipitation method, an electroporation method, and a liposome method, but is not limited thereto.

In the present invention, prevention means any action that delays fibrosis of liver tissue or abnormalities thereof by administration of the composition according to the present invention, and treatment means fibrosis of liver tissue by administration of the pharmaceutical composition according to the present invention and its It refers to all actions that improve or beneficially change the symptoms of metabolic abnormalities. In addition, the subject subject to administration of the pharmaceutical composition of the present invention is not limited as long as it is a mammal, but may preferably be a human, and more specifically, a non-alcoholic fatty liver disease patient.

In the present invention, the pharmaceutical composition (pharmaceutical composition) may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions.

In the present invention, "carrier" is also called a vehicle, and means a compound that facilitates the addition of proteins or peptides into cells or tissues. For example, dimethyl sulfoxide (DMSO) is or a commonly used carrier that facilitates the incorporation of many organisms into tissues.

In the present invention, "diluent" is defined as a compound diluted in water that not only stabilizes the biologically active form of a protein or peptide of interest, but also dissolves the protein or peptide. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline because it mimics the salt state of human solutions. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely modify the biological activity of a compound. Compounds containing azelaic acid as used herein may be administered to human patients by themselves or as pharmaceutical compositions mixed with other ingredients, as in combination therapy, or with suitable carriers or excipients.

In addition, the pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis according to the present invention is prepared in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and sterile injection solutions according to conventional methods, respectively. It may be formulated and used, and carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin , calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, decomposers, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose, etc. ) or by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions for oral use, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogeratin and the like may be used.

The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc. can

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity. It can be.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or can be prepared by incorporating into a large-capacity container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, since various changes can be made to the embodiments, the scope of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents or substitutes to the embodiments are included within the scope of rights.

Terms used in the examples are used only for descriptive purposes and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't

In addition, in the description with reference to the accompanying drawings, the same reference numerals are given to the same components regardless of reference numerals, and overlapping descriptions thereof will be omitted. In describing the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description will be omitted.

[Example]

Example 1. Confirmation of overexpression of miRNA-4449 in steatohepatitis patients with fibrosis

1-1. Production of heat map of miRNA expression targeting the raw sample

Serum and liver tissues of 24 patients diagnosed with non-alcoholic fatty liver disease through histological examination were collected and analyzed. When miRNA expression in serum was compared using RNA sequencing in 15 patients with simple fatty liver or non-alcoholic steatohepatitis without fibrosis and 9 patients with NAFLD with fibrosis, miR-4449 showed no significant difference in non-alcoholic steatohepatitis with fibrosis. It was significantly increased in patients with alcoholic steatohepatitis (FIG. 1).

1-2. Confirmation of miRNA-4449 overexpression in tissue samples

RT-RCT was performed using the liver tissue of the patient of Example 1 above. As a result, it was confirmed that the expression level of miRNA-4449 was higher in the liver tissue of non-alcoholic steatohepatitis patients with fibrosis than patients with simple fatty liver or non-alcoholic steatohepatitis without fibrosis (FIG. 2).

Example 2. Search for target molecules of miRNA-4449 and confirm relationship with miRNA-44449

Candidate target genes for miR-4449 are diverse. Among them, Moesin-Ezrin-Radixin-Like Protein (merlin) was identified and its secondary structure was predicted through interaction with miRNA-4449 (FIG. 3). Subsequently, as a result of confirming the expression level of the Merlin gene in the liver tissue of the patient of Example 1, compared to simple fatty liver or non-alcoholic steatohepatitis patients without fibrosis in the liver tissue of patients with non-alcoholic steatohepatitis accompanied by fibrosis A low level of Merlin expression was confirmed (FIG. 4).

Subsequently, to confirm whether the expression level of miRNA-4449 directly affects the expression level of Merlin, miR-4449 mimics (has-miR-4449 mimics, Ambion, ID: MC22101, Cat#) were introduced into Hep3B, a hepatocyte cell line. 4464066 / mirVana ^TM miRNA mimics Negative control #1 , Ambion, Cat# 4464058 ) or inhibitor ( has-miR-4449 inhibitor, Ambion, ID: MH22101, Cat# 4464084 / mirVana ^TM miRNA inhibitor Negative control #1, Cat# 4464076) After treatment with Nega, RT-PCR was performed to confirm the expression level of Merlin. As a control, palmitic acid was treated. As a result, it was confirmed that the expression of Merlin significantly decreased when the miR-4449 mimic was treated, and the expression of Merlin increased when the miR-4449 inhibitor was treated (FIG. 5).

Example 3. Confirmation of the relationship between miRNA-4449 and TAZ

Merlin is known to regulate TAZ phosphorylation in controlling liver fibrosis in NAFLD. Therefore, in order to confirm the mechanism by which miR-4449 participates in the progression from non-alcoholic fatty liver disease to fibrosis, the level of phosphorylated TAZ was examined in liver cells treated with miR-4449 mimics and inhibitors. As a result, it was confirmed through Western blot that TAZ phosphorylation was suppressed in miR-4449 mimic-treated liver cells, and TAZ phosphorylation increased in miR-4449 inhibitor-treated liver cells (FIG. 6). .

Example 4. Confirmation of relationship between miRNA-4449 and liver glandular cells

Hepatic stellate cells are known to be the most important cells in the progression of liver fibrosis in all liver diseases. Therefore, in order to confirm the mechanism by which miR-4449 regulates the progression from non-alcoholic fatty liver disease to fibrosis, hepatic stellate cells were treated with mimic or inhibitor-treated liver cell cultures of miR-4449 to generate genes related to liver fibrosis. Expression was confirmed. As a result, the expression of genes related to fibrosis, such as a-SMA, collagenase 1a1, and TGF-β, increased in hepatic stellate cells treated with the culture medium derived from hepatocytes treated with miR-4449 mimics, and miR-4449 The expression of genes related to fibrosis, such as a-SMA, collagenase 1a1, and TGF-β, was decreased in hepatic stellate cells treated with the culture medium derived from hepatocytes treated with inhibitors of (FIG. 7).

As described above, although the embodiments have been described with limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, the described techniques may be performed in an order different from the method described, and/or components of the described system, structure, device, circuit, etc. may be combined or combined in a different form than the method described, or other components may be used. Or even if it is replaced or substituted by equivalents, appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents of the claims are within the scope of the following claims.

Claims (7)

A pharmaceutical composition for preventing or treating liver fibrosis, comprising an agent that inhibits the expression of miRNA-4449. According to claim 1,
The pharmaceutical composition, characterized in that the agent is at least one selected from the group consisting of siRNA, aptamer, antisense oligonucleotide, and compound. According to claim 1,
The pharmaceutical composition, wherein the agent increases the level of merilin expression reduced by miRNA-4449 overexpressed in liver cells. According to claim 1,
The pharmaceutical composition, wherein the agent activates TAZ in liver cells. According to claim 1,
The composition is intended for use in the prevention or treatment of liver fibrosis for patients with non-alcoholic steatohepatitis, a pharmaceutical composition. A method for screening a drug for preventing or treating liver fibrosis, comprising the following steps:
(1) treating liver cells with a candidate substance and measuring the expression level of miRNA-4449; and
(2) selecting the candidate substance as a preventive or therapeutic agent for non-alcoholic steatohepatitis when the expression level of miRNA-4449 is decreased. Information provision method for predicting the risk of liver fibrosis progression in patients with non-alcoholic steatohepatitis disease, including the following steps:
(1) preparing a biological sample from patients with non-alcoholic steatohepatitis;
(2) checking the level of miRNA-4449 in the biological sample; and
(3) classifying into a high-risk group for liver fibrosis when the expression level of the miRNA-4449 is higher than that of the control group in patients with non-alcoholic steatohepatitis in which fibrosis has not progressed.