KR20220163046A - Composition for Preventing or Treating Non-Alcoholic SteatoHepatitis Comprising Peptide Nucleic Acid Complex - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising a nucleic acid composite as an active ingredient for treating non-alcoholic steatohepatitis (NASH) and, more specifically, to a pharmaceutical composition for preventing or treating NASH, which comprises a nucleic acid composite where bioactive peptide nucleic acid having a sequence binding to an NLRP3 gene and carrier peptide nucleic acid complementarily bind to each other. The nucleic acid composite according to the present invention has excellent cell penetration and intracellular activity and can effectively inhibit expression of the NLRP3 gene and a sub-gene thereof to be useful for preventing or treating NASH.

Description

[Composition for Preventing or Treating Non-Alcoholic SteatoHepatitis Comprising Peptide Nucleic Acid Complex}

The present invention relates to a pharmaceutical composition for the treatment of non-alcoholic steatohepatitis containing a nucleic acid complex as an active ingredient, and more particularly, to a bioactive peptide nucleic acid having a sequence binding to NLRP3 gene; And a pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis containing a nucleic acid complex in which a carrier peptide nucleic acid is complementarily bound.

Non-alcoholic steatohepatitis is a condition in which inflammation and cell damage occur in the liver due to accumulation of fat in the liver, and if the lesion worsens due to continuous liver damage, it is a disease that can progress to liver fibrosis, liver cirrhosis, and liver cancer. Currently, the prevalence of non-alcoholic steatohepatitis is increasing not only in Korea but also worldwide, causing major problems in terms of health, society, and economy (Povero et al., Hepatol Commun. Vol. 4(9), pp. 1263- 1278, 2020).

However, there are no drugs or treatments that have been proven effective in the treatment of non-alcoholic steatohepatitis. Therefore, most of the treatments are for the underlying disease that causes steatohepatitis, and lifestyle changes such as exercise, diet, and weight loss are required. In addition, treatment for diabetes and insulin resistance, treatment for hyperlipidemia, prevention and treatment of oxidative stress, and other liver protection It is being treated with medication.

Korean Patent No. 10-2177304 describes a compound effective for the treatment of nonalcoholic steatohepatitis, and Korean Patent Nos. 10-1837173 and 10-2239804 disclose a composition for preventing or treating nonalcoholic steatohepatitis containing a ginseng extract. Although described, the specific molecular mechanism has not been elucidated, and is only based on alleviation of symptoms.

NLRP3 is a signaling substance that induces inflammatory activity and is known to have increased expression in adipocytes of patients with non-alcoholic steatohepatitis. In addition, the expression of caspase-1, a subgene of NLRP3, is also increased in human and mouse adipose tissue, suggesting that adipocytes It has been reported to aggravate differentiation and nonalcoholic steatohepatitis (Claire H Wilson et al., Cell Death & Differentiation, Vol.25, pp. 1010-1024, 2018).

On the other hand, unlike traditional drugs, nucleic acid drugs inhibit the expression of target-specific messenger RNA (mRNA), so it is possible to address research areas that could not be treated with existing protein-targeting drugs (Kole R. et al. al., Nature Rev. Drug Discov. 11;125, 2012., Wilson C. et al., Curr. Opin. Chem. Bio. 2006; 10:607, 2006). Due to its performance and advantages as a drug, various clinical trials using nucleic acids are in progress, and despite the increasing use of nucleic acid-based therapeutics, the use of carriers for intracellular introduction or blood-brain barrier penetration is extremely limited.

Therefore, in recent years, in order to promote safer and more effective use of drugs, not only formulations suitable for drugs, but also methods for efficiently delivering drugs to biological sites targeted by drugs have attracted attention. In particular, there is a demand for practical use of a drug delivery system (DDS), which is a transport system that efficiently transports a drug to a required location in a required amount at a required time.

In this regard, the present inventors have found that a nucleic acid complex in which a bioactive nucleic acid and a carrier peptide nucleic acid modified to have a positive charge as a whole are complementarily coupled have surprisingly improved cell permeability, and using this It was confirmed that the expression of the target gene can be controlled very efficiently, and a patent was applied for a new construct with low cytotoxicity and improved cell permeability of bioactive nucleic acids and the ability to regulate gene expression (PCT/KR2017/008636 ). In addition, the present inventors have continuously conducted research on the function of the construct and developed a new construct having the ability to penetrate the blood-brain barrier with excellent efficiency (KR 10-2019-0128465).

Accordingly, the present inventors have made intensive efforts to apply a nucleic acid complex having excellent cell permeability to the treatment of non-alcoholic steatohepatitis, and as a result, a bioactive peptide nucleic acid targeting the NLRP3 gene (Bioactive Peptide Nucleic Acid); And carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) It was confirmed that the nucleic acid complex complementarily bound shows an excellent effect in the prevention or treatment of non-alcoholic steatohepatitis, and the present invention was completed.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis containing a nucleic acid complex as an active ingredient.

In order to achieve the above object, the present invention is a bioactive nucleic acid targeting the NLRP3 gene (Bioactive Nucleic Acid); And a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) provides a pharmaceutical composition for preventing, improving or treating non-alcoholic steatohepatitis containing a cell-permeable nucleic acid complex complementarily bound thereto as an active ingredient.

The nucleic acid complex according to the present invention has excellent cell penetrability and intracellular activity, and can effectively suppress the expression of the NLRP3 gene and subgenes thereof, so that it is useful for preventing or treating non-alcoholic steatohepatitis.

Figure 1 shows the results of confirming the expression of NLRP3 target genes and sub-pathway genes over time after treating human liver cancer cells (HepG2) with the peptide nucleic acid complex of the present invention.
Figure 2 (A) shows the result of confirming NLRP3 protein expression according to time for each combination after treating human liver cancer cells (HepG2) with the peptide-nucleic acid complex of the present invention.
Figure 3 shows the result of confirming body weight (upper panel) and liver weight (lower panel) for each combination after treating STZ (streptozotocin) and a high-fat diet animal model with the peptide-nucleic acid complex of the present invention.
Figure 4 shows the results of confirming the expression of the NLRP3 gene and subgenes for each combination after treating STZ (streptozotocin) and a high-fat diet animal model with the peptide-nucleic acid complex of the present invention.
Figure 5a shows the results of H&E staining for each combination of liver tissue obtained after treatment with STZ (streptozotocin) and a high-fat diet animal model with the peptide nucleic acid complex of the present invention (A) and lesions in liver tissue (steatosis (B), cell balloon) These are the confirmation results of inflammation (C), inflammatory cell infiltration (D), and NAFLD activity score (E)).
Figure 5b shows the results of Oil red O (ORO) staining for each combination of liver tissue obtained after treatment of STZ (streptozotocin) and a high-fat diet animal model with the peptide-nucleic acid complex of the present invention.
Figure 5c shows the results of picrosirius red staining for each combination of liver tissues obtained after treatment with STZ (streptozotocin) and a high-fat diet animal model with the peptide-nucleic acid complex of the present invention, (A) is a micrograph, and (B) is a fibrosis It is a graph that quantifies the degree.
Figure 6a is an immunostaining chemistry result (A) and a quantification graph (B) of the NLRP3 gene obtained after treating STZ (streptozotocin) and a high-fat diet animal model with the peptide-nucleic acid complex of the present invention.
Figure 6b is an immunostaining chemical result (A) and a quantification graph (B) of IL-1β, a sub-pathway gene of the NLRP3 gene, obtained after treating STZ (streptozotocin) and a high-fat diet animal model with the peptide-nucleic acid complex of the present invention. )to be.
Figure 7 is a photograph (A) and a graph (B) of weight change of liver tissue obtained after treating an MCD-fed animal model with the peptide-nucleic acid complex of the present invention.
8 is a result of confirming the expression of the NLRP3 gene and sub-pathway genes for each combination obtained after treating the MCD-fed animal model with the peptide-nucleic acid complex of the present invention.
Figure 9a shows the results of H&E staining for each combination of liver tissue obtained after treatment with the peptide nucleic acid complex of the present invention in an animal model fed with MCD (A) and lesions in the liver tissue (steatosis (B), cell ballooning (C), These are the confirmation results of inflammatory cell infiltration (D) and NAFLD activity score (E)).
Figure 9b shows the results of Oil red O (ORO) staining for each combination of liver tissues obtained after treatment of an MCD-fed animal model with the peptide-nucleic acid complex of the present invention.
Figure 9c shows the result of picrosirius red staining for each combination of liver tissues obtained after treatment with the peptide nucleic acid complex of the present invention in an animal model fed with MCD, (A) is a micrograph, and (B) is a graph quantifying the degree of fibrosis to be.
10 is a photograph (A), a weight change graph (B), and a liver weight (C) of liver tissue for each combination obtained after treatment of an animal model on the HFHC+F/G diet with the peptide-nucleic acid complex of the present invention.
Figure 11 shows the results of liver function analysis (ALT (A), AST (B) and TG (C)) for each combination obtained by ELISA after treatment with the peptide nucleic acid complex of the present invention in a HFHC + F / G diet animal model. to be.
12 is a result of confirming the expression of the NLRP3 gene and sub-pathway genes for each combination obtained after treating the HFHC+F/G diet animal model with the peptide nucleic acid complex of the present invention.
Figure 13a shows the results of H&E staining for each combination of liver tissue obtained after treatment with the peptide nucleic acid complex of the present invention in an animal model fed with HFHC + F / G diet (A) and lesions in liver tissue (steatosis (B), cell ballooning) (C), inflammatory cell infiltration (D) and NAFLD activity score (E)) are the confirmation results.
Figure 13b shows the results of Oil red O (ORO) staining for each combination of liver tissues obtained after treatment of the HFHC+F/G diet animal model with the peptide-nucleic acid complex of the present invention.
Figure 13c shows the results of picrosirius red staining for each combination of liver tissues obtained after treatment with the peptide nucleic acid complex of the present invention in an animal model fed with HFHC + F / G diet, (A) is a photomicrograph, (B) is a degree of fibrosis is a numerical graph of
Figure 14 is a photograph (A), weight change graph (B), and liver weight of liver tissues for each combination obtained after treatment with the peptide nucleic acid complex of the present invention for an additional 4 weeks in an HFHC + F / G diet animal model. C) is.
15 is an analysis of liver function by combination (ALT (A), AST (B) and TG (C)) is the result.
16 shows the results of confirming the expression of the NLRP3 gene and sub-pathway genes for each combination obtained after additionally treating the HFHC+F/G diet animal model with the peptide-nucleic acid complex of the present invention for 4 weeks.
Figure 17a shows the results of H&E staining (A) and intra-hepatic lesions (steatosis (B) for each combination of liver tissue obtained after treatment with the peptide nucleic acid complex of the present invention for an additional 4 weeks in an animal model on the HFHC + F/G diet. ), cell ballooning (C), inflammatory cell infiltration (D), and NAFLD activity score (E)).
Figure 17b shows the results of Oil red O (ORO) staining for each combination of liver tissues obtained after additionally treating the HFHC+F/G diet animal model with the peptide-nucleic acid complex of the present invention for 4 weeks.
Figure 17c shows the results of picrosirius red staining for each combination of liver tissues obtained after further treatment with the peptide nucleic acid complex of the present invention for 4 weeks in an HFHC + F / G diet animal model, (A) is a photomicrograph, ( B) is a graph digitizing the degree of fibrosis.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are those well known and commonly used in the art.

In the present invention, a bioactive peptide nucleic acid targeting the NLRP3 gene; And carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) When administering a nucleic acid complex complementarily bound, it was intended to determine whether it is effective in preventing and treating non-alcoholic steatohepatitis.

That is, in one embodiment of the present invention, a bioactive peptide nucleic acid having a sequence binding to the NLRP3 gene; and carrier peptide nucleic acid (Carrier Peptide Nucleic Acid), when administered to a non-alcoholic steatohepatitis cell line and a non-alcoholic steatohepatitis cell line orthotopic transplant model, the expression of the NLRP3 gene and its subgenes is efficiently It was confirmed that the treatment effect of non-alcoholic steatohepatitis was excellent.

Accordingly, the present invention, in one aspect, a bioactive peptide nucleic acid targeting the NLRP3 gene (Bioactive Peptide Nucleic Acid); and a pharmaceutical composition for preventing, improving, or treating non-alcoholic steatohepatitis, comprising a nucleic acid complex complementarily coupled to a carrier peptide nucleic acid as an active ingredient.

In the present invention, the bioactive nucleic acid may be a bioactive peptide nucleic acid.

In the present invention, a nucleic acid complex in which a bioactive peptide nucleic acid and a carrier peptide are complementaryly bonded may have a structure represented by the following structural formula (1).

[Structural formula (1)]

[A ≡C ⁽⁺⁾ ]

In the structural formula (1),

A is a sequence capable of binding to a gene of interest, or a bioactive nucleic acid having a sequence of a gene of interest,

C is a carrier peptide nucleic acid capable of binding to a bioactive nucleic acid;

'≡ means a complementary bond between a bioactive nucleic acid and a carrier peptide nucleic acid,

The bioactive nucleic acid represented by A is a peptide nucleic acid and has a negative charge as a whole.

The carrier peptide nucleic acid represented by C ⁽⁺⁾ has an overall positive charge,

A The nucleic acid complex of structural formula (1) represented by ≡ C ⁽⁺⁾ has a positive charge as a whole,

The carrier peptide nucleic acid includes one or more gamma- or alpha-backbone modified peptide nucleic acid monomers such that the carrier peptide nucleic acid is positively charged as a whole, and the gamma- or alpha-backbone modified peptide nucleic acid monomer is a positively charged amino acid. It is characterized in that the charge of the carrier peptide nucleic acid as a whole is positive because more monomers are included than monomers having amino acids having negative charges.

In the present invention, "biological nucleic acid" has a complementary sequence capable of binding to a target gene of interest whose expression is to be reduced, in particular a complementary sequence capable of binding to the mRNA of such a target gene of interest, A nucleic acid containing a nucleic acid containing a sequence that promotes the expression of a target gene to be expressed, and refers to a nucleic acid involved in the regulation of gene expression, such as inhibiting or promoting the expression of the gene, and decreasing or increasing the expression. It may be a nucleic acid having a sequence complementary to a target gene to be targeted, or a nucleic acid having a sequence complementary to a single-stranded RNA sequence such as pre-mRNA, miRNA, or mRNA.

In particular, the “Bioactive Peptide Nucleic Acid” in the present invention binds to a target gene and a nucleotide sequence containing the target gene in vitro or in vivo, thereby functioning as a unique function of the gene. Functions such as activating or inhibiting (eg, transcript expression or protein expression), regulating pre-mRNA splicing (eg, exon skipping) The nucleotide sequence may be a gene regulatory sequence, a gene coding sequence, or a splicing regulatory sequence. The gene regulatory region may include a promoter, Transcription enhancer, 5' untranslated region, 3' untranslated region, virus packaging sequence, and selectable marker.The gene region may be an exon or an intron, and the gene region It may be within 10, 5, 3, or 1 kb or 500, 300, or 200 bp of the initiation site, for example upstream or downstream of the initiation site. It may include sequences related to exon skipping, cryptic splicing, pseudo-splice site activation, intron retention, and alternative splicing deregulation.

Therefore, the "bioactive peptide nucleic acid" in the present invention is preferably an antisense peptide nucleic acid of NLRP3, a non-alcoholic steatohepatitis target gene, more preferably the sequence represented by the sequence of SEQ ID NO: 1 Including, but not limited to.

In the present invention, "carrier peptide nucleic acid" refers to a nucleic acid to which a bioactive nucleic acid and some or all of its bases complementarily bind to give functionality, and the carrier peptide nucleic acid used in the present invention is a peptide nucleic acid (PNA: Peptide Nucleic Acid), as well as modified nucleic acids similar thereto, and peptide nucleic acids are preferred, but are not limited thereto.

In particular, in the present invention, the carrier peptide nucleic acid preferably includes a sequence represented by a sequence selected from the group consisting of SEQ ID NOs: 4 to 7, but is not limited thereto.

In the present invention, the "nucleic acid complex" can penetrate the bioactive substance into the body, ultimately into cells, and specifically has the ability to pass through the blood-brain barrier and deliver into the body.

Accordingly, in the present invention, the nucleic acid complex may have blood-brain barrier penetrating ability.

In the present invention, the bioactive nucleic acid and the carrier peptide nucleic acid each comprise 2 to 50, preferably 5 to 30, more preferably 10 to 25, most preferably 15 to 17 nucleic acid monomers. that can be characterized.

In the present invention, the nucleic acid complex comprises a bioactive nucleic acid comprising the sequence represented by SEQ ID NO: 2; and a carrier peptide nucleic acid comprising a sequence represented by any one sequence selected from the group consisting of SEQ ID NOs: 4 to 7, but is not limited thereto.

The composition of the present invention comprises (i) a nucleic acid complex composed of a bioactive nucleic acid comprising the sequence represented by SEQ ID NO: 2 and a carrier peptide nucleic acid comprising the sequence represented by SEQ ID NO: 4;

(ii) a nucleic acid complex composed of a bioactive nucleic acid comprising the sequence represented by SEQ ID NO: 2 and a carrier peptide nucleic acid comprising the sequence represented by SEQ ID NO: 5;

(iii) a nucleic acid complex composed of a bioactive nucleic acid comprising the sequence represented by SEQ ID NO: 2 and a carrier peptide nucleic acid comprising the sequence represented by SEQ ID NO: 6; and

(iv) a nucleic acid complex composed of a bioactive nucleic acid comprising the sequence represented by SEQ ID NO: 2 and a carrier peptide nucleic acid comprising the sequence represented by SEQ ID NO: 7;

It is preferable to include a nucleic acid complex selected from the group consisting of as an active ingredient, but is not limited thereto.

In the present invention, the bioactive nucleic acid or the carrier peptide nucleic acid may be characterized by additionally binding a substance that helps endosome escape to the 5'-end or 3'-end of each nucleic acid. That is, it may be characterized in that it has a structure of the following Structural Formula (2) by further including a material that helps the endosome escape of the bioactive nucleic acid and the carrier peptide nucleic acid.

structural formula (2)

[ mA ≡ mC ⁽⁺⁾ ]

In the structural formula (2),

'm' means a substance that helps endosome escape of bioactive nucleic acid and carrier peptide nucleic acid.

In the present invention, "substances that help endosomes escape" can be characterized in that they help the escape of bioactive nucleic acids from endosomes by increasing the osmotic pressure inside the endosomes or by destabilizing the membranes of endosomes. have. It means that bioactive nucleic acids move more efficiently and quickly to the nucleus or cytoplasm to meet and act on target genes (D. W. Pack, A. S. Hoffman, S. Pun, P. S. Stayton, “and development of polymers for gene delivery,” Nat (Rev. Drug. Discov., Vol. 4, pp. 581-593, 2005).

In the present invention, the material that helps the endosome escape is a peptide, lipid nanoparticles, conjugate nanoparticles (polyplex nanoparticles), polymer nanospheres (polymer nanospheres), inorganic nanomaterials (inorganic nanoparticles), cationic lipids It may be characterized in that at least one selected from the group consisting of nanomaterials (cationic lipid-based nanoparticles), cationic polymers (cationic polymers) and pH sensitive polymers (pH sensitive polymers).

In the present invention, a peptide having the sequence of GLFDIIKKIAESF (SEQ ID NO: 8) may be linked to the bioactive nucleic acid as a material that helps the endosome escape through a linker, and to the carrier peptide nucleic acid, Histidine (10) is linked through a linker It may be characterized by combining in a way, but is not limited thereto.

In the present invention, the lipid nanoparticles may be selected from the group consisting of Lipid, phospholipids, acetyl palmitate, poloxamer 18, Tween 85, tristearin glyceride and Tween 80.

In the present invention, the polyplex nanoparticles may be poly(amidoamine) or polyethylenimine (PEI).

In the present invention, the polymer nanospheres are selected from the group consisting of polycaprolactone, poly(lactide-co-glycolide, polylactide, polyglycolide, poly(d,l-lactide), chitosan, and PLGA-polyethylene glycol. can be characterized.

In the present invention, the inorganic nanoparticles may be selected from the group consisting of Fe ₂ O ₃ Fe ₃ O ₄ , WO3 and WO2.9.

In the present invention, the cationic lipid-based nanoparticles are 1- (aminoethyl) iminobis [N- (oleicylcysteinyl-1-amino-ethyl) propionamide], N-alkylated derivative of PTA and 3, 5- It may be characterized in that it is selected from the group consisting of didodecyloxybenzamidine.

In the present invention, the cationic polymer may be selected from the group consisting of vinylpyrrolidone-N, N-dimethylaminoethyl methacrylate acid copolymer diethyl sulphate, polyisobutylene and poly(N-vinylcarbazole).

In the present invention, the pH sensitive polymers may be selected from the group consisting of polyacids, poly(acrylic acid), poly(methacrylic acid), and hydrolyzed polyacrylamide.

In the present invention, the bioactive nucleic acid may be characterized in that it consists of a natural nucleic acid base and/or a modified nucleic acid monomer, and the carrier peptide nucleic acid has a part or all of the base sequence complementary to the bioactive nucleic acid. It can be characterized as consisting of a unique sequence.

In particular, the carrier peptide nucleic acid may include one or more universal bases, and all of the carrier peptide nucleic acids may consist of universal bases.

In the present invention, the bioactive nucleic acid is DNA, RNA, or modified nucleic acids such as peptide nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO), locked nucleic acid (LNA), glycol nucleic acid (GNA), and three It may be selected from the group consisting of nucleic acid), antisense oligonucleotide, aptamer, small interfering RNA (siRNA), short hairpin RNA (shRNA), ribozyme and DNAzyme, Preferably, the bioactive nucleic acid is DNA, RNA, or a modified nucleic acid such as peptide nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO), locked nucleic acid (LNA), glycol nucleic acid (GNA), and three nucleic acid (TNA). ) It may be selected from the group consisting of.

In the present invention, when the monomer used for the bioactive nucleic acid is PNA, it is referred to as a bioactive peptide nucleic acid, and when other monomers are used, it is referred to in the same way.

In the present invention, the bioactive nucleic acid and the carrier peptide nucleic acid are phosphodiester, 2' 0-methyl (2' 0-methyl), 2' methoxy-ethyl (2' methoxy-ethyl), phosphor It may be characterized by further comprising at least one functional group selected from the group consisting of amidate, methylphosphonate, and phosphorothioate.

In the present invention, the carrier peptide nucleic acid may be characterized in that a part or all of the base sequence of the bioactive nucleic acid is composed of a complementary sequence. In particular, the carrier peptide nucleic acid may include one or more universal bases, and all of the carrier peptide nucleic acids may consist of universal bases.

In the present invention, each of the bioactive nucleic acid and the carrier peptide nucleic acid in the nucleic acid complex may be a complex characterized by having a positive charge (positive), negative charge (negative) or neutral charge as a whole.

In the expression of the electrical properties, the meaning of "overall" means the electrical properties of the entire bioactive nucleic acid or carrier peptide nucleic acid as a whole, not the electrical properties of individual bases, when viewed from the outside. Even if some of the monomers in the sexual nucleic acid have a positive charge, if there are more monomers with a negative charge, the bioactive nucleic acid will have a negative charge when looking at the electrical properties “as a whole”, and some bases and/or Even if the backbone has a negative charge, when a larger number of bases and/or backbones having a positive charge are present, the carrier peptide nucleic acid has a positive charge when looking at the electrical characteristics “as a whole”.

Accordingly, the nucleic acid complex of the present invention may be characterized as having a positive charge as a whole. In the nucleic acid complex, preferably, the bioactive nucleic acid has negative or neutral electrical characteristics as a whole, and the carrier peptide nucleic acid has positive electrical characteristics as a whole. It is not limited thereto.

In the present invention, the electrical properties of the bioactive nucleic acid and the carrier peptide nucleic acid can be imparted using a modified peptide nucleic acid monomer, and the modified peptide nucleic acid monomer is a carrier peptide nucleic acid having a positive charge, such as lysine (Lysine, Lys, K) , arginine (Arg, R), histidine (Histidine, His, H), diamino butyric acid (DAB), ornithine (Orn), and any one or more positive charges selected from the group consisting of amino acid analogs It is a carrier peptide nucleic acid that includes amino acids of and has a negative charge, and may be characterized in that it includes a negatively charged amino acid such as glutamic acid (Glu, E) or an amino acid analog that is negatively charged.

In the present invention, the carrier peptide nucleic acid may be characterized by including one or more gamma- or alpha-backbone modified peptide nucleic acid monomers so as to have a positive charge as a whole.

The gamma- or alpha-backbone modified peptide nucleic acid monomer has lysine (Lysine, Lys, K), arginine (Arginine, Arg, R), histidine (His, H), diamino butyric acid (Diamino butyric acid) to have an electrical positivity. acid, DAB), ornithine (Orn), and amino acids having at least one positive charge selected from the group consisting of amino acid analogs.

In the present invention, the modification of the peptide nucleic acid monomer for charge imparting may use a peptide nucleic acid monomer having a modified nucleobase in addition to the backbone modification. Preferably, an amine, triazole, or imidazole moiety may be included in the nucleobase to have an electronegative property, or a carboxylic acid may be included in the base to have an electronegative property.

In the present invention, the modified peptide nucleic acid monomer of the carrier peptide nucleic acid may further contain a negative charge in the backbone or nucleobase, but the modified peptide nucleic acid monomer contains more positively charged monomers than monomers having negative charges, so that the carrier peptide as a whole is formed. It is preferred that the charge of the nucleic acid be positive.

In the nucleic acid complex according to the present invention, at least one material selected from the group consisting of a hydrophobic moiety, a hydrophilic moiety, a target antigen-specific antibody, an aptamer, or a fluorescent/luminescent marker is a bioactive nucleic acid And / or may be characterized in that it is bound to a carrier peptide nucleic acid, preferably the hydrophobic moiety, the hydrophilic moiety, a target antigen-specific antibody, an aptamer, and a fluorescent / luminescent marker for imaging One or more substances selected from the group consisting of and the like may be bound to the carrier peptide nucleic acid.

In the present invention, at least one material selected from the group consisting of a hydrophobic moiety, a hydrophilic moiety, a target antigen-specific antibody, an aptamer, a quencher, a fluorescent marker, and a luminescent marker, and a bioactive nucleic acid and/or The binding of the carrier peptide nucleic acid may be characterized as simple covalent bond or linker mediated covalent bond, but is not limited thereto. Preferably, substances related to cell permeation, solubility, stability, transport and imaging (eg, hydrophobic residues, etc.) bound to the nucleic acid carrier exist independently of the bioactive nucleic acid that regulates the expression of the target gene.

In the present invention, as described above, the complementary binding form of the bioactive nucleic acid and the carrier peptide nucleic acid may be largely characterized by having the form of antiparallel binding and parallel binding. . The complementary binding form has a structure that separates in the presence of a target sequence of a bioactive nucleic acid (a sequence complementary to the bioactive nucleic acid).

The antiparallel bond and the parallel bond are determined according to the 5'-direction and the 3'-direction in the binding method of DNA-DNA or DNA-PNA. Antiparallel coupling is a general DNA-DNA or DNA-PNA coupling method. Taking the nucleic acid complex according to the present invention as an example, the bioactive nucleic acid is in the 5' to 3' direction and the carrier peptide nucleic acid is in the 3' to 5' direction. It means that the shape is connected to each other in the direction. Parallel binding is a form in which binding force is somewhat lower than that of anti-parallel binding, and refers to a form in which both the bioactive nucleic acid and the carrier peptide nucleic acid are bonded to each other in the 5' to 3' direction or the 3' to 5' direction.

In the nucleic acid complex according to the present invention, preferably, the binding force of the bioactive nucleic acid and the carrier peptide nucleic acid is lower than the binding force of the bioactive nucleic acid and the target gene, particularly the mRNA of the target gene. . The binding force is determined by melting temperature, melting temperature or Tm.

As an example of a specific method for making the binding strength (melting temperature, Tm) of the bioactive nucleic acid and the carrier peptide nucleic acid lower than the binding strength of the bioactive nucleic acid and the bioactive nucleic acid to the target gene, in particular, the mRNA of the target gene, the above Parallel binding or partial specific binding between the bioactive nucleic acid and the carrier peptide nucleic acid may be characterized, but is not limited thereto.

As another example, the carrier peptide nucleic acid has at least one peptide nucleic acid base selected from the group consisting of a linker, a universal base, and a peptide nucleic acid base having a base that is not complementary to a corresponding base of a bioactive nucleic acid. It can be, but is not limited thereto.

In the present invention, the universal base binds to natural bases such as adenine, guanine, cytosine, thymine, and uracil without selectivity, and is complementary to One or more bases selected from the group consisting of inosine PNA, indole PNA, nitroindole PNA, and abasic may be used as a base having a binding force lower than the binding force, preferably. It can be characterized by using inosine PNA.

In the present invention, a combination of the binding form and electrical properties of nucleic acids for function control of the nucleic acid complex is provided, and the combination of the binding form and electrical properties of the nucleic acids controls the particle size and the time point of action, cell permeability, solubility and specificity It can be characterized as improving the degree.

In the present invention, the binding force of the bioactive peptide nucleic acid and the carrier peptide nucleic acid is adjusted, in the presence of the target gene, when the bioactive peptide nucleic acid binds to the target sequence (when the bioactive nucleic acid is substituted with the target sequence, Target specific separation and binding time) etc. can be adjusted.

In the nucleic acid complex according to the present invention, the control of the strand displacement time point and the target specific release and bind time point of the bioactive nucleic acid into the target gene is a carrier peptide for non-specific binding of the complex It can be characterized in that it can be controlled by the presence, number, and location of non-specific bases, universal bases, and linkers in nucleic acids. It may be characterized in that control is possible by a combination of the above conditions, such as a parallel or antiparallel bond, which is a form of complementary bond of the peptide complex.

As used herein, the term "disease" or "disorder" are used interchangeably herein, which impairs or disrupts the performance of a function and/or causes symptoms such as discomfort, dysfunction, difficulty, or even disease. Any change in the state of the body or some organ that results in the death of a person or the death of a person who comes into contact with it.

In the present invention, the term “therapeutic composition” may be used interchangeably with “pharmaceutical composition” and includes the bioactive nucleic acid of the present invention and a carrier peptide nucleic acid binding to the nucleic acid. It includes a nucleic acid complex as an active ingredient, and additionally, a therapeutic drug for treating a desired disease may be combined with the nucleic acid complex.

The term “physiologically acceptable” refers to the property of not compromising the biological activity and physical properties of a compound.

The term "carrier" is defined as a compound that facilitates the addition of a nucleic acid complex into a cell or tissue. For example, dimethylsulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic compounds into the cells or tissues of living organisms.

The term “diluent” is defined as a compound that is diluted in water which will dissolve the compound as well as stabilize the biologically active form of the subject compound. 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.

A substance containing a nucleic acid complex in the present invention can be administered to a patient by itself or as a mixed pharmaceutical composition together with other active ingredients, such as in combination therapy, or with suitable carriers or excipients.

Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredients are contained in effective amounts to achieve their intended purpose. More specifically, a therapeutically effective amount refers to an amount of a compound effective to prolong the survival of the subject being treated or to prevent, alleviate or ameliorate symptoms of a disease. Determination of a therapeutically effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein.

The term "prevention" used in the present invention refers to all actions that prevent the onset of a disease by administering a therapeutic composition containing the nucleic acid complex or inhibit its progression.

The term "improvement" of the present invention refers to all actions that at least reduce the parameters related to the condition to be treated, for example, the severity of symptoms, by administration of the therapeutic composition containing the nucleic acid complex.

In addition, the term "treatment" used in the present invention refers to any activity in which symptoms of a disease are improved or cured by administration of a composition for treatment containing the nucleic acid complex.

The composition comprising the nucleic acid complex according to the present invention can be applied in a pharmaceutically effective amount to treat non-alcoholic steatohepatitis or to suppress (or alleviate) the symptoms of non-alcoholic steatohepatitis. It can vary depending on various factors such as the type of non-alcoholic steatohepatitis, the patient's age, weight, characteristics and severity of symptoms, type of current treatment, number of treatments, application form and route, etc. can be determined The composition of the present invention may be applied together or sequentially with the pharmacological or physiological components described above, and may be applied in combination with additional conventional therapeutic agents, and may be applied sequentially or simultaneously with conventional therapeutic agents. These applications may be single or multiple applications.

In the present invention, "subject" means a mammal suffering from or at risk of a condition or disease that can be alleviated, inhibited or treated by administering a nucleic acid complex according to the present invention, and preferably means a human.

In addition, the dose of the compound of the present invention to the human body may vary depending on the patient's age, weight, sex, administration form, health condition and disease degree, and when based on an adult patient weighing 70 kg, generally It is 0.001 to 1,000 mg/day, preferably 0.01 to 500 mg/day, and may be administered in divided doses once a day to several times at regular time intervals according to the judgment of a doctor or pharmacist.

Toxicity and therapeutic efficacy of compositions comprising the nucleic acid complexes described herein can be measured by, for example, the LD50 (lethal dose to 50% of the population), the ED50 (the dose that is therapeutically effective in 50% of the population), the IC50 It can be estimated by standard pharmaceutical procedures in cell culture or laboratory animals to determine (the dose that has a therapeutically inhibitory effect on 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between the LD50 and the ED50 (or IC50). Compounds exhibiting high therapeutic indices are preferred. Data obtained from these cell culture assays can be used to estimate a range of human doses. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 (or IC50) with little or no toxicity.

The term "administration" of the present invention refers to the act of introducing the pharmaceutical composition of the present invention to a subject by any appropriate method, and the route of administration may be administered through various oral or parenteral routes as long as it can reach the target tissue. have.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" of the present invention is a reasonable benefit / risk ratio applicable to medical treatment or prevention to treat or prevent a disease It means a sufficient amount, and the effective dose level is the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the administration time of the composition of the present invention used, the route of administration and the excretion rate It may be determined according to the treatment period, factors including drugs used in combination or simultaneous use with the composition of the present invention used, and other factors well known in the medical field.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors.

The dosage of the pharmaceutical composition of the present invention can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the patient's age, weight, sex, history, or the type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention can be administered to mammals including humans at 10 to 100 mg/kg, more preferably 1 to 30 mg/kg, for a day, and the frequency of administration of the composition of the present invention is It is not particularly limited thereto, but may be administered once to three times a day or administered several times by dividing the dose.

In another aspect, the present invention provides a bioactive peptide nucleic acid having a sequence that binds to the NLRP3 gene; and a method for preventing or treating non-alcoholic steatohepatitis comprising administering to a subject a nucleic acid complex to which a carrier peptide nucleic acid is complementarily bound and/or a composition comprising the nucleic acid complex.

In another aspect, the present invention provides a bioactive peptide nucleic acid having a sequence that binds to the NLRP3 gene for the preparation of a drug for preventing or treating non-alcoholic steatohepatitis (Bioactive Peptide Nucleic Acid); and the use of a nucleic acid complex to which a carrier peptide nucleic acid is complementarily bound and/or a composition comprising the nucleic acid complex.

In another aspect, the present invention provides a bioactive peptide nucleic acid having a sequence that binds to the NLRP3 gene; and a nucleic acid complex to which a carrier peptide nucleic acid is complementarily bound and/or a composition comprising the nucleic acid complex is provided for preventing or treating non-alcoholic steatohepatitis.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Bioactive Peptide Nucleic Acid and Carrier Peptide Nucleic Acid, and Preparation of Complexes Using These

In this example, among diseases that develop in the liver, it is a term that includes all of the series of processes leading to fatty liver and liver cirrhosis due to fat deposition in the liver, which is a form of liver tissue similar to alcoholic steatohepatitis despite not drinking much alcohol. To test the effect on alcoholic steatohepatitis (NASH), NLRP3 was used as a target gene, and to confirm the therapeutic effect of non-alcoholic steatohepatitis, a bioactive peptide nucleic acid (Bioactive Peptide Nucleic Acid Acid) was used as an antisense peptide nucleic acid (antisense PNA).

The bioactive peptide nucleic acid (antisense PNA) used as a control of the present invention is composed of the sequence represented by SEQ ID NO: 1, and the bioactive peptide nucleic acid (antisense PNA) used to confirm the therapeutic effect of nonalcoholic steatohepatitis is sequence It consists of the sequence represented by number 2.

The carrier peptide nucleic acids used in this Example are all composed of the sequences shown in the table below in the form of no peptides to help the endosomes escape function. Base sequences, monomer modifications and structures are shown in Table 1 below.

All peptide nucleic acids used in the present invention were synthesized by HPLC purification method at PANAGENE (Korea) (Table 1).

Sequences of bioactive nucleic acids and carrier peptide nucleic acids for verification of non-alcoholic steatohepatitis therapeutic effect division order
number endosome escape
peptide base sequence monomer
transform bioactive nucleic acid One - 5'-AC ^(-) CT ⁽⁺⁾ CTA ^(-) CGC ⁽⁺⁾ AAT ^(-) CC-OK-3' -+-+- 2 - 5'-ACG ^(-) TGC ⁽⁺⁾ ATT ^(-) AT ⁽⁺⁾ CT ^(-) GA-OK-3' -+-+-
carrier peptide
nucleic acid 3 - 5'-TG ⁽⁺⁾ GAGAT ⁽⁺⁾ GCGTTA ⁽⁺⁾ GG-OK-3' +++ 4 - 5'-TGC ⁽⁺⁾ ACGTAA ⁽⁺⁾ TAGA ⁽⁺⁾ CT-OK-3' +++ 5 - 5'-KO-TC ⁽⁺⁾ AGAT ⁽⁺⁾ AATGCA ⁽⁺⁾ CGT-3' +++ 6 - 5′-A ⁽⁺⁾ GA ⁽⁺⁾ CT-OK-3′ ++ 7 - 5'-KO-TC ⁽⁺⁾ AG ⁽⁺⁾ A-3' ++

Table 1 above shows the sequence information of the bioactive peptide nucleic acid and carrier peptide nucleic acid used to confirm the effect as a non-alcoholic steatohepatitis targeting NLRP3 and the carrier peptide nucleic acid used as a control and the bioactive peptide nucleic acid and carrier peptide nucleic acid. .

Modification of monomers converts the backbone of peptide nucleic acid to electrically positive lysine (represented by Lysine, Lys, K, ⁽⁺⁾ ) and electronegatively to glutamic acid (Glu, E, ^(-)) to impart electrical properties. notation) was constructed to have a modified peptide backbone.

Each combination of the bioactive nucleic acid and the carrier peptide nucleic acid was hybridized in the presence of DMSO, and as a result, a complex composed of the bioactive nucleic acid and the carrier peptide nucleic acid was prepared.

Example 2: Analysis of NLRP3 protein expression using Lipopolysaccharide (LPS)

According to Example 1, the inhibitory effect of non-alcoholic steatohepatitis was analyzed using a nucleic acid complex comprising a carrier peptide nucleic acid and a bioactive peptide nucleic acid having NLRP3 as a target gene, prepared according to the structure shown in Table 2 below.

Example 2-1: Cell culture

Human liver cancer cell HepG2 (ATCC, HB-8065) obtained from ATCC (American Type Culture Collection, USA) was cultured in DMEM culture medium (Dulbecco Modified Eagle Medium, Wellgene, Korea), 10% (v/v) fetal bovine serum, and penicillin. 100 units/ml and 100 μg/ml of streptomycin were added and cultured under the conditions of 37°C and 5% (v/v) CO ₂ .

Example 2-2: Analysis of gene expression by Western blot assay

The human liver cancer cell line HepG2 was seeded in 1x10 ⁵ cells in a 6-well plate, cultured for 24 hours, and the experiment was performed under the conditions of Example 2-1 to treat a complex containing a bioactive peptide nucleic acid and a carrier peptide nucleic acid, and LPS 2 μg / ㎖ (Sigma, USA) was induced and cultured for 24, 48, 72, 96, and 120 hours, and 30 μl of RIPA buffer was added to each well to obtain protein lysate.

Protein lysate was quantified using a BCA assay kit (Thermo Fisher, USA), and 30 μg of protein was separated by size through electrophoresis, and the protein was transferred to a PVDF membrane, followed by primary antibody, NLRP3 (Abcam, UK). , ASC (Cell Signaling Technology, USA), IL-1β (Abcam, UK), and Caspase-1 (Abcam, UK) were treated at a ratio of 1:1000 and left at 4° C. for one day.

After washing with 1X TBS-T, secondary antibodies, Goat Anti-Rabbit (Cell signaling Technology, USA) and Mouse Anti-Goat (Santa cruz Biotechnology, USA) were treated at a ratio of 1:2000 and allowed to stand at room temperature for 1 hour. The cells were treated with Supersignal ^TM West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA) and analyzed for target gene expression inhibition efficiency in human liver cancer cell lines using Image600 (Amersham, Germany).

Nucleic acid complex combinations used in this Example are shown in Table 2 below.

Combination of Nucleic Acid Complexes for Gene Expression Analysis in Human Liver Cancer Cell Lines Combination nucleic acid complex One SEQ ID NOs 1 and 3 2 SEQ ID NOs 2 and 4 3 SEQ ID NOs 2 and 5 4 SEQ ID NOs 2 and 6 5 SEQ ID NOs 2 and 7

As a result, as shown in FIG. 1, in HepG2 cells in which inflammation was induced by LPS, the nucleic acid complex of the combination of SEQ ID NOs: 2 and 5 (combination 3) suppressed both the target gene, NLRP3, and subgenes for up to 96 hours. It was confirmed (Fig. 1).

Example 2-3: Analysis of protein expression through immunocytochemistry

Human liver cancer cell lines were seeded in 5x10 ³ cells in an 8-well plate, cultured for 24 hours, and the experiment was conducted under the conditions of Example 2-2 to treat a complex containing a bioactive peptide nucleic acid and a carrier peptide nucleic acid, and after 4 hours LPS After treatment with 2 μg/ml (Sigma, USA) and incubation for 24 and 72 hours, fixation in 4% paraformaldehyde, 0.1% Triton X-100 and normal donkey serum After blocking with ), the primary antibody, NLRP3 (abcam, USA) was treated at 1:200 and left at 4 ° C for one day.

Then, the secondary antibody, Donkey Anti-Goat IgG H&L (Alexa Fluor®647) (abcam, USA) was treated at a ratio of 1:200, left at room temperature for 1 hour, fixed using a mounting agent containing DAPI, and the result was observed under a microscope. confirmed.

In this example, the NLRP3 expression change in the non-alcoholic steatohepatitis cell model using LPS was analyzed through immunocytochemical staining, and the nucleic acid complex combinations used are shown in Table 3 below.

Combination of Nucleic Acid Complexes for Protein Expression Analysis in Human Liver Cancer Cell Lines Combination nucleic acid complex One SEQ ID NOs 1 and 3 3 SEQ ID NOs 2 and 5 5 SEQ ID NOs 2 and 6

As a result, as shown in FIG. 2, the nucleic acid complex combination of SEQ ID NO: 2 and SEQ ID NO: 5 (combination 3) in the human liver cancer cell line in which the immune response was activated by treatment with LPS (combination 3) expressed NLRP3 protein in the non-alcoholic steatohepatitis cell model. It was confirmed that the most inhibited according to this time.

Example 3: Analysis of nonalcoholic steatohepatitis phenotypic effect in nonalcoholic steatohepatitis animal model using high-fat diet and STZ (streptozotocin)

The STAM mouse model, which is an animal model for non-alcoholic steatohepatitis used in Example 3, is a mouse in which diabetes was induced using primary Streptozotocin (STZ), and non-alcoholic steatohepatitis was induced through a secondary high-fat diet. This results in non-alcoholic fatty liver disease (NAFLD) in mice, induction of hepatocytotoxicity, inflammatory infiltration of the portal vein and lobules, fibrosis with nodular formation, sclerosis, and ultimately hepatocellular carcinoma in some individuals. -Alcoholic SteatoHepatitis), a proven model that progresses to HCC (Hepatocellular Carcinoma) (Takakura K et al., World j Gastroenterol. Vol. 24,18 pp. 1989-1994. 2018).

In the STAM model used in this study, streptozotocin (STZ) was injected as a single subcutaneous injection (200 μg / 20 μl) on day 2 after birth, and a high-fat diet was fed 4 weeks after administration.

After lesion induction, nucleic acid complexes were administered by subcutaneous injection twice a week using three animals in each experimental group, and in the case of a positive control group, NLRP3 inhibitor MCC950 (Sigma, USA) was orally administered once daily at a dose of 10 mg/kg did After 2 weeks of administration of the nucleic acid complex and positive control, all animals were euthanized.

In this example, nucleic acid combinations whose effects were verified were selected and histological findings and NLRP3 gene expression changes were analyzed in a non-alcoholic steatohepatitis animal model using a high-fat diet and STZ (streptozotocin). The nucleic acid complex combinations used are shown in the table below. Same as 4.

Nucleic acid complex combination for effect analysis in non-alcoholic steatohepatitis animal model using high-fat diet and STZ (streptozotocin) Combination nucleic acid complex One SEQ ID NOs 1 and 3 3 SEQ ID NOs 2 and 5

Example 3-1: Analysis of liver weight and body weight change in non-alcoholic steatohepatitis animal model (STAM mouse model) using high-fat diet and STZ (streptozotocin)

After the animal experiment was performed under the above conditions, the body weight and the appearance of the liver of the biopsied animal were compared, and differences in size and weight were observed.

As a result, as shown in Figure 3, the body weight and It was confirmed that the weight of the liver decreased, and in particular, it was confirmed that the group treated with the nucleic acid complex (combination 3, 1 mg/kg) and the positive control group (MCC950) showed similar values (Fig. 3).

Example 3-2: Analysis of gene expression by Western blot assay

After animal experiments were performed under the above conditions, protein lysate was obtained by adding 200 μl of RIPA buffer to a portion of mouse liver tissue. Protein lysate was quantified using a BCA assay kit (Thermo Fisher, USA), and 30 μg of protein was separated by size through electrophoresis, and the protein was transferred to a PVDF membrane, followed by primary antibody, NLRP3 (Abcam, UK). , ASC (Cell Signaling Technology, USA), caspase-1 (Abcam, UK), and IL-1β (Abcam, UK) were treated at a ratio of 1:1000 and left at 4° C. for one day.

After washing with 1X TBS-T, secondary antibodies, Goat Anti-Rabbit (Cell signaling Technology, USA) and Mouse Anti-Goat (Santa cruz Biotechnology, USA) were treated at a ratio of 1:2000 and allowed to stand at room temperature for 1 hour. The liver tissue was treated with Supersignal ^TM West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA) and the efficiency of suppression of target gene expression was analyzed using Image600 (Amersham, Germany) equipment.

As a result, as shown in FIG. 4, in the group treated with the nucleic acid complex (PNA3, combination 3) compared to the control group, the expression of NLRP3 target genes and the downstream genes ASC, pro-caspase-1, and pro-IL were similar to the positive control group (MCC950). It was confirmed that the expression of -1β was reduced (FIG. 4).

Example 3-3: Analysis of phenotype in non-alcoholic steatohepatitis animal model (STAM mouse model) using high-fat diet and STZ (streptozotocin) using H&E staining

A portion of the liver of the animal tissues tested under the above conditions was cut, fixed in 4% formalin solution for 24 hours, paraffin-embedded, and the tissue was sectioned into 5 μm and attached to a slide glass. The liver tissue attached to the slide glass was stained with Hematoxylin:Eoin staining solution for a certain period of time, washed with 1X PBS, and morphologically analyzed under a microscope.

The pathological analysis of non-alcoholic steatohepatitis was first presented as a criterion for determining the degree of pathological severity by grading necroinflammatory activity (grading) and structural alterations related to fibrosis (staging) (Brunt et al., the Americal Journal of Gastroentrology 1999, 94(9).

The histopathologic criteria for determining the grade were 10 items: 1) microvesicular steatosis, 2) ballooning of hepatocytes, 3) inflammation of lobules, 4) inflammation of hepatic fibrillation, 5) Mallory hyaline, 6) eosinophils, 7) Kupffer cells, 8) glycogenation nuclei, 9) lipogranuloma, and 10) iron levels in hepatocytes. Using these criteria, two pathologists classified NASH and simple fat deposition without knowing the patient's information and past diagnosis. were reclassified according to three criteria (Kleiner et al., Hepatology, 2005).

1) steatosis (0: less than 5%, 1: 5-33%, 2: 33-66%, 3: when more than 66% of hepatocyte infiltration is observed), 2) intralobular inflammation (0: No foci, 1 : 4 fociper 200x field), 3) Ballooning (0: none, 1: few, 2:many cells/prominent ballooning). By summing the scores of the three criteria, a score of 5 or more was considered NASH, and a score of less than 3 was considered non-NASH, and scores of 3 and 4 were defined as the borderline. Non-alcoholic steatohepatitis score criteria are shown in Table 5 below.

Nonalcoholic Steatohepatitis Animal Model Clinician Criteria Histologic features score Definition Seatosis 0 <5% One 5-33% 2 34-66% 3 >66% Hepatocyte ballooning 0 None One Few 2 many Lobular inflammation 0 None One 1-2 foci per 20 X field 2 2-4 foci per 20 X field 3 >4 foci per 20 X field NAFLD activity score (NAS); range 0-8 fibrosis 0 <5% One 5-33% 2 34-66% 3 >66%

As a result, as shown in FIG. 5a, lesions in liver tissue appearing in non-alcoholic steatohepatitis, such as steatosis, cell ballooning, and inflammatory cell infiltration induced through the STAM mouse model, compared to the control group (PNA1, combination 1), nucleic acid complex (PNA3, It decreased in the group treated with combination 3), and it was confirmed that the effect was equivalent to that of the positive control group (MCC950), an NLRP3 inhibitor.

Example 3-4: Analysis of lipid deposition inhibitory effect in the liver in a STAM mouse model using a high-fat diet and STZ (streptozotocin) using ORO staining

Liver tissue was isolated on the final day of the experiment after the animal experiment was conducted under the above conditions. Tissues were fixed in a 4% formalin solution for 24 hours, then dehydrated in a 30% sucrose solution for 24 hours, and the OCT compound was embedded. It was sectioned to 5 μm and attached to a slide glass. After staining in Oil red O staining solution for a certain period of time and washing with 1X PBS, morphological analysis was performed under a microscope.

As a result, as shown in FIG. 5b, the distribution and size of lipid deposition in the liver induced by the STAM mouse model were reduced in the group treated with the nucleic acid complex (PNA3, combination 3) compared to the control group (PNA1, combination 1), and the positive control group (MCC950), it is expected that the nucleic acid complex (PNA3, combination 3) inhibits fat accumulation in liver tissue to prevent or treat the occurrence of non-alcoholic steatohepatitis (FIG. 5b).

Example 3-5: Analysis of liver fibrosis inhibition effect in non-alcoholic steatohepatitis animal model (STAM mouse model) using high-fat diet and STZ (streptozotocin) using picrosirius red staining

Liver tissue isolated from animal experiments under the above conditions was fixed in a 4% formalin solution for 24 hours, embedded in paraffin, and the tissue was sectioned into 5 μm and attached to a slide glass. Picrosirius red staining was performed to confirm the production of collagen. The mounted tissue was stained with a picrosirius red staining solution for a certain period of time, washed with 1X PBS, and the effect of inhibiting liver fibrosis was analyzed under a microscope.

As a result, as shown in FIG. 5c, it was confirmed that fibrosis in liver tissue induced through the STAM mouse model was reduced to a level similar to that of the positive control group (MCC950) in the group treated with the nucleic acid complex (PNA3, combination 3) compared to the control group (Fig. 5c).

Example 3-6: Expression change analysis of NLRP3 and IL-1β, a downstream gene, in tissue in a non-alcoholic steatohepatitis animal model (STAM mouse model) using a high-fat diet and STZ (streptozotocin) using immunostaining

Animal experiments were performed under the above conditions, and liver tissues isolated at the end of the final experiment were fixed in a 4% formalin solution for one day, and the fixed tissues were paraffin-embedded, sectioned into 5 μm, and attached to slide glass. In order to identify the NLRP3 Inflammasome, which is a major factor in fibrosis, changes in the expression of NLRP and IL-1β, a subgene, were analyzed. The mounted tissue was blocked in 0.5% BSA solution for 1 hour, and NLRP3 (Abcam, UK) and IL-1β primary antibody (Abcam, UK) were left at 4°C for one day.

Then, the primary antibody solution was removed, washed with 1X TBS, treated with secondary antibody solution Goat Anti-Rabbit (Cell signaling Technology, USA) at a ratio of 1:2000 at room temperature for 2 hours, and then analyzed by DAB staining.

As a result, as shown in Figures 6a and b, the expression of NLRP3 and IL-1β stained brown in the control tissue was confirmed, and the expression of NLRP3 and IL-1β was reduced in the group treated with the nucleic acid complex (combination 3). was observed in all subjects. When compared by quantification, it was confirmed that the expression of each gene was reduced by the nucleic acid complex (combination 3) at a level similar to that of the positive control (Fig. 6a, b).

Example 4: Analysis of non-alcoholic steatohepatitis phenotype effect in non-alcoholic steatohepatitis animal model (MCD mouse model) using methionine/choline deficient diet

In Example 4, among animal models showing a phenotype similar to non-alcoholic steatohepatitis disease, an animal model using a MCD (Methionine-Choline Deficient diet) diet was used, and the diet was high in sucrose and fat and showed liver beta-oxidation and lack of methionine and choline, which are essential for the production of very low density lipoprotein (VLDL). Animal models subjected to this diet are known to result in lipid accumulation in the liver and decrease in VLDL synthesis, and when the diet progresses up to 2-4 weeks, it not only induces hepatic steatosis (mainly macrovesicular steatosis) in rodents within a short period of time. It has been reported that it progresses to fibrosis afterwards.

However, although it is known that the representative phenotype of the disease in non-alcoholic steatohepatitis appears in the animal model using the MCD diet, it does not show weight loss or insulin resistance, which is different from the phenotype of actual NASH patients with obesity and insulin resistance. There may be some differences (Anstee et al., Int J Exp Pathol, Vol. 87(1), pp. 1-16, 2006).

In order to verify the efficacy in non-alcoholic steatohepatitis animal model using the MCD diet, 7-week-old male mice (Coretech, Korea) were used. After mooring for a week, a methionine/choline deficient diet (Dooyeol Biotech, Korea) was conducted to induce a non-alcoholic steatohepatitis model, and after 4 weeks of ingestion, six animals per experimental group were used for control twice a week (SC, combination). 1) and nucleic acid complexes (Combination 3-1: 0.5 mg/kg, Combination 3-2: 1 mg/kg, Combination 3-3: 2 mg/kg) were administered by subcutaneous injection.

In the case of the positive control group, MCC950 (Sigma, USA), an NLRP3 inhibitor, was orally administered once daily at a dose of 10 mg/kg. The nucleic acid complex was administered a total of 4 times for 2 weeks, and then all animals were euthanized.

In this example, nucleic acid combinations whose effects were verified were selected and histological findings and NLRP3 gene expression changes were analyzed in a non-alcoholic steatohepatitis animal model using a methionine/choline deficient diet. The nucleic acid complex combinations used are shown in Table 6 below. .

Nucleic acid complex combination for effect analysis in non-alcoholic steatohepatitis animal model using methionine/choline deficient diet Combination nucleic acid complex One SEQ ID NOs 1 and 3 3 SEQ ID NOs 2 and 5

Example 4-1: Analysis of body weight change in non-alcoholic steatohepatitis animal model (MCD mouse model) using a methionine/choline deficient diet

In animal experiments conducted under the above conditions, differences in size and weight were observed by comparing body weight and liver appearance after the experiment was completed.

As a result, as shown in FIG. 7, the size of the external liver in the group treated with the nucleic acid complex of SEQ ID NOs: 2 and 5 (combination 3) compared to the induction group (MCD) and control group (Scramble control, SC, combination 1). It was confirmed that the browning reaction was restored as the treatment concentration increased, and in particular, it was confirmed that the liver was restored to red color than the group treated with the positive control group (Gemcitabine, Positive control, PC). Due to the characteristics of the MCD diet, the model induction group could confirm a pattern of weight loss compared to the negative control (NC), and it was confirmed that the nucleic acid complex (combination 3) administration group showed slight recovery from weight loss compared to the control group (Fig. 7).

Example 4-2: Analysis of gene expression by Western blot assay

After animal experiments were performed under the above conditions, protein lysate was obtained by adding 200 μl of RIPA buffer to a part of the biopsied mouse liver tissue. Protein lysate was quantified using a BCA assay kit (Thermo Fisher, USA), and 30 μg of protein was separated by size through electrophoresis, and the protein was transferred to a PVDF membrane, followed by primary antibody, NLRP3 (Abcam, UK). , ASC (Cell Signaling Technology, USA), caspase-1 (Abcam, UK), and IL-1β (Abcam, UK) were treated at a ratio of 1:1000 and left at 4° C. for one day.

After washing with 1X TBS-T, secondary antibodies, Goat Anti-Rabbit (Cell signaling Technology, USA) and Mouse Anti-Goat (Santa cruz Biotechnology, USA) were treated at a ratio of 1:2000 and allowed to stand at room temperature for 1 hour. The liver tissue was treated with Supersignal ^TM West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA) and the efficiency of suppression of target gene expression was analyzed using Image600 (Amersham, Germany) equipment.

As a result, as shown in FIG. 8, expression of NLRP3 target genes and downstream genes ASC, pro-caspase-1, pro-IL- It was confirmed that the expression of 1β was reduced, and in particular, in the group treated with the concentrations of the nucleic acid complex combinations 3-1, 3-2 and 3-3, it was confirmed that the expression of the target gene decreased similarly to the positive control group (MCC950) ( Fig. 8).

Example 4-3: Analysis of phenotype in non-alcoholic steatohepatitis animal model (MCD mouse model) using methionine/choline deficient diet using H&E staining

Animal experiments were performed under the above conditions, and liver tissues isolated on the final day of the experiment were fixed in 4% formalin solution for 24 hours, embedded in paraffin, and sectioned into 5 μm and attached to slide glass. The mounted tissues were stained with Hematoxylin:Eosin staining solution for a certain period of time, washed with 1X PBS, and morphological findings were analyzed under a microscope.

As a result, as shown in FIG. 9a, the disease phenotype (steatosis, cell ballooning, inflammatory cell infiltration, etc.) in non-alcoholic steatohepatitis induced by the MCD diet was higher in the group treated with the nucleic acid complex (combination 3) compared to the control group. It was confirmed that the lesion was improved by decreasing. In the NAFLD activity score described in Example 3-3, it was confirmed that the concentration of nucleic acid complexes 3-1 and 3-2 was recovered to a level similar to that of the NLRP3 inhibitor positive control group (FIG. 9a).

Example 4-4: Analysis of lipid deposition inhibitory effect in the liver in a non-alcoholic steatohepatitis animal model (MCD mouse model) using a methionine/choline deficient diet using ORO staining

After the experiment was performed under the above conditions, the liver tissue of the mouse biopsied was fixed in a 4% formalin solution for 24 hours, then dehydrated in a 30% sucrose solution for 24 hours, and then embedded in OCT compound. It was sectioned to 5 μm and attached to a slide glass. The mounted tissues were stained with Oil red O staining solution for a certain period of time, washed with 1X PBS, and morphological findings were analyzed under a microscope.

As a result, lipid deposition in the form of red droplets in the liver tissue induced by the MCD diet was confirmed in the disease induction group (MCD), compared to the induction group and the control group (PNA1, combination 1), as shown in FIG. 9b. In the group treated with the nucleic acid complex (PNA3, combination 3), it was confirmed that the distribution or size of red-stained fat droplets decreased (FIG. 9b).

Example 4-5: Analysis of liver fibrosis inhibition effect in nonalcoholic steatohepatitis animal model (MCD mouse model) using methionine/choline deficient diet using picrosirius red staining

Tissues fixed, paraffin-embedded, and sectioned under the same conditions as in Example 4-3 and mounted on slide glass were stained with picrosirius red staining solution for a certain period of time, washed with 1X PBS, and morphological findings were analyzed under a microscope.

As a result, as shown in FIG. 9c , it was confirmed that the characteristics of liver fibrosis, which were stained red by the MCD diet, were reduced in the group treated with the nucleic acid complex (PNA3, combination 3) compared to the control group, and at all concentrations (PNA3) treated with the nucleic acid complex. -1, 3-2, 3-3), it was confirmed that the effect was equivalent to that of the NLRP3 inhibitor positive control group (FIG. 9c).

Example 5: Analysis of non-alcoholic steatohepatitis phenotypic effects in a non-alcoholic steatohepatitis animal model (HFHC+F/G mouse model) using a high-fat/high-cholesterol diet and fructose/glucose negative water

The HFHC+F/G (High Fat High Cholesterol + Fructose/Glucose) diet, which is an animal model used to induce a phenotype similar to non-alcoholic steatohepatitis disease, increases body weight and body fat in rodent models and rapidly reduces liver fat. Not only can it accumulate, but it can also increase hepatic insulin resistance (Liu, XJ. et al., Lab. Invest. Vol. 98, pp. 1184-1199, 2018).

In order to verify the efficacy in non-alcoholic steatohepatitis animal model through HFHC + F / G diet, 7-week-old male mice (Coretech, Korea) were used. To induce non-alcoholic steatohepatitis model, high-fat/high-cholesterol diet (Raon Bio, Korea) and fructose/glucose (sigma, USA) were consumed for 16 weeks, and then nucleic acid complexes were used three times a week using five animals in each experimental group. was administered by subcutaneous injection. For a positive control, MCC950 (Sigma, USA), an NLRP3 inhibitor, was orally administered once daily at a dose of 10 mg/kg. After 4 weeks of administration of the nucleic acid complex and positive control, all animals were euthanized.

In this example, nucleic acid combinations whose effects have been verified were selected to analyze histological findings and NLRP3 gene expression changes in a non-alcoholic steatohepatitis animal model using a high-fat, high-cholesterol diet and fructose/glucose negative water. The nucleic acid complex combination used is as follows: As shown in Table 7.

Combination of Nucleic Acid Complexes for Effect Analysis in Nonalcoholic Steatohepatitis Animal Model Using High-Fat/High-Cholesterol Diet and Fructose/Glucose Negative Water Combination nucleic acid complex 3 SEQ ID NOs 2 and 5

Example 5-1: Analysis of liver phenotype and weight change in non-alcoholic steatohepatitis animal model (HFHC+F/G mouse model) using a high-fat/high-cholesterol diet and fructose/glucose negative water

In the animal experiment conducted under the above conditions, the color of the liver was observed and the size and weight were measured to compare the body weight and appearance of the liver after the experiment was completed.

As a result, as shown in FIG. 10, when comparing the color of the liver with the naked eye, the non-alcoholic steatohepatitis control group induced by the HFHC + F / G diet and the positive control group (MCC950, MCC950, Positive control, PC) group was observed to change color to yellow and enlarge. On the other hand, in the group treated with the nucleic acid complexes of SEQ ID NOs: 2 and 5 (combination 3, 1 mg/kg), it was observed that the size and weight of the liver decreased compared to the control group, and the color of the liver changed to red. In addition, a statistically significant weight loss was observed in the nucleic acid complex (combination 3) administration group compared to the control group.

Example 5-2: Analysis of liver function improvement effect using enzyme-linked immunosorbent assay (ELISA) analysis

The experiment was conducted under the above conditions, and after blood was collected before the end of the animal experiment, serum was separated using a centrifuge at 13000 rpm and 4 ° C. Separated serum is alanine aminotransferase (ALT or SGPT) Activity Colorimetric/Fluorometric Assay Kit (biovision, USA), Aspartate Aminotransferase (AST or SGOT) Activity Colorimetric Assay Kit (biovision, USA), Triglyceride Assay Kit (Abcam, UK), Cholesterol OD (optical density) was measured and analyzed with a spectrophotometer using an Assay Kit (Abcam, UK).

As a result, as shown in FIG. 11, the levels of acute liver injury marker ALT (Alanine Aminotransferase) and triglyceride marker TG (Triglyceride) in the group treated with the nucleic acid complex (combination 3) compared to the control group were significant compared to the control group and the positive control group. It was found that there was a significant decrease in On the other hand, changes in AST (Aspartate Aminotransferase) were not observed.

Example 5-3: Analysis of gene expression by Western blot assay

After the animal experiment under the above conditions was completed, protein lysate obtained by adding 200 μl of RIPA buffer to a part of the mouse liver tissue biopsied was quantified using a BCA assay kit (Thermo Fisher, USA). 30 μg of protein was separated by size through electrophoresis, transferred to a PVDF membrane, and then primary antibodies NLRP3 (Abcam, UK), ASC (Cell signaling Technology, USA), caspase-1 (Abcam, UK), IL -1β (Abcam, UK) was treated at 1:1000 and left at 4 °C for one day.

After washing with 1X TBS-T, secondary antibodies, Goat Anti-Rabbit (Cell signaling Technology, USA) and Mouse Anti-Goat (Santa cruz Biotechnology, USA) were treated at a ratio of 1:2000 and allowed to stand at room temperature for 1 hour. Supersignal ^TM West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA) was treated, and the expression inhibition efficiency of the target gene was analyzed using Image600 (Amersham, Germany) equipment.

As a result, as shown in FIG. 12, expression of NLRP3 target genes and expression of sub-pathway genes ASC, pro-caspase-1, and pro-IL-1β in the group treated with the nucleic acid complex (combination 3) compared to the model induction group and the control group It was confirmed that all of them decreased (FIG. 12).

Example 5-4: Phenotypic analysis through H&E staining in a non-alcoholic steatohepatitis animal model (HFHC+F/G mouse model) using a high-fat/high-cholesterol diet and fructose/glucose negative water

Animal experiments were conducted under the above conditions, and liver tissue was isolated on the final day of the experiment. The tissue was fixed in 4% formalin solution for 24 hours, embedded in paraffin, and the tissue was sectioned to 5 μm and attached to a slide glass. The mounted tissue was stained with Hematoxylin:Eoin staining solution for a certain period of time, washed with 1X PBS, and morphological findings were analyzed under a microscope.

As shown in FIG. 13a , steatosis, cell ballooning, inflammatory cell infiltration, etc., which are disease phenotypes of steatohepatitis shown by the HFHC+F/G diet, were analyzed by NAFLD activity score described in Example 3-3. As a result, it was confirmed that it decreased in the group treated with the nucleic acid complex (combination 3) compared to the control group, and showed an effect equivalent to that of the positive control group, which is an NLRP3 inhibitor.

Example 5-5: Analysis of the effect of inhibiting lipid deposition in the liver in a non-alcoholic steatohepatitis animal model (HFHC+F/G mouse model) using a high-fat/high-cholesterol diet and fructose/glucose negative water using ORO staining

Animal experiments were conducted under the above conditions, and liver tissue was isolated on the final day of the experiment. Tissues were fixed in 4% formalin solution for 24 hours, then dehydrated in 30% sucrose solution for 24 hours, and embedded in OCT compound. It was sectioned to 5 μm and attached to a slide glass. The mounted tissues were stained with Oil red O staining solution for a certain period of time, washed with 1X PBS, and morphological findings were analyzed under a microscope.

As a result, as shown in FIG. 13B , it was confirmed that lipid deposition in the liver tissue induced by the HFHC+F/G diet was red and distributed throughout the liver tissue. In the group treated with the nucleic acid complex (combination 3), the distribution of lipid clumps was rarely observed compared to the control group, and the reduced effect was confirmed to a level similar to that of the positive control group.

Example 5-6: Analysis of the effect of inhibiting liver fibrosis in a non-alcoholic steatohepatitis animal model (HFHC+F/G mouse model) using a high-fat/high-cholesterol diet and fructose/glucose negative water using picrosirius red/fast green staining

Animal experiments were conducted under the above conditions, and liver tissue was isolated on the final day of the experiment. The tissue was fixed in 4% formalin solution for 24 hours, embedded in paraffin, and the tissue was sectioned to 5 μm and attached to a slide glass. The mounted tissues were stained with picrosirius red/fast green staining solution for a certain period of time, washed with 1X PBS, and morphological findings were analyzed under a microscope.

As a result, as shown in FIG. 13c, liver fibrosis induced by the HFHC + F/G diet was reduced in the group treated with the nucleic acid complex (combination 3) compared to the control group, and it was confirmed that the positive control group, which is an NLRP3 inhibitor, showed an equivalent effect. could

Example 6: Analysis of non-alcoholic steatohepatitis phenotype effect according to increasing administration period in non-alcoholic steatohepatitis animal model (HFHC+F/G mouse model) using high-fat/high-cholesterol diet and fructose/glucose negative water

As an animal model having a phenotype similar to non-alcoholic steatohepatitis, the efficacy was verified after administration of the nucleic acid complex (combination 3) for 4 weeks in the HFHC + F / G dietary model used in Example 5, in non-alcoholic steatohepatitis Based on this, the administration period of the nucleic acid complex (combination 3) was extended to 8 weeks in the same animal model, and then, to verify the treatment effect, 7-week-old male mice ( Coretech, Korea) was used.

To induce non-alcoholic steatohepatitis model, high-fat/high-cholesterol diet (Raon Bio, Korea) and fructose/glucose (sigma, USA) were consumed for 16 weeks, and then nucleic acid complexes were used three times a week using five animals in each experimental group. was administered by subcutaneous injection. For a positive control, MCC950 (Sigma, USA), an NLRP3 inhibitor, was orally administered once daily at a dose of 10 mg/kg. After 8 weeks of administration of the nucleic acid complex and positive control, all animals were euthanized.

In this example, the effect of the nucleic acid complex verified in Example 5 was extended to the same animal model (HFHC+F/G mouse model), and the effect was verified by examining the improvement of lesions such as histological findings and changes in NLRP3 gene expression. It was intended, and the nucleic acid complex combinations used are shown in Table 8 below.

Combination of Nucleic Acid Complexes for Effect Analysis in Nonalcoholic Steatohepatitis Animal Model Using High-Fat/High-Cholesterol Diet and Fructose/Glucose Negative Water Combination nucleic acid complex 3 SEQ ID NOs 2 and 5

Example 6-1: Analysis of liver phenotype and weight change in a non-alcoholic steatohepatitis animal model (HFHC+F/G mouse model) using a high-fat/high-cholesterol diet and fructose/glucose negative water

In the animal experiment conducted under the above conditions, the color of the liver was observed and the size and weight were measured to compare the body weight and appearance of the liver after the experiment was completed.

As a result, as shown in FIG. 14, when comparing the color of the liver with the naked eye, the non-alcoholic steatohepatitis control group induced by the HFHC + F / G diet and the positive control group (MCC950, MCC950, Positive control, PC) group was observed to change color to yellow and enlarge. On the other hand, in the group treated with the nucleic acid complex of SEQ ID Nos. 2 and 5 (PNA3, combination 3), it was observed that the size and weight of the liver decreased compared to the control group, and the color of the liver changed to red.

Example 6-2: Analysis of liver function improvement effect using enzyme-linked immunosorbent assay (ELISA) analysis

The experiment was conducted under the above conditions, and after blood was collected before the end of the animal experiment, serum was separated using a centrifuge at 13000 rpm and 4 ° C. Separated serum is alanine aminotransferase (ALT or SGPT) Activity Colorimetric/Fluorometric Assay Kit (biovision, USA), Aspartate Aminotransferase (AST or SGOT) Activity Colorimetric Assay Kit (biovision, USA), Triglyceride Assay Kit (Abcam, UK), Cholesterol OD (optical density) was measured and analyzed with a spectrophotometer using an Assay Kit (Abcam, UK).

As a result, as shown in FIG. 15, acute liver damage marker ALT (Alanine Aminotransferase), chronic liver damage marker AST (Aspartate Aminotransferase) and neutral fat marker TG ( It was confirmed that the level of triglyceride) was significantly decreased compared to the control group and the positive control group.

Example 6-3: Analysis of gene expression by Western blot assay

After the animal experiment under the above conditions was completed, protein lysate obtained by adding 200 μl of RIPA buffer to a part of the mouse liver tissue biopsied was quantified using a BCA assay kit (Thermo Fisher, USA). 30 μg of protein was separated by size through electrophoresis, transferred to a PVDF membrane, and then primary antibodies NLRP3 (Abcam, UK), ASC (Cell signaling Technology, USA), caspase-1 (Abcam, UK), IL -1β (Abcam, UK) was treated at 1:1000 and left at 4 °C for one day.

After washing with 1X TBS-T, secondary antibodies, Goat Anti-Rabbit (Cell signaling Technology, USA) and Mouse Anti-Goat (Santa cruz Biotechnology, USA) were treated at a ratio of 1:2000 and allowed to stand at room temperature for 1 hour. Supersignal ^TM West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA) was treated, and the expression inhibition efficiency of the target gene was analyzed using Image600 (Amersham, Germany) equipment.

As a result, as shown in FIG. 16, expression of NLRP3 target genes and expression of sub-pathway genes ASC, pro-caspase-1, and pro-IL-1β in the group treated with the nucleic acid complex (combination 3) compared to the model induction group and the control group It was confirmed that all of these decreased (FIG. 16).

Example 6-4: Phenotypic analysis through H&E staining in a non-alcoholic steatohepatitis animal model (HFHC+F/G mouse model) using a high-fat/high-cholesterol diet and fructose/glucose negative water

Animal experiments were conducted under the above conditions, and liver tissue was isolated on the final day of the experiment. The tissue was fixed in 4% formalin solution for 24 hours, embedded in paraffin, and the tissue was sectioned to 5 μm and attached to a slide glass. The mounted tissue was stained with Hematoxylin:Eoin staining solution for a certain period of time, washed with 1X PBS, and morphological findings were analyzed under a microscope.

As shown in FIG. 17a, the disease phenotypes of steatohepatitis, such as steatosis, cell ballooning, and inflammatory cell infiltration, which are shown by the HFHC+F/G diet, were analyzed by the NAFLD activity score described in Example 3-3. As a result, it was confirmed that it decreased in the group treated with the nucleic acid complex (combination 3) compared to the control group, and showed an effect equivalent to that of the positive control group, which is an NLRP3 inhibitor.

Example 6-5: Analysis of the effect of inhibiting lipid deposition in the liver in a non-alcoholic steatohepatitis animal model (HFHC+F/G mouse model) using a high-fat/high-cholesterol diet and fructose/glucose negative water using ORO staining

Animal experiments were conducted under the above conditions, and liver tissue was isolated on the final day of the experiment. Tissues were fixed in 4% formalin solution for 24 hours, then dehydrated in 30% sucrose solution for 24 hours, and embedded in OCT compound. It was sectioned to 5 μm and attached to a slide glass. The mounted tissues were stained with Oil red O staining solution for a certain period of time, washed with 1X PBS, and morphological findings were analyzed under a microscope.

As a result, as shown in FIG. 17B , it was confirmed that lipid deposition in the liver tissue induced by the HFHC+F/G diet was red and distributed throughout the liver tissue. In the group treated with the nucleic acid complex (combination 3), the distribution of lipid deposition was rarely observed compared to the control group, and the reduced effect was confirmed to a level similar to that of the positive control group.

Example 6-6: Analysis of the effect of inhibiting liver fibrosis in a non-alcoholic steatohepatitis animal model (HFHC+F/G mouse model) using a high-fat/high-cholesterol diet and fructose/glucose negative water using picrosirius red/fast green staining

Animal experiments were conducted under the above conditions, and liver tissue was isolated on the final day of the experiment. The tissue was fixed in 4% formalin solution for 24 hours, embedded in paraffin, and the tissue was sectioned to 5 μm and attached to a slide glass. The mounted tissues were stained with picrosirius red/fast green staining solution for a certain period of time, washed with 1X PBS, and morphological findings were analyzed under a microscope.

As a result, as shown in FIG. 17c, liver fibrosis induced by the HFHC+F/G diet was reduced in the group treated with the nucleic acid complex (combination 3) compared to the control group, and it was confirmed that the effect was equivalent to that of the NLRP3 inhibitor positive control group. could

Based on the above results, when the administration period of nucleic acid complex 8 was increased, the lesions worsened in the non-alcoholic steatohepatitis control group induced by the HFHC + F / G diet, whereas the nucleic acid complexes of SEQ ID NOs: 2 and 5 (PNA3 , In the group treated with combination 3), it was confirmed that the disease phenotype of nonalcoholic steatohepatitis, such as liver fibrosis and lipid deposition in the liver, was more effectively improved, and the expression of the target gene NLRP3 and the expression of subgenes were also effectively suppressed. could confirm that

Having described specific parts of the present invention in detail above, it will be clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> SEASUN THERAPEUTICS <120> Composition for Preventing or Treating Non-Alcoholic SteatoHepatitis Comprising Peptide Nucleic Acid Complex <130> P21-B108 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 15 <212> DNA <213> artificial sequence <220> <223> control <400> 1 acctctacgc aatcc 15 <210> 2 <211> 15 <212> DNA <213> artificial sequence <220> <223> Bioactive PNA <400> 2 acgtgcatta tctga 15 <210> 3 <211> 15 <212> DNA <213> artificial sequence <220> <223> carrier 1 <400> 3 tggagatgcg ttagg 15 <210> 4 <211> 15 <212> DNA <213> artificial sequence <220> <223> carrier 2 <400> 4 tgcacgtaat agact 15 <210> 5 <211> 15 <212> DNA <213> artificial sequence <220> <223> carrier 3 <400> 5 tcagataatg cacgt 15 <210> 6 <211> 5 <212> DNA <213> artificial sequence <220> <223> carrier 4 <400> 6 Agact 5 <210> 7 <211> 5 <212> DNA <213> artificial sequence <220> <223> carrier 5 <400> 7 tcaga 5 <210> 8 <211> 13 <212> PRT <213> artificial sequence <220> <223> endosome escaping helper <400> 8 Gly Leu Phe Asp Ile Ile Lys Lys Ile Ala Glu Ser Phe 1 5 10

Claims (10)

Bioactive Peptide Nucleic Acid targeting NLRP3 gene; and a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) complementary cell-permeable nucleic acid complex containing as an active ingredient a pharmaceutical composition for preventing, improving or treating non-alcoholic steatohepatitis.
The composition according to claim 1, wherein the bioactive peptide nucleic acid comprises the sequence represented by the sequence of SEQ ID NO: 2.
The composition according to claim 1, wherein the carrier peptide nucleic acid comprises a sequence represented by a sequence selected from the group consisting of SEQ ID NOs: 4 to 7.
The method of claim 1, wherein the nucleic acid complex
(i) a nucleic acid complex composed of a bioactive nucleic acid comprising the sequence represented by SEQ ID NO: 2 and a carrier peptide nucleic acid comprising the sequence represented by SEQ ID NO: 4;
(ii) a nucleic acid complex composed of a bioactive nucleic acid comprising the sequence represented by SEQ ID NO: 2 and a carrier peptide nucleic acid comprising the sequence represented by SEQ ID NO: 5;
(iii) a nucleic acid complex composed of a bioactive nucleic acid comprising the sequence represented by SEQ ID NO: 2 and a carrier peptide nucleic acid comprising the sequence represented by SEQ ID NO: 6; and
(iv) a nucleic acid complex composed of a bioactive nucleic acid comprising the sequence represented by SEQ ID NO: 2 and a carrier peptide nucleic acid comprising the sequence represented by SEQ ID NO: 7;
A composition characterized in that selected from the group consisting of.
The composition according to claim 1, wherein the bioactive peptide nucleic acid or the carrier peptide nucleic acid has a 5'-terminus or a 3'-terminus of each nucleic acid, in which a material that helps endosome escape is additionally bound.
The method of claim 5, wherein the substance that helps escape the endosomes is a peptide, lipid nanoparticles, conjugate nanomaterials (polyplex nanoparticles), polymer nanospheres, inorganic nanoparticles, cations A composition, characterized in that at least one selected from the group consisting of cationic lipid-based nanoparticles, cationic polymers and pH sensitive polymers.
The composition according to claim 6, wherein the peptide helping endosomes escape is GLFDIIKKIAESF (SEQ ID NO: 8) or Histidine (10).
The composition according to claim 1, wherein the bioactive peptide nucleic acid has an overall negative charge or neutral charge.
2. The composition of claim 1, wherein the carrier peptide nucleic acid has an overall positive charge.
The composition according to claim 1, wherein the nucleic acid complex has a positive charge as a whole.

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