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WO2023132673A1 - Composition comprenant un complexe d'acide nucléique pour la prévention ou le traitement d'une maladie cérébrale dégénérative - Google Patents

Composition comprenant un complexe d'acide nucléique pour la prévention ou le traitement d'une maladie cérébrale dégénérative Download PDF

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WO2023132673A1
WO2023132673A1 PCT/KR2023/000259 KR2023000259W WO2023132673A1 WO 2023132673 A1 WO2023132673 A1 WO 2023132673A1 KR 2023000259 W KR2023000259 W KR 2023000259W WO 2023132673 A1 WO2023132673 A1 WO 2023132673A1
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nucleic acid
disease
acid complex
bioactive
carrier peptide
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Korean (ko)
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옥예진
박민정
김혜주
유지연
박희경
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Seasun Therapeutics Inc
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Seasun Therapeutics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to a nucleic acid complex having blood-brain barrier penetrating ability and a pharmaceutical composition for preventing or treating degenerative brain disease containing the complex as an active ingredient, and more specifically, to a bioactive nucleic acid targeting NLRP3 gene; And a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) relates to a complementary nucleic acid complex and a pharmaceutical composition for preventing or treating degenerative brain disease containing the same as an active ingredient.
  • Carrier Peptide Nucleic Acid Carrier Peptide Nucleic Acid
  • Parkinson's disease is a degenerative brain disease caused by a decrease in dopamine, a neurotransmitter, in the striatum and substantia nigra. .
  • the prevalence is second only to Alzheimer's disease, and it is reported that about 1% of the population aged 60 or older suffer from this disease (Ali Samii, et al., Lancet. 2004 May 29; 363(9423):1783-93).
  • Parkinson's disease is characterized by the loss of dopaminergic neurons due to the accumulation and diffusion of Lewy bodies composed of misfolded ⁇ -synuclein. .
  • Lewy bodies which are the main cause of Parkinson's disease, are neurotoxic substances that propagate to peripheral areas of the brain and cause dopaminergic degeneration (Werner Poewe, et al., Nat Rev Dis Primers. 2017 Mar 23;3:17013). It is known that chronic microglia neuroinflammation occurs in the substantia nigra region of patients with Parkinson's disease in the early stages of onset, and this feature is also known to be prominent in the brains of patients with Parkinson's disease confirmed postmortem (Alexander Gerhard, et al., Neurobiol Dis.
  • NLRP3 NLR family pyrin domain containing 3; NACHT, leucine-rich repeat, and pyrin domain (PYD)-containing protein 3 (NALP3)
  • NACHT nucleotide-binding and oligomerization domain-like receptors
  • PYD pyrin domain
  • NALP3 pyrin domain-containing protein 3
  • the NLRP3 inflammasome is a NLRP3 sensor and signal adapter ASC (apoptosis-associated speck-like protein containing a CARD (caspase activation and recruitment) domain)) and pro-caspase-1.
  • ASC apoptosis-associated speck-like protein containing a CARD (caspase activation and recruitment) domain
  • caspase-1 is activated, triggering the release of interleukin-1 ⁇ (IL-1 ⁇ ) and IL-18, which are inflammatory cytokines, to initiate an inflammatory response.
  • alpha-synuclein aggregation activates the NLRP3 inflammasome and induces dopamine degeneration due to an increase in the inflammatory response of microglia (Richard Gordon, et al., Sci Transl Med. 2018 Oct. 31;10(465):eaah4066).
  • a significant number of drugs developed for the treatment of brain diseases have a problem in that they do not pass through the blood-brain barrier well.
  • the penetration mechanism of the blood-brain barrier has not yet been elucidated, and even if a disorder occurs in the central nervous system, it is a reality that drugs cannot reach the target area of the central nervous system, and effective treatment methods have not yet been developed.
  • nucleic acid drugs suppress 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 (Ryszard Kole, et al. al., Nat Rev Drug Discov. 2012 Jan 20;11(2):125-40). 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.
  • mRNA messenger RNA
  • 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 has been registered for a new structure with low cytotoxicity and improved cell permeability of bioactive nucleic acids and the ability to regulate gene expression (Korean Registered Patent No. 10). -1963885). In addition, the present inventors have continuously conducted research on the function of the construct to develop a new construct having the ability to penetrate the blood-brain barrier with excellent efficiency (Korean Patent Application No. 10-2019-0128465).
  • the present inventors have made diligent efforts to use nucleic acid complexes with excellent blood-brain barrier penetration ability to apply them to the treatment of degenerative brain diseases. It was found that it exhibits excellent effects in preventing or treating brain diseases, and the present invention was completed.
  • An object of the present invention is to provide a nucleic acid complex having blood-brain barrier penetrating ability and a pharmaceutical composition for preventing or treating degenerative brain disease containing the same as an active ingredient.
  • 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 complementary nucleic acid complex.
  • the present invention also provides a pharmaceutical composition for preventing or treating degenerative brain disease containing the nucleic acid complex as an active ingredient.
  • the present invention also provides a method for preventing or treating a degenerative brain disease comprising administering the nucleic acid complex.
  • the present invention also provides the use of the nucleic acid complex for the prevention or treatment of degenerative brain diseases.
  • the present invention also provides the use of the nucleic acid complex for the preparation of a drug for preventing or treating degenerative brain disease.
  • 1 is a diagram confirming the ability of nucleic acid complexes to inhibit the expression of target genes and subgenes in an LPS-induced Parkinson's disease-like cell model.
  • FIG. 2 is a diagram confirming the ability of a nucleic acid complex to suppress intracellular expression of a target gene in a Parkinson's disease-like cell model induced by LPS.
  • Figure 3 is a diagram confirming the efficacy of the nucleic acid complex in a primary microglia model isolated from tissue.
  • Figure 4 is a view confirming the ability to improve animal behavior according to the administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.
  • 5 is a diagram confirming the ability to inhibit target gene expression and subgene expression by brain region according to the administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.
  • 6a is a view showing changes in expression of Tyrosine Hydroxylase (TH) in the striatum of the brain according to the administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.
  • TH Tyrosine Hydroxylase
  • 6B is a diagram showing changes in expression of Tyrosine Hydroxylase (TH) in the substantia nigra of the brain according to administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.
  • TH Tyrosine Hydroxylase
  • Figure 6c is a view confirming the expression suppression ability of ⁇ -Synuclein in the striatum (striatum) of the brain region according to the administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.
  • Figure 6d is a view confirming the expression suppression ability of ⁇ -Synuclein in the substantia nigra of the brain region according to the administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.
  • Figure 6e is a view confirming the expression inhibition ability of ionized calcium-binding adapter molecule 1 (IBA-1) in the striatum (striatum) of the brain region according to the administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.
  • IBA-1 ionized calcium-binding adapter molecule 1
  • 6f is a diagram confirming the expression inhibition ability of ionized calcium-binding adapter molecule 1 (IBA-1) in the substantia nigra of the brain according to the administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.
  • IBA-1 ionized calcium-binding adapter molecule 1
  • Parkinson's disease causes of Parkinson's disease are very diverse. About 5% of all patients are caused by genetic factors, and external environmental factors such as inflammation and oxidative stress play an important role. Parkinson's disease is caused by abnormal protein aggregation in dopaminergic neurons, which is the aggregation of alpha-synuclein protein. Aggregation of these proteins is further promoted by oxidative stress. Until now, the treatment of Parkinson's disease has been made by supplementing the dopamine neurotransmitter, but this is not a fundamental treatment for Parkinson's disease.
  • the number of patients with Parkinson's disease in Korea was 61,565 in 2010 and increased to 85,888 in 2014 with an average annual growth rate of 8.7%.
  • the proportion of patients aged 60 years or older was 95.7%, and the prevalence showed a correlation with the patient's age. Parkinson's disease patient and treatment cost increase, young doctors, 2015).
  • the onset of Parkinson's disease is very early, around 60 years of age, and because of this, the progression of the disease lasts for more than 20 years, so a treatment that slows the disease progression through early detection is actively required.
  • nucleic acid complex in which a bioactive nucleic acid targeting the NLRP3 gene and a carrier peptide nucleic acid are complementaryly bound can be used for the prevention and treatment of Parkinson's disease.
  • the present invention in one aspect, a bioactive nucleic acid targeting the NLRP3 gene (Bioactive Nucleic Acid); and a nucleic acid complex in which a carrier peptide nucleic acid is complementarily bound.
  • a nucleic acid complex in which a bioactive nucleic acid targeting the NLRP3 gene and a carrier peptide are complementaryly bound may have a structure of the following structural formula (1).
  • A is a bioactive nucleic acid having a sequence capable of binding to 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 has an overall negative charge or neutral charge
  • the carrier peptide nucleic acid includes one or more peptide nucleic acid monomers modified to have a positive charge throughout the carrier peptide nucleic acid.
  • the bioactive nucleic acid and the carrier peptide nucleic acid in the nucleic acid complex according to the present invention may have anti-parallel binding or parallel binding.
  • the complementary binding form of the nucleic acid can be separated in the presence of a target sequence of the bioactive nucleic acid (a sequence complementary to the bioactive nucleic acid).
  • Bioactive Nucleic Acid binds to a target gene and a nucleotide sequence containing the target gene in vitro or in vivo to determine the unique function of the gene (e.g., For example, activating or inhibiting transcript expression or protein expression), or regulating pre-mRNA splicing (eg, exon skipping), etc.
  • the nucleotide sequence may be characterized as a gene regulatory sequence, a gene coding sequence, or a splicing regulatory sequence.
  • the bioactive nucleic acid is expressed
  • a nucleic acid having a complementary sequence capable of binding to a target gene of interest to be reduced in particular, a complementary sequence capable of binding to the mRNA of such a target gene of interest, and suppressing the expression of the gene. It means a nucleic acid involved in regulation, and may be a nucleic acid having a sequence complementary to a target gene whose expression is to be reduced.
  • the bioactive nucleic acid in the present invention is preferably an antisense peptide nucleic acid of the NLRP3 (NLR family pyrin domain containing 3) gene, a target gene related to Parkinson's disease, and more preferably the nucleotide sequence represented by the sequence of SEQ ID NO: 2 It may include, but is not limited thereto.
  • the bioactive nucleic acids include DNA, RNA, or modified nucleic acids such as PNA (peptide nucleic acid), PMO (phosphorodiamidate morpholino oligonucleotide), LNA (locked nucleic acid), GNA (glycol nucleic acid) and TNA (threose nucleic acid), antisense It may be selected from the group consisting of oligonucleotide, aptamer, small interfering RNA (siRNA), short hairpin RNA (shRNA), ribozyme and DNAzyme, preferably the bioactive
  • the nucleic acid is selected from the group consisting of DNA, RNA, or modified nucleic acid PNA (peptide nucleic acid), PMO (phosphorodiamidate morpholino oligonucleotide), LNA (locked nucleic acid), GNA (glycol nucleic acid) and TNA (threose nucleic acid) it may be selected from the group consisting of DNA, RNA
  • Carrier Peptide Nucleic Acid refers to a nucleic acid to which a bioactive nucleic acid and some or all bases are complementaryly combined to impart functionality, and the carrier peptide nucleic acid used in the present invention is In addition to peptide nucleic acid (PNA), modified nucleic acids similar thereto may be used, and peptide nucleic acids are preferred, but are not limited thereto.
  • PNA peptide nucleic acid
  • the carrier peptide nucleic acid preferably includes one or more gamma- or alpha-backbone modified peptide nucleic acid monomers so that the entire carrier peptide nucleic acid is positively charged, and the gamma- or alpha-backbone modified peptide nucleic acid It is more preferable that monomers having amino acids having a positive charge are included more than monomers having amino acids having a negative charge so that the overall charge of the carrier peptide nucleic acid is positive.
  • the carrier peptide nucleic acid preferably includes the nucleotide sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5, but is not limited thereto.
  • the "nucleic acid complex” can penetrate the bioactive substance into the body and ultimately into the cell through extracellular treatment, and specifically has the ability to deliver the bioactive nucleic acid targeting the NLRP3 gene into the cell. .
  • the nucleic acid complex is a bioactive nucleic acid represented by the sequence of SEQ ID NO: 2; and a carrier peptide nucleic acid represented by the sequence of SEQ ID NO: 4 or 5, but is not limited thereto.
  • the binding ability (melting temperature, Tm) of the bioactive nucleic acid targeting the NLRP3 gene and the carrier peptide nucleic acid is lower than that of the bioactive nucleic acid and the target NLRP3 gene.
  • the binding force is obtained by parallel binding or partial specific binding between the bioactive nucleic acid and the carrier peptide nucleic acid according to the 5'-direction and the 3'-direction of each nucleic acid, so that the bioactive nucleic acid and the carrier peptide nucleic acid
  • the binding force (Tm) of may be lower than the binding force between the bioactive nucleic acid and the target gene of the bioactive nucleic acid.
  • the bioactive nucleic acid or 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.
  • 'm' means a substance that helps endosome escape of bioactive nucleic acid and carrier peptide nucleic acid.
  • “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. there is. It means that bioactive nucleic acids move more efficiently and rapidly to the nucleus or cytoplasm to meet and act on target genes (Daniel W Pack, et al., Nat Rev Drug Discov. 2005 Jul;4(7):581-93 ).
  • 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).
  • a peptide (GLFDIIKKIAESF, SEQ ID NO: 6) may be linked to the bioactive nucleic acid via a linker, and Histidine (10) to the carrier peptide nucleic acid via a linker. It may be characterized by combining, but is not limited thereto.
  • the lipid nanoparticles may be selected from the group consisting of Lipid, phospholipids, acetyl palmitate, poloxamer 18, Tween 85, tristearin glyceride and Tween 80.
  • the polyplex nanoparticles may be poly(amidoamine) or polyethylenimine (PEI).
  • 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.
  • the inorganic nanoparticles may be selected from the group consisting of Fe2O3, Fe3O4, WO3 and WO2.9.
  • 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.
  • 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).
  • the pH sensitive polymers may be selected from the group consisting of polyacids, poly(acrylic acid), poly(methacrylic acid), and hydrolyzed polyacrylamide.
  • 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.
  • the bioactive nucleic acid may be characterized in that it consists of natural nucleic acid bases and/or modified nucleic acid monomers.
  • 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.
  • the bioactive nucleic acid and the carrier peptide nucleic acid are phosphodiester, 2' O-methyl, 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.
  • 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.
  • 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.
  • 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.
  • 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”.
  • the nucleic acid complex of the present invention may be characterized as having a positive charge as a whole.
  • the bioactive nucleic acid has negative or neutral electrical characteristics as a whole
  • the carrier peptide nucleic acid has positive electrical characteristics as a whole. It is not limited thereto.
  • the electrical properties of the bioactive nucleic acid and the carrier peptide nucleic acid can be imparted using a modified peptide nucleic acid monomer
  • 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
  • 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.
  • 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.
  • 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.
  • a peptide nucleic acid monomer having a modified nucleobase in addition to the backbone modification.
  • 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.
  • 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.
  • the nucleic acid complex according to the present invention has a positive charge as a whole.
  • 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
  • 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.
  • the binding of the carrier peptide nucleic acid may be characterized as a simple covalent bond or a covalent bond mediated by a linker, but is not limited thereto.
  • substances related to cell permeation, solubility, stability, delivery 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 can be used as a base having a lower binding force than the binding force, preferably. It can be characterized by using inosine PNA.
  • 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.
  • the binding force of the bioactive nucleic acid and the carrier peptide nucleic acid is controlled, in the presence of the target gene, when the bioactive nucleic acid binds to the target sequence (when the bioactive nucleic acid is substituted with the target sequence, the target specific It is possible to adjust the timing of enemy separation and joining).
  • 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.
  • the particle size of the nucleic acid complex may be characterized in that it is controlled by adjusting the charge balance between the bioactive nucleic acid and the carrier peptide nucleic acid. Specifically, when the positive charge of the carrier peptide nucleic acid increases, the particle size decreases, but when the positive charge of the carrier peptide nucleic acid exceeds a certain level, the particle size increases.
  • the particle size is determined by an appropriate charge balance with the overall carrier peptide nucleic acid according to the charge of the bioactive nucleic acid forming the complex as another important factor determining the particle size.
  • the positive charge of the carrier peptide nucleic acid according to the present invention is 1 to 7 (meaning that 1 to 7 monomers having a positive charge are included), preferably 2 to 5, most preferably 2 to 3
  • the charge of the bioactive nucleic acid may be characterized in that the net charge of the charge balance is 0 to 5 negative charges, preferably 0 to 3.
  • the nucleic acid complex can be prepared by hybridizing a bioactive nucleic acid and a carrier peptide nucleic acid under appropriate conditions.
  • Hybridization in the present invention means that complementary single-stranded nucleic acids form a double-stranded nucleic acid. Hybridization can occur when the complementarity between the two nucleic acid strands is perfect (perfect match) or even when some mismatch bases are present. The degree of complementarity required for hybridization may vary depending on hybridization conditions, and may be particularly controlled by binding temperature.
  • the nucleic acid complex may have blood-brain barrier penetrating ability.
  • blood-brain barrier or "BBB (Blood-Brain Barrier)” is used interchangeably herein, which closely regulates and severely restricts the exchange between blood and brain tissue and is circulated through brain tissue. Therefore, it is used to refer to the permeability barrier present in the blood.
  • Components of the blood brain barrier include the endothelial cells that form the deepest lining of all blood vessels, the dense junctions between adjacent endothelial cells that are structurally correlated with the BBB, the basement membrane of endothelial cells, and almost all of the exposed outer surface of blood vessels. An enlarged foot process of the overlying astrocytes is included.
  • the BBB prevents most substances in the blood, including most large molecules, such as Ig, antibodies, complement, albumin, and drugs and small molecules, from entering brain tissue.
  • the nucleic acid complex suggests that it has BBB penetrating ability.
  • target gene refers to a nucleic acid sequence (base sequence) to be activated, inhibited, or labeled, and is not different from the term “target nucleic acid”, and is used interchangeably herein.
  • bioactive nucleic acid when a target nucleic acid (base sequence) containing a target gene is contacted (bound) with the complex in vitro or in vivo , bioactive nucleic acid is separated from the carrier peptide nucleic acid and exhibit biological activity.
  • diseases that can be prevented or treated using the nucleic acid complex may be determined according to the target gene of the bioactive nucleic acid in the nucleic acid complex.
  • the target gene of the bioactive nucleic acid may be characterized in that NLRP3.
  • the present invention from another point of view, a bioactive nucleic acid targeting the NLRP3 gene (Bioactive Nucleic Acid); And a pharmaceutical composition for preventing or treating degenerative brain disease containing a nucleic acid complex complementary to a carrier peptide nucleic acid as an active ingredient.
  • a bioactive nucleic acid targeting the NLRP3 gene Bioactive Nucleic Acid
  • a pharmaceutical composition for preventing or treating degenerative brain disease containing a nucleic acid complex complementary to a carrier peptide nucleic acid as an active ingredient.
  • the disease that can be prevented and treated using the nucleic acid complex may preferably be a degenerative brain disease
  • the degenerative brain disease includes Parkinson's disease, Alzheimer's disease, Niemann-Pick disease, Creutzfeldt-Jakob disease, Huntington's disease, amyotrophic lateral sclerosis (amyotrophic lateral sclerosis), multiple sclerosis or dementia ( dementia), but is not limited thereto.
  • prevention refers to any action that prevents the onset of a disease or delays its progression by administering a composition containing the nucleic acid complex.
  • treatment used in the present invention refers to all activities in which symptoms of a disease are improved, symptoms are alleviated, or cured by administration of a composition containing the nucleic acid complex.
  • a pharmaceutical composition comprising a nucleic acid complex according to the present invention may include a pharmaceutically effective amount of the nucleic acid complex alone, or may include one or more pharmaceutically acceptable carriers, excipients or diluents.
  • the pharmaceutically effective amount refers to an amount sufficient to prevent, improve, and treat symptoms of degenerative brain disease.
  • pharmaceutically acceptable refers to a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions when administered to humans.
  • the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • the pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers and preservatives.
  • carrier is defined as a compound that facilitates the addition of a nucleic acid complex into a cell or tissue.
  • DMSO dimethylsulfoxide
  • carrier facilitates the introduction of many organic compounds into the cells or tissues of living organisms.
  • diot 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.
  • compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration.
  • the dosage form may be in the form of a powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, or sterile powder.
  • composition of the present invention can be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient depends on various factors such as the route of administration, age, sex, weight and severity of the patient. can be selected appropriately.
  • 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.
  • any nucleic acid delivery method known in the art may be used.
  • suitable delivery reagents include, for example, Mirus Transit TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, polycations (eg, polylysine), atelocollagen, nanoplexes, and liposomes.
  • atelocollagen as a delivery vehicle for nucleic acid molecules has been described by Minakuchi et al. Nucleic Acids Res., 32(13):e109 (2004); Hanai et al. Ann NY Acad Sci., 1082:9-17 (2006); and Kawata et al. Mol Cancer Ther., 7(9):2904-12 (2008).
  • Exemplary interfering nucleic acid delivery systems are provided in U.S. Patent Nos. 8,283,461, 8,313,772, and 8,501,930.
  • the nucleic acid complex may be administered using a delivery system such as a liposome.
  • a delivery system such as a liposome.
  • the liposome can help target the complex to a specific tissue, such as lymphoid tissue, or selectively target infected cells, and can also help increase the half-life of a composition containing the complex.
  • Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like.
  • the complex to be delivered is a liposome.
  • a molecule that binds to a receptor prevalent in lymphocytes such as a monoclonal antibody that binds to the CD45 antigen, or in combination with other therapeutic compositions, is a liposome.
  • liposomes filled or decorated with a given complex of the present invention to deliver the nucleic acid complex composition can be directed to the site of lymphocytes.
  • Liposomes for use in accordance with the present invention are generally formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids and sterols such as cholesterol.
  • lipids are selected in consideration of, for example, stability of liposomes in the blood stream, acid lability, and size of liposomes.
  • a variety of methods can be used to prepare liposomes. See, eg, Szoka, et al., Ann. Rev. Biophys. Bioeng., 9:467, 1980), and US Pat. Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369.
  • the present invention provides a bioactive nucleic acid targeting the NLRP3 gene; And a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) It relates to a method for preventing or treating degenerative brain disease comprising administering to a subject a nucleic acid complex complementary thereto.
  • the present invention provides a bioactive nucleic acid targeting the NLRP3 gene for the prevention or treatment of degenerative brain diseases; And it relates to the use of a nucleic acid complex in which a carrier peptide nucleic acid is complementarily bound.
  • the present invention provides a bioactive nucleic acid targeting the NLRP3 gene for the preparation of drugs for preventing or treating degenerative brain diseases; And it relates to the use of a nucleic acid complex in which a carrier peptide nucleic acid is complementaryly bound.
  • subject means a mammal suffering from or at risk of a condition or disease that can be alleviated, suppressed or treated by administering a nucleic acid complex according to the present invention, and preferably means a human.
  • the dose of the nucleic acid complex of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health condition and disease severity.
  • 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 or applied amount of such compounds lies preferably within a range of circulating concentrations that include the ED50 (or IC50) with little or no toxicity.
  • administration 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. there is.
  • the administration route of the pharmaceutical composition of the present invention may be administered through any general route as long as it can reach the target tissue.
  • the pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonaryly, or intrarectally, as desired.
  • the composition may be administered by any device capable of transporting an active substance to a target cell.
  • 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.
  • Example 1 Preparation of bioactive peptide nucleic acids and carrier peptide nucleic acids, and complexes using them
  • NLRP3 was used as a target gene to test the effect of the nucleic acid complex on Parkinson's disease, and to confirm the therapeutic effect of Parkinson's disease, antisense peptide nucleic acid (Bioactive Peptide Nucleic Acid) for NLRP3 was used. antisense PNA) was used.
  • the bioactive nucleic acid (antisense PNA) used as a control of the present invention includes the sequence represented by SEQ ID NO: 1, and the bioactive peptide nucleic acid (antisense PNA) used to confirm the therapeutic effect of Parkinson's disease has the sequence represented by SEQ ID NO: 2 includes
  • the carrier peptide nucleic acids used in the examples of the present invention are composed of sequences represented by SEQ ID NOs: 3 to 5 (Table 1). All peptide nucleic acids used in the present invention were synthesized by HPLC purification method from PANAGENE (Korea).
  • Modification of the monomers converts the backbone of the peptide nucleic acid to lysine (indicated by Lysine, Lys, K, (+) ) for electrical properties and glutamic acid (Glutamic acid, Glu, E, (-)) for electrical negative to impart electrical properties. ) 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 1 the therapeutic effect of Parkinson's disease was analyzed using a nucleic acid complex comprising a carrier peptide nucleic acid and a bioactive peptide nucleic acid targeting NLRP3, which was prepared according to the structure shown in Table 2 below.
  • Example 2-1 Cell culture
  • HMC-3 Human-derived microglia obtained from ATCC (American Type Culture Collection, USA) were mixed with 10% (v/v) fetal bovine serum, penicillin 100 units/ml, and streptomycin in MEM culture medium (Wellgene, Korea). 100 ⁇ g/ml was added and cultured under conditions of 37°C and 5% (v/v) CO 2 .
  • Example 2-2 Analysis of gene expression using Western blot assay
  • Human-derived microglial cell lines were seeded in 1x10 5 cells in a 6-well plate and cultured for 24 hours, and treated with 1 ⁇ g/ml of Lipopolysaccharide (LPS, Sigma, USA) to induce an immune response. After 4 hours of LPS treatment, the experiment was conducted under the conditions of Example 2-1, and the complex containing the bioactive peptide nucleic acid and the carrier peptide nucleic acid was treated and cultured for 24, 48, 72, 96, and 120 hours, respectively, and RIPA buffer Protein lysate was obtained by adding 30 ⁇ L to each well.
  • LPS Lipopolysaccharide
  • Protein lysate was quantified using a BCA assay kit (Thermo Fisher, USA), and 30 ⁇ g of protein was separated by size through electrophoresis, and the protein was transferred to a PVDF membrane.
  • Pro Cas-1 (abcam, USA) and Pro IL-1 ⁇ (abcam, USA) were treated at a ratio of 1:1000 and left at 4°C for one day. It was washed with 1X TBS-T, treated with a secondary antibody, Goat Anti-Rabbit (Cell signaling Technology, USA) at a ratio of 1:2000, and left at room temperature for 1 hour.
  • Supersignal TM West Femto Maximum Sensitivity Substrate was treated, and the expression inhibition efficiency of the target gene was analyzed using Image600 (Amersham, Germany) equipment.
  • Human-derived microglial cell lines were seeded in 3x10 3 cells in an 8-well plate and cultured for 24 hours, and treated with 1 ⁇ g/ml of Lipopolysaccharide (LPS, Sigma, USA) to induce an immune response. After 4 hours of LPS treatment, the experiment was conducted under the conditions of Example 2-1, and the complex containing the bioactive peptide nucleic acid and the carrier peptide nucleic acid was treated, cultured for 24 and 72 hours, respectively, and cells were treated with 4% paraformaldehyde (Sigma, USA). was fixed.
  • LPS Lipopolysaccharide
  • the fixed cells were permeabilized with 0.1% Triton X-100 (Sigma, USA) dissolved in PBS for 10 minutes, blocked with 1% bovine serum albumin (BSA, Sigma, USA) for 1 hour, and then The antibody, NLRP3 (ABclonal, USA) was treated at a ratio of 1:100 and left at 4° C. for one day. After washing with 1X PBS, the secondary antibody, Alexa 488-goat Anti-Mouse (Abcam Technology, USA) was treated at a ratio of 1:200 and allowed to stand at room temperature for 1 hour. After diluting the DAPI solution (Sigma, USA) to 1x and leaving it for 10 minutes, the expression inhibition efficiency of the target gene was analyzed using a fluorescence microscope (Leica, Germany).
  • Example 3 Analysis of Parkinson's disease treatment effect using nucleic acid complex in primary cultured microglial cells isolated from brain tissue
  • Example 2 After selecting the nucleic acid combination whose effect was verified through Example 2, the treatment effect of Parkinson's disease was analyzed using primary microglia isolated from neonatal rats having the most similar characteristics to the body in culture conditions. .
  • Human-derived neuroblastoma obtained from KCLB (Korean Cell Line Bank, Korea) was mixed with 10% (v/v) fetal bovine serum, penicillin 100 units/ml, and streptomycin 100 in DMEM culture medium (Wellgene, Korea). [mu]g/ml was added and cultured under the conditions of 37°C and 5% (v/v) CO 2 .
  • Example 3-2 Isolation and culture of primary microglia from mouse brain tissue
  • mice In order to isolate and culture primary microglia from brain tissues of mice, 1-day-old neonatal SD rats (Nara Biotech, Korea) were purchased and brain tissues were isolated. The cortex from which the meninges and other tissue parts were removed was transferred to a Petri dish containing iced 1x HBSS (gibco, USA), and then the tissue was dissociated using a pipette and centrifuged at 1200 rpm for 5 minutes.
  • the supernatant containing only floating cells was collected and centrifuged at 1200 rpm for 8 minutes. After centrifugation, the cells were seeded in 1x10 5 in a 6-well plate, and cultured under conditions of 37°C and 5% (v/v) CO 2 .
  • 100 ng/mL of LPS was treated for 4 hours, followed by nucleic acid complex treatment and culture.
  • Example 3-3 Analysis of gene expression using Western blot assay
  • Example 3-2 Primary microglia cultured under the culture conditions of Example 3-2 were treated with 100 ng/ml of Lipopolysaccharide (LPS, sigma, USA) to induce an immune response. After 4 hours of LPS treatment, complexes containing bioactive peptide nucleic acids and carrier peptide nucleic acids were treated, and after incubation for 24, 48, and 72 hours, respectively, 30 ⁇ L 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 ⁇ g of protein was separated by size through electrophoresis, and the protein was transferred to a PVDF membrane.
  • LPS Lipopolysaccharide
  • Pro Cas-1 (abcam, USA) and Pro IL-1 ⁇ (abcam, USA) were treated at a ratio of 1:1000 and left at 4°C for one day. It was washed with 1X TBS-T, treated with a secondary antibody, Goat Anti-Rabbit (Cell signaling Technology, USA) at a ratio of 1:2000, and left 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.
  • Example 3-4 Analysis of cell viability of neuroblastoma cell lines using MTT assay
  • Example 3-2 Primary microglia cultured under the culture conditions of Example 3-2 were treated with a complex containing a bioactive peptide nucleic acid and a carrier peptide nucleic acid, and 4 hours later, LPS was treated with 100 ng/ml and cultured for 24, 48, and 72 hours, respectively. did After filtering the culture medium obtained at the end of each culture using a syringe filter (Millipore, USA), it was treated with human-derived neuroblastoma. Human-derived neuroblastoma was seeded in 2x10 4 in a 96-well plate, and after 24 hours, the medium was replaced with the medium obtained from primary microglia, and cultured for 24, 48, and 72 hours, respectively.
  • MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide, sigma, USA) solution was prepared at a concentration of 5 mg/mL in 1X PBS, treated at 20 ⁇ L per well and incubated for 4 hours, then the OD (optical density) was measured and analyzed with a spectrophotometer.
  • Example 4 Analysis of Parkinson's disease treatment effect using nucleic acid complex in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/probenecid-induced mouse model
  • Example 4-1 Production of MPTP/probenecid-induced Parkinson's disease animal model
  • levodopa levodopa, sigma, USA
  • carbidopa carbidopa, sigma, USA
  • Example 4-2 Analysis of motor function change using rotarod test analysis
  • a rotarod test was performed to evaluate the effect of improving motor deficits and balance maintenance, which are characteristics of lesions in animal models of Parkinson's disease, by nucleic acid complexes.
  • a rotarod apparatus (Jeongdo B&P, Korea) with a rotatable cylindrical rod composed of 5 compartments with a diameter of 7 cm and a height of 60 cm at 15 cm intervals was used.
  • the speed of the rotarod used in the test was set to increase constantly from 0 to 40 rpm, and the latency time taken until the animal was placed on the rotating rod and dropped was measured.
  • the maximum measurement time was limited to 300 seconds, and the therapeutic effect of the nucleic acid complex was analyzed using the average value after three repeated measurements.
  • a pole test was performed to evaluate the recovery of akinesia and spastic response using the Parkinson's disease animal disease model prepared under the conditions of Example 4-1.
  • the pole test was performed by placing the mouse facing the sky using a stick with a width of 0.8 cm and a height of 55 cm, and then rotating 180° to measure the total time required to reach the floor. The same number of training sessions were conducted for all experimental animals before the test, and this experiment was conducted after the administration of the nucleic acid complex was completed. The experiment was repeated three times, and the average value was used for analysis of the results.
  • Example 4-4 Analysis of motor function changes using cylinder test analysis
  • Example 4-1 In order to confirm the effect of sensorimotor function treatment in an animal model induced with Parkinson's disease by the method of Example 4-1, a cylinder test was performed.
  • the group of animals exposed to MPTP, a neurotoxin, and administered with only MPTP/probenecid decreased the number of times of using the paws compared to normal mice.
  • the group treated with the complex (PNA 2) increased the number of times of using the forelimbs, confirming the therapeutic efficacy of sensorimotor impairment in an animal model of Parkinson's disease (FIG. 4c).
  • Example 5 Gene expression analysis in Parkinson's disease induced animal model using MPTP/probenecid
  • Parkinson's disease animal model induced by the method of Example 4-1 changes in the expression of Parkinson's disease-related genes were confirmed in the striatum and substantia nigra regions of brain tissue where dopamine production and metabolism occur. .
  • Example 5-1 Analysis of gene expression in Parkinson's disease animal model tissue using Western blot assay
  • the striatum and substantia nigra were separated from the brain tissue extracted on the end day after the experiment was conducted by the method of Example 4-1, and protein lysate was obtained by adding RIPA buffer. Protein lysate was quantified using a BCA assay kit (Thermo Fisher, USA), and 30 ⁇ g of protein was separated by size through electrophoresis, and the protein was transferred to a PVDF membrane. Pro IL-1 ⁇ (abcam, USA), Tyrosine Hydroxylase (TH, abcam, USA), and alpha-synuclein ( ⁇ -synuclein, abcam, USA) were treated at a ratio of 1:1000 and left at 4°C for one day.
  • NLRP3 and lower-order gene expression changes and the expression levels of dopaminergic neurons and alpha-synuclein in Parkinson's disease-induced animal models were analyzed, and the nucleic acid complex combinations used are the same as those in Table 3 above.
  • the expression in the substantia nigra region is shown in FIG. 5B, and the expression of the target gene NLRP3 and IL-1 ⁇ , a sub-pathway gene, increased in the disease induction group (MPTP/p) in the second combination of nucleic acid complexes (PNA). 2), and the decreased dopaminergic neurons in the disease model were recovered with increased expression in the group treated with the nucleic acid complex of No. 2 (combination of SEQ ID NOs: 2 and 5, PNA 2) confirmed that In addition, the expression of alpha-synuclein decreased after treatment with the nucleic acid complex in combination No. 2, confirming the therapeutic efficacy of the nucleic acid complex of SEQ ID NOs: 2 and 5 (FIG. 5b).
  • Example 5-2 Analysis of expression changes in tissues in Parkinson's disease animal models using immunostaining
  • mouse brain tissue was separated on the end day, fixed in 4% formalin solution for one day, immersed in 30% sugar solution, dehydrated for 3 days, and then embedded in OCT (Leica, USA) solution (embedding) was done.
  • the embedded tissue was stored and frozen at -20°C, and then the tissue was sectioned at 20 ⁇ m using a cryostat (Cryostat, Leica, USA) and then mounted on a slide glass.
  • the mounted tissues were blocked for 2 hours using 1% BSA solution, and primary antibodies were used to detect Tyrosine Hydroxylase (abcam, USA), alpha-synuclein ( ⁇ -synuclein, abcam, USA), and IBA1 (wako, Japan).
  • the expression levels of dopaminergic neurons, alpha-synuclein, and immune cells, which are onset factors, were analyzed in brain tissues of Parkinson's disease-induced animal models, and comparative analysis was performed in the striatum and substantia nigra, which are brain regions important for onset.
  • the nucleic acid complex of the present invention in which a bioactive nucleic acid targeting NLRP3 and a carrier peptide nucleic acid are complementaryly bound, has the ability to penetrate the blood-brain barrier and can efficiently inhibit the expression of NLRP3, thereby preventing degenerative brain diseases, particularly Parkinson's disease. Useful for prevention or treatment.

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Abstract

La présente invention concerne un complexe d'acide nucléique ayant une capacité à pénétrer dans la barrière hémato-encéphalique et une composition pharmaceutique le comprenant en tant que principe actif pour la prévention ou le traitement de maladies cérébrales dégénératives et, plus spécifiquement, un complexe d'acide nucléique dans lequel un acide nucléique bioactif ciblant le gène NLRP3 se lie de manière complémentaire à un acide nucléique peptidique porteur, ainsi qu'une composition pharmaceutique le comprenant en tant que principe actif pour la prévention ou le traitement de maladies cérébrales dégénératives. Le complexe d'acide nucléique dans lequel l'acide nucléique bioactif ciblant NLRP3 se lie de manière complémentaire à l'acide nucléique peptidique porteur a une capacité à pénétrer dans la barrière hémato-encéphalique et peut inhiber efficacement l'expression de NLRP3 et, en tant que tel, est utile pour prévenir ou traiter des maladies cérébrales dégénératives, en particulier la maladie de Parkinson.
PCT/KR2023/000259 2022-01-06 2023-01-06 Composition comprenant un complexe d'acide nucléique pour la prévention ou le traitement d'une maladie cérébrale dégénérative Ceased WO2023132673A1 (fr)

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KR20180018405A (ko) * 2016-08-09 2018-02-21 주식회사 시선바이오머티리얼스 세포투과성이 향상된 펩티드 핵산 복합체 및 이를 포함하는 약학적 조성물
KR20190096148A (ko) * 2018-02-08 2019-08-19 주식회사 시선테라퓨틱스 엔도좀 탈출능을 갖는 펩티드 핵산 복합체 및 이의 용도
KR20210088446A (ko) * 2020-01-06 2021-07-14 주식회사 시선테라퓨틱스 세포 투과성 핵산 복합체를 유효성분으로 함유하는 황반변성의 예방 또는 치료용 조성물
KR20220041397A (ko) * 2020-09-25 2022-04-01 주식회사 시선테라퓨틱스 펩티드 핵산 복합체를 유효성분으로 함유하는 치매 예방 또는 치료용 조성물

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WO2025159525A1 (fr) * 2024-01-24 2025-07-31 주식회사 카인사이언스 Utilisation d'un peptide en tant qu'agent thérapeutique pour des maladies cérébrales dégénératives

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