WO2019156366A1 - 핵산 복합체를 함유하는 피부 투과성 전달체 및 이의 용도 - Google Patents
핵산 복합체를 함유하는 피부 투과성 전달체 및 이의 용도 Download PDFInfo
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- WO2019156366A1 WO2019156366A1 PCT/KR2019/000246 KR2019000246W WO2019156366A1 WO 2019156366 A1 WO2019156366 A1 WO 2019156366A1 KR 2019000246 W KR2019000246 W KR 2019000246W WO 2019156366 A1 WO2019156366 A1 WO 2019156366A1
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- nucleic acid
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- carrier peptide
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Definitions
- the present invention relates to a skin permeable delivery vehicle containing a nucleic acid complex capable of introducing a viable nucleic acid into a cell, and a composition for diagnosing, preventing or treating a disease comprising the same, and more particularly, to a viable nucleic acid and a carrier peptide nucleic acid.
- the present invention relates to a skin permeable delivery agent containing the complementary bound nucleic acid complex and having skin permeability and skin residualness, and a composition for diagnosing, preventing or treating a disease comprising the same.
- nucleic acid drugs inhibit the expression of target-specific messenger RNA (mRNA), allowing them to address areas of research that have not been curable with conventional drugs that target proteins (Kole R. et al. Nature Rev. Drug Discov. 2012; 11; 125-140., Wilson C. et al. Curr. Opin. Chem. Bio. 2006; 10: 607-614.
- mRNA messenger RNA
- oligonucleotides are at risk of damage due to nucleases, etc., and cell membrane permeation by passive diffusion is impossible due to the electrical properties (charges) and sizes of the oligonucleotides.
- efforts have been made to secure biological stability through modification of nucleic acids, and in the case of modified Artificial Nucleic Acid, it is possible to increase affinity with a target nucleic acid without losing biological activity. Was done.
- PNA Peptide Nucleic Acid
- a type of modified nucleic acid is an artificial nucleic acid into which (2-aminoethyl) -glycine peptide backbone is introduced.
- the peptide nucleic acid has a strong binding to RNA and DNA having a.
- the peptide nucleic acid is resistant to nucleases and has a high biological stability, and researches on therapeutic agents based on various oligonucleotides have been conducted.
- the peptide nucleic acid has the disadvantage of being difficult to introduce into cells due to its electric neutrality (Joergensen M. et al. Oligonucleotides 2011, 21; 29-37.).
- the cell membrane permeability of the oligo nucleic acid itself is considerably low, especially since DNA or RNA is negatively charged, and thus does not penetrate the hydrophobic phospholipid bilayer of the cell, making intracellular delivery through simple diffusion difficult.
- viral carriers such as retroviruses or adeno-associated viruses, allows for the introduction of oligonucleotides into cells, but there are risks such as unintended immunity and recombination of oncogenes. (Couto LB et al. Curr. Opin. Pharmacol. 2010, 5; 534-542.).
- nucleic acid carriers based on non-viral oligonucleotides with low cytotoxicity and low immunological activity is becoming more important, resulting in cationic lipids, liposomes, and stable nucleic acid lipid particles.
- Cellular transduction techniques using stable nucleic acid lipid particles (SNALP), polymers, and cell-penetrating peptides are being developed (Zhi D. et al. Bioconjug. Chem. 2013, 24; 487-519., Buyens K. et al. J. Control Release, 2012, 158; 362-70., ROSSI, JJ et al Gene Ther. 2006, 13: 583-584., Yousefi A. et al. J. Control Release, 2013, 170; 209-18. , Trabulo S. et al. Curr. Pharm. Des. 2013, 19; 2895-923.).
- nucleic acid delivery techniques have functional residues as direct bonds, include steps for complex formation, and have problems such as endosomal escape efficiency and biotoxicity of liposome structures. There is a need to improve the nucleic acid transduction function and to solve problems related to manufacturing procedures and side effects.
- the skin is the organ with the largest surface area in the human body, which provides a route for effective drug delivery when used properly. Therefore, administration of physiologically active agents such as therapeutic drugs through the skin, commonly referred to as transdermal delivery, has received great attention due to features such as relatively simple drug administration regimens.
- the skin consists of two main parts: the relatively thin outermost epidermal layer (epidermis, epidermal layer), and the thicker inner area, the dermis (dermis, dermal layer).
- the stratum corneum the outermost layer of the epidermis, consists of flat dead cells filled with keratin.
- the area between the flat dead cells of the stratum corneum consists of lipids that form a lamellar phase that contributes to the natural barrier properties of the skin.
- the stratum corneum, etc. present at the outermost side of the skin acts as a natural barrier, and the skin permeability of external substances such as therapeutic drugs is extremely low, making it difficult to deliver substances having high molecular weight and hydrophilicity.
- nucleic acid complexes complementarily complemented with carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) modified to have a positive charge as a whole have a surprisingly improved cell permeability. It has been confirmed that the expression of target genes can be controlled very efficiently, and a patent for a new construct having low cytotoxicity and improved cell permeability and gene expression control ability of viable nucleic acid has been applied (PCT / KR2017 / 008636). ).
- nucleic acid complexes complementary to the peptide peptide have the property of very efficiently passing through the skin, preferably the stratum corneum and / or epidermal layer, such that the nucleic acid complex can be used as a skin permeable carrier.
- Another object of the present invention to provide a composition for diagnosing, preventing or treating a disease comprising the skin permeable delivery system.
- a skin permeable carrier containing a nucleic acid complex having a structure of the following formula (1) containing a nucleic acid complex having a structure of the following formula (1).
- A is a bioactive nucleic acid having a sequence capable of binding a gene of interest or a gene sequence of interest
- C is a Carrier Peptide Nucleic Acid capable of binding to a live nucleic acid
- ' ⁇ ' means complementary binding between a living nucleic acid and a carrier peptide nucleic acid
- the viable nucleic acid represented by A is negatively charged or neutral as a whole.
- Carrier peptide nucleic acids include one or more peptide nucleic acid monomers modified to be positively charged throughout the carrier peptide nucleic acid.
- the present invention also provides a composition for diagnosing a disease and a prophylactic or therapeutic composition comprising the skin permeable delivery vehicle.
- the present invention also provides a method for preventing or treating a disease comprising administering the skin permeable delivery agent.
- the present invention also provides the use of said skin permeable carrier for the prevention or treatment of a disease.
- the present invention also provides the use of said skin permeable carrier for the manufacture of a medicament for the prevention or treatment of a disease.
- FIG. 1A to 1E schematically show a structure in which a live peptide nucleic acid and a carrier peptide nucleic acid are combined in a skin permeable delivery vehicle.
- 2 is a diagram showing the stratum corneum, epidermal layer and dermis layer.
- Figure 3 (A) is a diagram showing the skin delivery capacity of siRNA single and duplex, showing that siRNA itself was not confirmed from the epidermis to the dermis, (B) is a skin delivery capacity of the nucleic acid complex The figure shows that the nucleic acid complex is delivered from the epidermis to the dermis, and after 24 hours it is delivered to the dermis.
- 4A-4L show the results of transdermal ability testing of cy3-labeled siRNAs.
- Figures 5a to 5c is a view showing the skin delivery ability of the nucleic acid complex containing a low molecular weight material.
- 6A to 6F are diagrams showing the therapeutic effect in in vitro and animal tests on psoriasis, a skin disease of nucleic acid complexes having IFI16 as a target gene.
- FIGS. 7A and 7B are diagrams showing the effect of inhibiting cell metastasis capacity of metastatic skin melanoma cell lines of nucleic acid complexes targeting Mechanism study of anti-metastatic effect with TGF ⁇ R2 targeted PNA in vitro TGF ⁇ R2.
- 8A to 8J are diagrams showing the therapeutic effect of atopic dermatitis induced analog cell models and atopic dermatitis induced animal models of nucleic acid complexes with TLR2 as a target gene.
- FIGS. 9A and 9B are diagrams showing a cell damage recovery effect of a human-derived keratinocyte line of a nucleic acid complex containing smad3 as a target gene.
- FIGS. 10A and 10B are diagrams showing cell growth inhibitory effects in keloid fibroblasts of a nucleic acid complex containing TIEG1 as a target gene.
- the nucleic acid complex complementary to the living nucleic acid and carrier peptide nucleic acid is confirmed that the skin permeability and skin residual, can be utilized in the treatment of diseases through skin surface application Confirmed.
- the present invention relates to a skin permeable delivery vehicle containing a nucleic acid complex having a structure of the following structural formula (1).
- A is a bioactive nucleic acid having a sequence capable of binding a gene of interest or a gene sequence of interest
- C is a Carrier Peptide Nucleic Acid capable of binding to a live nucleic acid
- ' ⁇ ' means complementary binding between a living nucleic acid and a carrier peptide nucleic acid
- the viable nucleic acid represented by A is negatively charged or neutral as a whole.
- Carrier peptide nucleic acids include one or more peptide nucleic acid monomers modified to be positively charged throughout the carrier peptide nucleic acid.
- the bioactive nucleic acid and the carrier peptide nucleic acid in the nucleic acid complex according to Structural Formula (1) may have an anti-parallel binding or parallel binding (see FIGS. 1A and 1B). ).
- the "skin permeable carrier” means a delivery material capable of infiltrating the bioactive material into the body and ultimately into the cell through contact with the skin.
- the term refers to a substance having the ability to pass a desired drug to the epidermal or dermal layer through the stratum corneum and / or the epidermal layer, which is the outermost layer of the skin, or to deliver the desired drug into the body through the dermal layer. .
- the skin permeable carrier according to the present invention may be applied to the stratum corneum, epidermal layer or dermis layer according to the total net charge in the nucleic acid complex according to formula (1) and / or the number of viable nucleic acids and / or carrier peptide nucleic acids in the nucleic acid complex. It can remain or pass through the dermis to deliver the desired drug into the body.
- the skin permeation carrier comprising the nucleic acid complex may be characterized as having skin persistence.
- the living nucleic acid is present in the stratum corneum, epidermal layer and dermis layer.
- the bioactive nucleic acid itself in the "skin permeable carrier” may function as a therapeutic drug (see FIGS. 1A and 1B), and also in addition to the "skin permeable carrier", a therapeutic drug for the treatment of a disease, or the like. This may be in a combined form (see FIG. 1C).
- nucleic acid complex according to Structural Formula 1 itself may be used as a skin permeable carrier and a therapeutic agent itself, or may be used in a form in which a therapeutic drug is bound to the nucleic acid complex.
- the "skin permeable delivery vehicle" has a property that can be delivered into the desired cell after transdermal delivery into the body through the skin, and can be used in any form containing the nucleic acid complex. Can be.
- a "living nucleic acid” has a complementary sequence capable of binding to a target gene of interest to reduce expression, in particular a complementary sequence capable of binding to mRNA of such a target gene of interest
- Nucleic acid comprising a nucleic acid comprising a sequence that promotes the expression of the target gene to be expressed means a nucleic acid involved in gene expression control, such as inhibiting or promoting the expression of the gene, and decreases or increases the expression It may be a nucleic acid having a sequence complementary to the target gene to be, or a nucleic acid having a sequence complementary to a single-stranded RNA sequence such as pre-mRNA, miRNA, mRNA.
- the "bioactive nucleic acid" in the present invention binds to a target gene and a nucleotide sequence including the same in vitro or in vivo (for example, intrinsic function of the gene) Activating or inhibiting transcript expression or protein expression), or regulating the splicing of pre-mRNA (e.g. exon skipping). It may be characterized by.
- the base sequence may be a gene regulatory sequence or a gene coding sequence or a splicing regulatory sequence.
- the gene regulatory region may be selected from a promoter, a transcriptional enhancer, a 5 'untranslated region, a 3' untranslated region, a viral packaging sequence, and a selection marker.
- the gene site may be an exon or intron, and the gene site is within 10, 5, 3, 1 kb or 500, 300, 200 bp of the transcription initiation site of a gene, for example upstream or downstream of the initiation site ( downstream).
- the splicing regulatory region may include exon skipping, cryptic splicing, pseudo-splice site activation, intron retention, alternative splicing deregulation related sequences.
- carrier peptide nucleic acid refers to a nucleic acid that is functionally bonded to the living nucleic acid and some or all of the bases complementary
- the carrier peptide nucleic acid used in the present invention is a peptide nucleic acid (PNA: Peptide Nucleic Acid) as well as similar modified nucleic acids can be used, with peptide nucleic acids being preferred, but not limited to these.
- PNA Peptide Nucleic Acid
- the nucleic acid complex contained in the skin permeation carrier is characterized in that it has a structure of the following structural formula (2) further comprising a substance to help endosomal escape (endosome escape) of the live nucleic acid and carrier peptide nucleic acid can do.
- the "skin permeable carrier" according to the present invention may be in the form of a combination of substances that help endosome escape (see Structural Formula (2), FIGS. 1D and 1E).
- 'm' refers to a substance that helps endosomal escape of bioactive nucleic acids and carrier peptide nucleic acids.
- the bioactive nucleic acid and the carrier peptide nucleic acid may be characterized in that it comprises a substance to help the endosomes escape at the 5'-end and 3'-end of each nucleic acid.
- the "helping material to escape the endosomes” may be characterized in that to help escape from the endosomes of the living nucleic acid by increasing the osmotic pressure inside the endosomes, or by destabilizing the membrane of the endosomes have. It means that live nucleic acids move more efficiently and quickly into the nucleus or cytoplasm to help them meet and act on target genes (DW Pack, AS Hoffman, S. Pun, PS Stayton, “Design and development of polymers for gene delivery,” Nat. Rev. Drug.Dovov., 4, 581-593 (2005)).
- a peptide having a sequence of GLFDIIKKIAESF may be linked to the bioactive nucleic acid as a substance to help the endosomal escape, and a carrier peptide nucleic acid may also be linked to the carrier peptide nucleic acid by linker-mediated linkage. It may be characterized in that, but is not limited thereto.
- the material that helps escape the endosomes are peptides, lipid nanoparticles (lipid nanoparticles), conjugated nanoparticles (polyplex nanoparticles), polymer nanospheres (polymer nanospheres), inorganic nanoparticles (inorganic nanoparticles), cationic lipids Cationic lipid-based nanoparticles, cationic polymer (cationic polymer) and pH sensitive polymers (pH sensitive polymers) may be characterized in that any one or more selected from the group consisting of.
- the peptide may be selected from the group consisting of GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 48), GLFDIIKKIAESF (SEQ ID NO: 49) and Histidine (10).
- the lipid nanoparticles may be selected from the group consisting of Lipid, phospholipids, cetyl palmitate, poloxamer 18, Tween 85, tristearin glyceride, and Tween 80.
- the conjugate nanomaterials may be characterized in that the 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 It 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 nucleic acid complex included in the skin permeable delivery agent as described above, therapeutic drugs, for example, therapeutic proteins, therapeutic compounds, cancer chemotherapeutic agents, toxins, cytotoxic substances, anti-inflammatory agents, therapeutic agents for arthritis , Growth factors, cytokines, chemokines, antibodies, RNAi such as siRNA or miRNA, antisense, nucleic acids, nucleic acid analogs, cells, viruses, phages, viral particles, phage particles, virus capsids, phage capsids, virus analogs, liposomes, micelles, Beads, nanoparticles, microparticles, chemotherapeutic agents, contrast agents, imaging agents, labels (label), it may be characterized in that it further comprises any one or more selected from the group consisting of labeling agents or combinations thereof.
- therapeutic drugs for example, therapeutic proteins, therapeutic compounds, cancer chemotherapeutic agents, toxins, cytotoxic substances, anti-inflammatory agents, therapeutic agents for arthritis , Growth factors, cytokines, chem
- Nucleic acid complexes and therapeutic drugs included in the skin permeable delivery vehicle according to the present invention may be bound in a covalent or linker-mediated form.
- 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. It may be characterized by.
- the bioactive nucleic acid can be characterized as consisting of natural nucleic acid bases and / or modified nucleic acid monomers.
- the bioactive nucleic acid is DNA, RNA or modified nucleic acid PNA (peptide nucleic acid), PMO (phosphorodiamidate morpholino oligonucleotide), LNA (locked nucleic acid), GNA (glycol nucleic acid), TNA (threose nucleic acid) acid), antisense oligonucleotide, aptamer, small interfering RNA, siRNA, short hairpin RNA, ribozyme, and DNAzyme.
- PNA peptide nucleic acid
- PMO phosphorodiamidate morpholino oligonucleotide
- LNA locked nucleic acid
- GNA glycol nucleic acid
- TNA threose nucleic acid
- antisense oligonucleotide aptamer
- small interfering RNA small interfering RNA
- siRNA short hairpin RNA
- ribozyme threose nucleic acid
- the monomer used in the bioactive nucleic acid is PNA, it is called a bioactive peptide nucleic acid, and when other monomers are used, it is called in the same manner.
- the bioactive nucleic acid and the carrier peptide nucleic acid is phosphodiester (phosphodiester), 2 '0-methyl (2' 0-methyl), 2 'methoxy-ethyl (2' methoxy-ethyl), phosphor Amidodate (phosphoramidate), methylphosphonate (methylphosphonate) and phosphorothioate (phosphorothioate) may be characterized in that it further comprises any one or more functional groups selected from the group consisting of.
- the carrier peptide nucleic acid may be composed of a sequence complementary to some or all of the bioactive nucleic acid and the base sequence.
- the carrier peptide nucleic acid may comprise 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 carrier peptide nucleic acid in the nucleic acid complex included in the skin permeable carrier is a complex characterized in that the electrical properties as a whole, positive charge (positive), negative charge (negative) or neutral charge Can be.
- the term “total” means not the electrical properties of the individual bases, but rather the overall electrical properties of the whole living nucleic acid or carrier peptide nucleic acid when viewed from the outside. Even if some monomers in the active nucleic acid are positive, if there are more negative monomers, the viable nucleic acid will have a negative charge in view of the “overall” electrical properties, and some bases and / or in the carrier peptide nucleic acid. If the backbone is negative but there are more positive bases and / or more backbones, then the carrier peptide nucleic acid will be positively charged when viewed "in whole" electrical properties.
- the nucleic acid complex having the structure of Structural Formula (1) of the present invention may be characterized as having a positive charge as a whole.
- the bioactive nucleic acid has a negative charge or a neutral property when looking at the electrical properties as a whole
- the carrier peptide nucleic acid has a positive charge property when looking at the electrical properties as a whole. Characterized in, but is not limited thereto.
- the assignment of the electrical properties of the bioactive nucleic acid and the carrier peptide nucleic acid can use a modified peptide nucleic acid monomer
- the modified peptide nucleic acid monomer is a carrier peptide nucleic acid having a positive charge (Lysine, Lys, K) At least one positive charge selected from the group consisting of arginine (Arginine, Arg, R), histidine (Hisidine, His, H), diamino butyric acid (DAB), ornithine (Ornithine, Orn) and amino acid analogs
- a carrier peptide nucleic acid containing an amino acid of and a negatively charged carrier peptide nucleic acid may be characterized by containing a negatively charged amino acid of glutamic acid (Glutamic acid, Glu, E) or an amino acid analog.
- the carrier peptide nucleic acid may include at least one gamma- or alpha-backbone modified peptide nucleic acid monomer so as to have a positive charge as a whole.
- the gamma- or alpha-backbone modified peptide nucleic acid monomer is lysine (Lysine, Lys, K), arginine (Arginine, Arg, R), histidine (Histidine, His, H), diamino butyric to have an electrical positive acid, DAB), ornithine (Ornithine, Orn) and amino acid having one or more positive charges selected from the group consisting of amino acid analogs may be characterized in that it comprises a backbone.
- modification of the peptide nucleic acid monomer for charge assignment may use a peptide nucleic acid monomer modified with nucleobase.
- nucleobase Preferably, amine, triazole, and imidazole moeity may be included in the nucleobase to have electrical positiveness, or carboxylic acid may be included in the base to have electrical negativeness.
- the modified peptide nucleic acid monomer of the carrier peptide nucleic acid may further include a negative charge in the backbone or nucleobase, but the modified peptide nucleic acid monomer is contained more than the monomer having a positive charge as a monomer having a positive charge as a whole carrier peptide It is preferable that the charge of the nucleic acid be positive.
- the nucleic acid complex of formula (1) according to the present invention is characterized as having a positive charge as a whole.
- nucleic acid complex of the formula (1) at least one selected from the group consisting of hydrophobic moiety, hydrophilic moiety, target antigen specific antibody, aptamer or fluorescent / luminescent marker
- the substance may be bound to bioactive nucleic acids and / or carrier peptide nucleic acids, preferably the hydrophobic moiety, hydrophilic moiety, target antigen specific antibody, aptamer and imaging.
- At least one material selected from the group consisting of fluorescent / luminescent markers, etc. may be bound to the carrier peptide nucleic acid.
- the binding of the carrier peptide nucleic acid may be characterized in that the covalent bonds are simple covalent bonds or linkers, but is not limited thereto (see Table 1).
- cell permeation, solubility, stability, transport and imaging related materials (eg, hydrophobic residues, etc.) bound to the nucleic acid carrier will be present independently of the viable nucleic acid that regulates expression of the target gene.
- the complementary binding form of the bioactive nucleic acid and the carrier peptide nucleic acid may be characterized as having a form of antiparallel binding and parallel binding.
- the complementary binding forms have a structure that is separated in the presence of the target sequence (complementary sequence with the living nucleic acid) of the living nucleic acid.
- the antiparallel binding and parallel binding are determined according to the 5'-direction and the 3'-direction in the DNA-DNA or DNA-PNA binding mode.
- Anti-parallel binding is a general DNA-DNA or DNA-PNA binding method.
- the nucleic acid complex of structural formula (1) according to the present invention is described as an example, the live nucleic acid is in the 5 'to 3' direction, and the carrier peptide nucleic acid is It means a form that is coupled to each other in the 3 'to 5' direction.
- Parallel binding refers to a form in which binding force is slightly lower than antiparallel binding, in which both live nucleic acid and carrier peptide nucleic acid are bonded to each other in a 5 'to 3' direction or a 3 'to 5' direction.
- the binding force of the bioactive nucleic acid and the carrier peptide nucleic acid is lower than that of the target gene of the living nucleic acid and the living nucleic acid, in particular, the mRNA of the target gene. It can be characterized.
- the bonding force is determined by melting temperature, melting temperature or Tm.
- the bioactive nucleic acid and the carrier peptide nucleic acid may be characterized by parallel binding or partial specific binding, but are 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 the corresponding base of the bioactive nucleic acid. This may be, but is not limited thereto (see Table 1).
- the universal base binds without complementary binding to natural bases such as adenine, guanine, cytosine, thymine, uracil, and the like.
- natural bases such as adenine, guanine, cytosine, thymine, uracil, and the like.
- a base having a lower binding force than the binding force at least one selected from the group consisting of inosine PNA, indole PNA, nitroindole PNA, and abasic may be used. It may be characterized by the use of inosine PNA.
- N is a base of the nucleic acid (ATGC)
- * is a sequence not complementary to the antisense nucleic acid sequence
- $ is a universal base
- is a linker
- 5 '-, 3'- indicates the orientation of the nucleic acid (base).
- the present invention provides a combination of the binding form and the electrical properties of the nucleic acids for the function of the nucleic acid complex, the combination of the binding form and the electrical properties of the nucleic acid to control the particle size and time of action, cell permeability, solubility and specificity It may be characterized by improving the degree.
- the binding time between the bioactive peptide nucleic acid and the carrier peptide nucleic acid, in the presence of the target gene the point of time when the bioactive peptide nucleic acid is combined with the target sequence (the time of substitution of the bioactive nucleic acid to the target sequence, Target specific separation and binding time).
- the regulation of the point of displacement displacement, the target specific release and the binding point of the bioactive nucleic acid to the target gene is non-specific of the complex. Characterized by the presence, number and location of the non-specific base, universal base and linker of the carrier peptide nucleic acid for binding may be characterized.
- the peptide complex may be controlled by a combination of the above conditions, such as parallel or antiparallel forms of complementary binding.
- the particles of the nucleic acid complex of formula (1) may be characterized in that the size of 5 nm to 300 nm, preferably 10 nm to 80 nm, most preferably 15 nm to 70 nm. .
- the particle size of the nucleic acid complex may be controlled by controlling the charge balance of the bioactive peptide nucleic acid and the carrier peptide nucleic acid. Specifically, the particle size decreases when the positive charge of the carrier peptide nucleic acid increases, but when the positive charge of the carrier peptide nucleic acid exceeds a certain level, the particle size increases. Another important factor in determining particle size is also the particle size, which is determined by the proper charge balance with the overall carrier peptide nucleic acid depending on the bioactive peptide nucleic acid charge complexing.
- 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 is included), preferably 2 to 5, most preferably 2 to 3
- the charge of the viable nucleic acid is 0 to 5 negative charges, preferably 0 to 3, of the charge balance.
- the nucleic acid complex of the above formula (1) can be prepared by hybridizing the bioactive nucleic acid and the carrier peptide nucleic acid under appropriate conditions.
- Hybridization of the present invention means that the complementary single stranded nucleic acids form a double-stranded nucleic acid. Hybridization can occur when the complementarity between two nucleic acid strands is perfect or even when some mismatch base is present. The degree of complementarity required for hybridization may vary depending on the hybridization conditions, and in particular, may be controlled by the bonding temperature.
- the present invention relates to a composition for diagnosing a disease comprising a skin permeable delivery vehicle containing a nucleic acid complex having the structure of Formula (1) or (2).
- the reporter and the reporter fluorescence are quenched at both ends of the bioactive nucleic acid or carrier peptide nucleic acid according to Structural Formula (1).
- Quenchers can be combined.
- the reporter (reporter) in the group consisting of FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein) and CY5 It may be one or more selected, preferably CY5.
- the quencher may be any one or more selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, but is not limited thereto.
- the present invention relates to a composition for preventing or treating a disease comprising a skin permeable delivery vehicle containing a nucleic acid complex having a structure of Formula (1) or Formula (2).
- the present invention provides a method for preventing or treating a disease, which comprises administering a skin permeable delivery agent containing a nucleic acid complex having the structure of Formula (1) or (2) to a patient in need of prevention or treatment. To provide.
- the present invention relates to the use of a skin permeable carrier containing a nucleic acid complex having a structure of the above formula (1) or (2) for the prevention or treatment of a disease.
- the present invention relates to the use of a skin permeable carrier containing a nucleic acid complex having a structure of the above formula (1) or (2) for the manufacture of a medicament for the prevention or treatment of a disease.
- Target gene of the present invention means 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.
- the target nucleic acid (base sequence) including the target gene is contacted (bound) with the complex in vitro or in vivo, the viable nucleic acid is separated from the carrier peptide nucleic acid, thereby biologically active. Will be displayed.
- a disease that can be diagnosed, prevented or treated using a nucleic acid complex having a structure of the above formula (1) or (2) is a target gene or nucleic acid complex to which a living nucleic acid in the nucleic acid complex binds. Can be determined according to the combined therapeutic drug,
- skin diseases such as psoriasis, atopic disease including atopic dermatitis, skin cancer such as melanoma, keloid diseases, It may be used for the treatment of diseases such as skin damage and pigmentation, tumor or cancer, inflammatory disease, senile macular degeneration, diabetic retinopathy, rare disease and severe disease, cardiovascular disease, metabolic disease and the like, but is not limited thereto.
- the term “therapeutic composition” can be mixed with “pharmaceutical composition” (pharmaceutical composition), the active nucleic acid of the present invention and a carrier peptide nucleic acid binding to the nucleic acid It comprises a nucleic acid complex comprising an as an active ingredient, and may be in addition to the nucleic acid complex is combined with a therapeutic drug for treating a desired disease.
- the disease that can be prevented or treated with the therapeutic composition comprising the skin transit carrier is atopic disease including skin diseases such as psoriasis and atopic dermatitis. ), Skin cancers such as melanoma, keloid diseases, skin damage and pigmentation, etc., tumors or cancers, inflammatory diseases, senile macular degeneration, diabetic retinopathy, rare diseases, and It may be used for the treatment of severe diseases, cardiovascular diseases, metabolic diseases, but is not limited thereto.
- the therapeutic composition of the present invention may be formulated in a formulation of skin delivery according to standard medical practice. These formulations may contain, in addition to the active ingredient, additives such as carriers, excipients, adjuvants or diluents, which are suitable for formulation in a pharmaceutically acceptable, in particular, form applicable to the skin.
- additives such as carriers, excipients, adjuvants or diluents, which are suitable for formulation in a pharmaceutically acceptable, in particular, form applicable to the skin.
- the therapeutic composition according to the invention may be formulated in the form of an aqueous solution, gel, ointment, cream, lotion, paste, smear or patch.
- aqueous solutions can be used in the form of distilled water and buffer solutions.
- physiologically acceptable means a property that does not impair the biological activity and the properties of the compound.
- carrier is defined as a compound that facilitates the addition of a nucleic acid complex into a cell or tissue.
- DMSO dimethyl sulfoxide
- carrier facilitates the incorporation of many organic compounds into cells or tissues of an organism.
- diot is defined as a compound that not only stabilizes the biologically active form of the compound of interest, but also is diluted in water to dissolve the 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.
- the substance containing the nucleic acid complex in the present invention may be administered to the patient as it is or as a pharmaceutical composition mixed with other active ingredients, such as in combination therapy, or with a suitable carrier or excipient.
- compositions suitable for use in the present invention include compositions in which the active ingredients are contained in an amount effective to achieve their intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prolong the survival of the subject to be treated or to prevent, alleviate or alleviate the symptoms of a disease. Determination of a therapeutically effective amount is within the capabilities of one of ordinary skill in the art, particularly in view of the detailed disclosure provided herein.
- prevention means any action that prevents the development of a disease or inhibits its progression by administration (or application) of a therapeutic composition comprising the skin permeable delivery vehicle.
- treatment used in the present invention means all the actions that improve or cure the symptoms of the disease by administration (or application) of the therapeutic composition comprising the skin permeable delivery vehicle.
- the composition for preventing or treating a disease comprising the skin permeable delivery agent may be preferably a composition for preventing or treating a skin disease.
- the target gene to which the bioactive nucleic acid contained in the nucleic acid complex binds may be any one or more selected from the group consisting of IFI16, TGF ⁇ R2, TLR2, smad3, and TIEG1, but is not limited thereto.
- the skin diseases include, but are not limited to, psoriasis, skin cancer, atopic diseases or keloid diseases and pigmentation.
- the present invention provides a composition for treating psoriasis.
- the composition for treating psoriasis according to the present invention comprises an IFI16 specific bioactive peptide nucleic acid represented by SEQ ID NO: 22 and a carrier peptide nucleic acid complementary thereto.
- the carrier peptide nucleic acid may preferably have a sequence of SEQ ID NO: 23 or 24, and some of the sequences may be substituted with a universal base to be used.
- the present invention also provides a composition for treating malignant melanoma.
- the composition for treating malignant melanoma according to the present invention comprises a TGFR ⁇ 2 specific bioactive peptide nucleic acid of SEQ ID NO: 25 or 26 or a carrier peptide nucleic acid complementary thereto.
- the carrier peptide nucleic acid may preferably have a sequence of SEQ ID NOs: 27 to 30, and some of the sequences may be substituted with a universal base to be used.
- the present invention also provides a composition for treating atopic dermatitis.
- the composition for treating atopic dermatitis according to the present invention includes a TLR2 specific bioactive peptide nucleic acid having a sequence of SEQ ID NO: 31 or 32 and a carrier peptide nucleic acid complementary thereto.
- the carrier peptide nucleic acid may preferably have a sequence of SEQ ID NOs: 33 to 35, and some of the sequences may be substituted with a universal base to be used.
- the present invention also provides a composition for treating skin damage.
- the therapeutic composition is for skin regeneration including, but not limited to, skin wound healing.
- the composition for treating skin damage according to the present invention comprises a smad3 specific bioactive peptide nucleic acid having a sequence of SEQ ID NO: 36 or 37 and a carrier peptide nucleic acid complementary thereto.
- the carrier peptide nucleic acid may preferably have a sequence of SEQ ID NOs: 38 to 41, and some of the sequences may be substituted with a universal base to be used.
- the present invention also provides a composition for treating keloids.
- the composition for treating keloids according to the present invention comprises a TIEG1 specific bioactive peptide nucleic acid having a sequence of SEQ ID NO: 42 or 43 and a carrier peptide nucleic acid complementary thereto.
- the carrier peptide nucleic acid may preferably have a sequence of SEQ ID NOs: 44 to 47, and some of the sequences may be substituted with a universal base to be used.
- the nucleic acid complex contained in the skin permeable delivery agent may be administered (or applied) using a delivery agent such as a liposome.
- a delivery agent such as a liposome.
- the liposomes can help target the complex to specific tissues, such as lymphoid tissue, or selectively target infectious cells, and can also help increase the half-life of the 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 alone or in combination with other therapeutic compositions, or in combination with other therapeutic compositions for specific cells, the molecule that binds the predominant receptor in lymphoid cells, such as monoclonal antibodies that bind to the CD45 antigen.
- liposomes that are filled or decorated with certain complexes of the present invention to deliver the nucleic acid complex composition can be directed to the site of lymphatic cells.
- Liposomes for use according to the present invention are generally formed from standard vesicle-forming lipids, including sterols such as neutral and negatively charged phospholipids and cholesterol.
- lipids are selected in consideration of, for example, the stability of liposomes in the bloodstream, acid instability and size of liposomes.
- Various methods can be used for liposome preparation. 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 method for treating and inhibiting (or alleviating) a disease by administering (or applying) a skin permeable delivery vehicle containing a nucleic acid complex having a structure of Formula (1) or (2) to a subject. To provide.
- Diseases that can be treated using the carriers according to the invention are determined by the nature of the bioactive nucleic acid used, and are not particularly limited.
- composition comprising the carrier according to the present invention may be applied to the skin in a medicamentally effective amount for treating the above diseases or for inhibiting (or alleviating) the symptoms of the above diseases. It can vary depending on various factors such as the type of skin disease, the age, weight of the patient, the nature and extent of symptoms, the type of current treatment, the number of treatments, the type and route of treatment, and can be easily determined by experts in the field. .
- the compositions of the present invention may be applied together or sequentially applied to the pharmacological or physiological components described above, and may also be applied in combination with additional conventional therapeutic agents and may be applied sequentially or simultaneously with conventional therapeutic agents. Such an application may be a single or multiple application.
- “individual” means a mammal suffering from or at risk of a condition or disease that can be alleviated, inhibited or treated by administering (coating) a skin permeable delivery agent according to the invention, preferably human do.
- the dosage (coating amount) of the compound of the present invention to the human body may vary depending on the age, weight, sex, type of administration (coating), health condition and degree of disease of the patient, and is based on an adult patient having a weight of 70 kg. In general, it is generally 0.001 to 1,000 mg / day, preferably 0.01 to 500 mg / day, and may be dividedly administered once a day to several times at regular intervals according to the judgment of a doctor or a pharmacist (coating). have.
- Toxicity and therapeutic efficiencies of the skin permeable delivery agents or compositions comprising the same described herein can be, for example, LD50 (lethal dose for 50% of the population), ED50 (dose having a therapeutic effect for 50% of the population).
- LD50 lethal dose for 50% of the population
- ED50 dose having a therapeutic effect for 50% of the population
- IC 50 dose with a therapeutic 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 LD50 and ED50 (or IC50).
- Compounds showing high therapeutic indices are preferred.
- the data obtained from these cell culture assays can be used to estimate the range of doses used in humans.
- the dosage or dosage of such compounds is preferably in the range of circulating concentrations comprising ED 50 (or IC 50) in the absence or little toxicity.
- IFI16 a target gene for psoriasis diseases
- IFI16 is a protein commonly expressed in the skin of patients with psoriasis disease and is expected to be an important target in the treatment of psoriasis (Up-regulation of Interferon-inducible protein 16 contributes to psoriasis by modulating chemokine production in keratinocytes 6 : 25381).
- Antisense peptide nucleic acids (antisense PNA) and RNA were used as Bioactive Nucleic Acid for IFI16 to confirm skin permeation effect and skin residual effect.
- bioactive nucleic acids antisense PNA and RNA
- the bioactive nucleic acids (antisense PNA and RNA) of the present invention consist of the sequences set forth in SEQ ID NOs: 1-9.
- Peptide nucleic acid-based bioactive nucleic acid used in this example bound fluorescence (Cy3) for the 3 'end for imaging and the base sequence, monomer modification and structure are shown in Table 2 below.
- All peptide nucleic acids used in the present invention were synthesized by PANAGENE (PANAGENE, Korea) through HPLC purification method.
- All carrier peptide nucleic acids according to an embodiment of the invention consist of the sequences set forth in SEQ ID NOs: 10-21 (Table 2).
- Modification of the monomers gives the electrical properties of the backbone of the pepti nucleic acid, the positive lysine (Lysine, Lys, K, (+) ), the negative electron to glutamic acid (Glutamic acid, Glu, E, (-)) It was designed to have a peptide backbone modified).
- Each bioactive nucleic acid and carrier peptide nucleic acid combination was hybridized under DMSO, resulting in a complex consisting of bioactive nucleic acid and carrier peptide nucleic acid.
- Example 1 In order to confirm the skin permeation effect of the complex prepared in Example 1, a predetermined amount was applied to the nude mouse's back and left for 1, 3, 24 hours, and the tissue of the applied site was biopsied. The biopsied tissues were fixed in 4% formalin solution and left for one day, and then tissues such as mice were sectioned at 20 ⁇ m using a frozen sectioner and mounted on slides. The mounted tissue was checked for penetration of the complex by using a fluorescence microscope.
- the skin was applied to a nude mouse to a certain amount in order to confirm the skin permeation effect of the skin-permeable carrier in which the low-molecular substance was connected to the complex by linker media, and then left for 0, 3, 24 hours, and then applied.
- Tissue was biopsied. The biopsied tissue was fixed in 4% formalin solution and left for one day, and then the mouse back tissue was sectioned to 20 ⁇ m using a frozen sectioner and mounted on a slide. The mounted tissue was confirmed using a fluorescence microscope when the low molecular weight material was connected to the nucleic acid complex which is a skin permeable carrier.
- Example 4 Analysis of psoriasis treatment effect using a skin permeable carrier containing a complex having the structure of formula (1)
- Example 1 the effect of psoriasis treatment was analyzed using a skin permeable delivery vehicle comprising a bioactive peptide nucleic acid and a carrier peptide nucleic acid having IFI16 as a target gene prepared in the structure of Table 5 below.
- HaCaT Human keratinocytes obtained from CLS (CLS Cell Lines Service, Germany) were transferred to DMEM culture medium (Dulbecco Modified Eagle Medium, Weljin, Korea) 10% (v / v) fetal bovine serum, penicillin 100 units / ml, strepto 100 ⁇ g / ml of mycin was added and incubated under conditions of 37 ° C. and 5% (v / v) CO 2 .
- IL-17A was incubated at 10 ng / mL to create a psoriasis-like cell model.
- Example 1 the cells were seeded at 6 ⁇ 10 3 in a 96-well plate using a complex comprising a bioactive nucleic acid and a carrier peptide nucleic acid prepared in the structure of Table 5, and cultured for 24 hours under the conditions of Example 4-1.
- MTT 3- (4,5-Dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium Bromide
- OD optical density
- the nucleic acid complexes of SEQ ID NO: 22 and SEQ ID NO: 23 were used (PNA 4). As a result, as shown in FIG. 6A, it was confirmed that the cell viability was decreased in a concentration-dependent manner by the nucleic acid complex in the IL-17A-induced psoriasis-like cell model.
- Human seeded keratinocytes were seeded at 1 ⁇ 10 5 in 6-well plates, cultured for 24 hours, and subjected to the experiment of Example 4-1 to treat complexes containing bioactive peptide nucleic acids and carrier peptide nucleic acids. , 72 hours of incubation, RIPA buffer was added to each well 30 ⁇ L to obtain protein lysate. Protein lysate was quantified using a BCA assay kit (Thermo Fisher, USA), 30 ⁇ g of protein was isolated by size using electrophoresis, the protein was transferred to PVDF membrane, and the primary antibody IFI16 (Cell signaling Technology, P-NFkB (Cell signaling Technology, USA) was treated at 1: 1000 and left at 4 ° C.
- IFI16 Cell signaling Technology, P-NFkB (Cell signaling Technology, USA
- the nucleic acid complexes of SEQ ID NO: 22 and SEQ ID NO: 23 of Table 5 were analyzed for expression patterns of IFI16 protein and subpath genes in keratinocyte lines. As a result, as shown in FIG. 6B, it was confirmed that the expression of the target gene and the expression of the lower pathway gene were inhibited with time through the nucleic acid complex.
- a psoriasis-derived animal model was created for 7 days by applying 62.5 mg of 5% Imiquimod daily to the right ear of the Balb / C mouse, and the psoriasis model phenotype was photographed for the nucleic acid complexes of SEQ ID NO: 22 and SEQ ID NO: 23 in Table 5. Ear thickness was measured with a micrometer.
- mice ear tissues were biopsied at the end of the last experiment, and the tissues were fixed in 4% formalin solution for one day, and the tissues were paraffin embedded, and the tissues were sectioned at 5 ⁇ m and mounted on slide glass. Mounted tissues were stained in Hematoxylin: Eoin staining solution for a certain time, washed with 1X PBS and analyzed under a microscope.
- TGF ⁇ R2 was used as a target gene to analyze the effect of treating the malignant melanoma of the nucleic acid complex. Since the development of RNA-based therapeutics that inhibit the mechanism of TGF ⁇ -2 activation during cancer metastasis has been confirmed, we tried to verify the therapeutic effect using peptide nucleic acid complexes.
- Skin metastatic melanoma obtained from the American Type Culture Collection (ATCC) was collected in DMEM culture medium (Dulbecco Modified Eagle Medium, Weljin, Korea) at 10% (v / v) fetal bovine serum, penicillin 100 units / ml, 100 ⁇ g / ml of streptomycin was added and incubated under conditions of 37 ° C and 5% (v / v) CO 2 . TGF ⁇ -2 was incubated at 5 ng / mL to confirm cell metastasis.
- ATCC American Type Culture Collection
- the cells of the metastatic skin melanoma cell line were seeded at 1 ⁇ 10 5 in 6-well plates and cultured for 24 hours, and then subjected to the experiments in the conditions of Example 5-1 to treat complexes including bioactive peptide nucleic acids and carrier peptide nucleic acids. After 48 and 72 hours of incubation, RIPA buffer was added to each well by 30 ⁇ L to obtain protein lysate. Protein lysate was quantified using a BCA assay kit (Thermo Fisher, USA), 30 ⁇ g of protein was isolated by size using electrophoresis, the protein was transferred to PVDF membrane, and the primary antibody, TGF ⁇ R2 (Abcam, USA).
- MMP9 (SantaCruz Biotech., USA), p-Akt1 (Cell signaling Technology, USA) were treated 1: 1000 and left at 4 ° C. for one day. Washing with 1X TBS-T and the secondary antibodies Goat Anti-Rabbit, Goat Anti-mouse (SantaCruz Biotech., USA) was treated at 1: 2000 and left at room temperature for 2 hours. Supersignal TM West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA) was treated and Image600 (Amersham, Germany) was used to analyze the expression inhibition efficiency of target genes in metastatic skin melanoma cell lines.
- TGF ⁇ R2 protein and subpath genes in metastatic cutaneous melanoma cell lines were analyzed.
- the combination of the nucleic acid complexes used in this example is shown in Table 7 below.
- the metastatic skin melanoma cell line was seeded in a 24 well transwell plate with 2x10 4 cells in the upper chamber, and all the chambers were treated with a medium without fetal bovine serum, followed by incubation for 24 hours, and the experiment was carried out under the conditions of Example 5-1 After treatment with the complex containing the bioactive peptide nucleic acid and carrier peptide nucleic acid in the upper chamber and TGF ⁇ -2 in the lower chamber at 20 ng / mL, the cells were incubated for 24, 48 and 72 hours, and then 0.5% crystal Stained with violet. The stained cells were treated with methanol to dissolve crystal violet, and absorbance at 450 nm was measured.
- the nucleic acid complexes of Table 7 were analyzed for cell migration inhibition effect in metastatic skin melanoma cell line. As a result, as shown in FIG. 7B, it was confirmed that cell migration ability was inhibited with time through the nucleic acid complex.
- TLR2 (Toll-Like Receptor 2) was used as a target gene for the analysis of the atopic dermatitis therapeutic effect of the nucleic acid complex.
- TLR2 a gene expressed when allergens or bacteria penetrate into the skin, is overexpressed in patients with atopic dermatitis and exacerbates atopic dermatitis by increased inflammation caused by inflammatory cytokines in the skin. For this reason, it is expected to be an important target in atopic dermatitis.
- HaCaT Human keratinocytes obtained from CLS (CLS Cell Lines Service, Germany) were transferred to DMEM culture medium (Dulbecco Modified Eagle Medium, Weljin, Korea) 10% (v / v) fetal bovine serum, penicillin 100 units / ml, strepto 100 ⁇ g / ml of mycin was added and incubated under conditions of 37 ° C. and 5% (v / v) CO 2 . To prepare an atopic dermatitis-like cell model, 5 ng / mL of house dust mite extract and 5 ⁇ M of DNCB (2-dinitrochlorobenzene) were incubated for 24 hours.
- DMEM culture medium Dulbecco Modified Eagle Medium, Weljin, Korea
- penicillin 100 units / ml penicillin 100 units / ml
- strepto 100 ⁇ g / ml of mycin was added and incubated under conditions of 37 ° C. and 5%
- Example 6-1 seeded human keratinocytes line with 6x10 3 in 96 wells, and cultured for 24 hours, using a complex comprising a living nucleic acid and a carrier peptide nucleic acid prepared in the structure of Table 5
- MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium Bromide) solution was prepared at 5 mg / mL in 1X PBS and treated at 20 ⁇ L per well for 4 hours. After incubation, the optical density (OD) was analyzed by measuring with a spectrophotometer.
- Human-derived keratinocytes were seeded with 1 ⁇ 10 5 cells in 6-well plates and cultured for 24 hours, and then subjected to the experiments in the conditions of Example 6-1 to treat complexes containing bioactive peptide nucleic acids and carrier peptide nucleic acids. , 72 hours of incubation, RIPA buffer was added to each well 30 ⁇ L to obtain protein lysate.
- Protein lysate was quantified using a BCA assay kit (Thermo Fisher, USA), 30 ⁇ g of protein was separated by size by electrophoresis, the protein was transferred to PVDF membrane, and the primary antibody, TLR2 (SantaCruz Biotech., USA), MMP9 (SantaCruz Biotech., USA), p-NFkB (Cell signaling Technology, USA), MyD88 (Cell signaling Technology, USA), TARC (Abcam, USA) 1: 1: 1000 and treated at 4 °C for one day It was left.
- TLR2 SuraCruz Biotech., USA
- MMP9 SantaCruz Biotech., USA
- p-NFkB Cell signaling Technology, USA
- MyD88 Cell signaling Technology, USA
- TARC Abcam, USA
- NC / Nga mice The back of NC / Nga mice was depilated and AD cream (house dust mite extract cream, Biostir, Japan) was applied 100 mg twice a week for a total of 3 weeks to build an atopic dermatitis-induced animal model by house dust mite.
- AD cream house dust mite extract cream, Biostir, Japan
- the hair of Balb / C mice was depilated, and 50 ⁇ M of DNCB was applied twice a week for a total of 3 weeks to build an atopic dermatitis-induced animal model due to sick house syndrome.
- Nucleic acid complexes of a total of three cream formulations were treated per week, the atopic dermatitis animal model phenotype was analyzed by photos, and the back hair growth was measured by ImageJ.
- mice blood was collected through orbital blood collection at the end of the last experiment, and the serum was collected using a centrifuge (14,000 rpm, 15 min) after being left at room temperature for 2 hours or more.
- the concentration of the collected IgE ELISA kit (Goma Biotech, Korea) and TARC ELISA kit (R & D system, USA) was used to measure the concentration of IgE and TARC in serum.
- Example 6-4 Experiments were carried out under the conditions of Example 6-4, and the biopsy of mouse ears was performed at the end of the last experiment.
- the tissues were fixed in 4% formalin solution for one day, and the tissues were paraffin embedded, and the tissues were sectioned at 5 ⁇ m and mounted on slide glass. Mounted tissues were stained in Hematoxylin: Eoin staining solution for a certain time, washed with 1X PBS and analyzed under a microscope.
- Example 6-4 The experiments were carried out under the conditions of Example 6-4, and the tissues such as mice were biopsied at the end of the final experiment, fixed in 4% formalin solution for one day, and the fixed tissues were paraffin embedded, sectioned at 5 ⁇ m, and mounted on slide glass. Mounted tissues were blocked for 1 hour in 0.5% BSA solution and treated for one day by treatment with CD3, CD11c primary antibody solution. Subsequently, the primary antibody solution was removed, washed with 1X PBS, and then treated with a secondary antibody solution for 2 hours at room temperature and analyzed through DAB staining.
- Smad3 was used as a target gene. Smad3 is an overexpressed protein in wounded skin and is expected to be an important target for skin regeneration.
- HaCaT Human keratinocytes obtained from CLS (CLS Cell Lines Service, Germany) were transferred to DMEM culture medium (Dulbecco Modified Eagle Medium, Weljin, Korea) 10% (v / v) fetal bovine serum, penicillin 100 units / ml, strepto 100 ⁇ g / ml of mycin was added and incubated under conditions of 37 ° C. and 5% (v / v) CO 2 . TGF ⁇ -1 was incubated at 5 ng / mL to confirm cell migration capacity.
- DMEM culture medium Dulbecco Modified Eagle Medium, Weljin, Korea
- penicillin 100 units / ml penicillin 100 units / ml
- strepto 100 ⁇ g / ml of mycin was added and incubated under conditions of 37 ° C. and 5% (v / v) CO 2 .
- TGF ⁇ -1 was incubated at 5 ng / mL to confirm cell migration
- the cells were seeded at 2 ⁇ 10 4 in 12-well plates of human-derived keratinocyte lines and treated with a fetal bovine serum-free medium for 24 hours, followed by experiments under the conditions of Example 7-1 to obtain bioactive peptide nucleic acids and carrier peptide nucleic acids.
- the complex was treated and treated with TGF ⁇ -1 at 5 ng / mL and incubated for 24 and 48 hours, and then the cell migration capacity was analyzed under a microscope.
- Human seeded keratinocytes were seeded at 1 ⁇ 10 5 in 6-well plates and cultured for 24 hours, and the experiments were carried out under the conditions of Example 7-1 to treat complexes comprising bioactive peptide nucleic acids and carrier peptide nucleic acids. After incubation for time, RIPA buffer was added to each well 30 ⁇ L to obtain protein lysate. Protein lysate was quantified using a BCA assay kit (Thermo Fisher, USA), 30 ⁇ g of protein was isolated by size using electrophoresis, the protein was transferred to PVDF membrane, and the primary antibody, p-smad3 (Cell signaling). Technology, USA) was treated at 1: 1000 and left at 4 ° C. for one day.
- Example 8 Analysis of keloid therapeutic effect using a skin permeable carrier containing a complex having the structure of formula (1)
- TIEG1 was used as a target gene to analyze the effect of treatment of keloids after wounding of nucleic acid complexes. It is a protein overexpressed in the skin tissue of Keloid patients and is expected to be an important target for keloid treatment.
- Keloid fibroblasts obtained from the American Type Culture Collection (ATCC) were cultured in DMEM culture medium (Dulbecco Modified Eagle Medium, Weljin, Korea) at 10% (v / v) fetal bovine serum, penicillin 100 units / ml, 100 ⁇ g / ml of streptomycin was added and incubated under conditions of 37 ° C and 5% (v / v) CO 2 .
- DMEM culture medium Dulbecco Modified Eagle Medium, Weljin, Korea
- Example 8-1 Seed the keloid fibroblast line 6x10 3 in 96 wells in the experimental conditions in Example 8-1, and incubated for 24 hours, using a complex comprising a living nucleic acid and a carrier peptide nucleic acid prepared in the table structure MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium Bromide) solution was prepared at 5 mg / mL in 1X PBS and treated at 20 ⁇ L per well for 4 hours. OD (optical density) was then analyzed by measuring with a spectrophotometer.
- MTT 3- (4,5-Dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium Bromide
- the cells were seeded at 1 ⁇ 10 5 in 6-well plates of keloid fibroblasts, and cultured for 24 hours, the experiments were carried out under the conditions of Example 8-1 to treat complexes containing bioactive peptide nucleic acids and carrier peptide nucleic acids. After incubation for 72 hours, RIPA buffer was added to each well 30 ⁇ L 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 by electrophoresis.
- the protein was transferred to PVDF membrane, and then TIEG1 (SantaCruz Biotech., USA), p-smad2 (SantaCruz Biotech., USA), smad7 (Cell Signaling Technology, USA) were treated 1: 1000 and left at 4 ° C. for one day. Washing with 1X TBS-T and the secondary antibodies Goat Anti-Rabbit, Goat Anti-mouse (SantaCruz Biotech., USA) was treated at 1: 2000 and left at room temperature for 2 hours. Supersignal TM West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA) was treated and analyzed for the inhibition of expression of target genes in keloid fibroblast lines using Image600 (Amersham, Germany) instrument.
- the nucleic acid complexes of Table 14 were analyzed for expression patterns of TIEG1 protein and subpath genes in keratinocyte lines. As a result, as shown in FIG. 10B, it was confirmed that the expression of the target gene and the expression of the downstream pathway genes were inhibited with time through the nucleic acid complex.
- the skin permeable delivery agent containing the nucleic acid complex having the structure of formula (1) has both a skin permeation function and an effect on the body for effectively delivering a large molecular weight drug.
- the carrier according to the present invention enables the passage of the stratum corneum, epidermal and / or dermal layers of bioactive nucleic acids or various compounds, in particular for the external treatment of therapeutic drugs applied to the skin surface and for the body's circulatory system such as blood. Drugs can be delivered.
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Abstract
Description
Claims (23)
- 하기 구조식 (1)의 구조를 가지는 핵산 복합체를 함유하는 피부 투과성 전달체:구조식 (1)[ A ≡ C (+) ]상기 구조식 (1)에 있어서,A는 목적하는 유전자와 결합할 수 있는 서열 또는 목적하는 유전자 서열을 가지는 생활성 핵산(Bioactive Nucleic Acid)이고,C는 생활성 핵산과 결합할 수 있는 캐리어 펩티드 핵산(Carrier Peptide Nucleic Acid)이고,'≡'는 생활성 핵산과 캐리어 펩티드 핵산 간의 상보적인 결합을 의미하며,A로 표시되는 생활성 핵산은 전체적으로 음전하 또는 중성을 가지며,C(+)는 캐리어 펩티드 핵산이 전체적으로 양전하를 가진다는 것을 의미하며,캐리어 펩티드 핵산은 캐리어 펩티드 핵산 전체적으로 양전하를 띠도록 변형된 펩티드 핵산 단량체를 하나 이상 포함한다.
- 제1항에 있어서, 상기 핵산 복합체는 피부 잔류성을 갖는 것을 특징으로 하는 피부 투과성 전달체.
- 제1항에 있어서, 상기 핵산 복합체는 생활성 핵산과 캐리어 펩티드 핵산의 엔도좀 탈출(endosome escape)을 도와주는 물질을 더 포함하여 하기 구조식 (2)의 구조를 가지는 것을 특징으로 하는 피부 투과성 전달체:구조식 (2)[ mA ≡ mC (+) ]상기 구조식 (2)에 있어서,'m'은 생활성 핵산과 캐리어 펩티드 핵산의 엔도좀 탈출(endosome escape)을 도와주는 물질을 의미한다.
- 제3항에 있어서, 상기 생활성 핵산 또는 캐리어 펩티드 핵산은 각각의 핵산의 5'-말단 또는 3'-말단에 엔도좀 탈출을 도와주는 물질이 결합된 것을 특징으로 하는 피부 투과성 전달체.
- 제3항에 있어서, 상기 엔도좀 탈출을 도와주는 물질은 펩티드, 지질 나노물질(lipid nanoparticles), 접합체 나노물질(polyplex nanoparticles), 고분자 나노구(polymer nanospheres), 무기물 나노물질(inorganic nanoparticles), 양이온 지질 나노물질(cationic lipid-based nanoparticles), 양이온 고분자(cationic polymer) 및 pH 감응 고분자(pH sensitive polymers)로 이루어진 군에서 선택되는 어느 하나 이상인 것을 특징으로 하는 핵산 복합체.
- 제5항에 있어서, 상기 펩티드는 GIGAVLKVLTTGLPALISWIKRKRQQ(서열번호 48), GLFDIIKKIAESF(서열번호 49) 및 Histidine(10)으로 구성된 군에서 선택되고,상기 지질 나노물질(lipid nanoparticles)은 Lipid, phospholipids, cetyl palmitate, poloxamer 18, Tween 85, tristearin glyceride 및 Tween 80로 구성된 군에서 선택되고,상기 접합체 나노물질(polyplex nanoparticles)은 poly(amidoamine) 또는 polyethylenimine (PEI)이고,상기 고분자 나노구(polymer nanospheres)는 polycaprolactone, poly(lactide-co-glycolide, polylactide, polyglycolide, poly(d,l-lactide), chitosan 및 PLGA-polyethylene glycol로 구성된 군에서 선택되고,상기 무기물 나노물질(inorganic nanoparticles)은 Fe2O3 Fe3O4, WO3 및 WO2.9로 구성된 군에서 선택되고,상기 양이온 지질 나노물질(cationic lipid-based nanoparticles)은 1-(aminoethyl)iminobis[N-(oleicylcysteinyl-1-amino-ethyl)propionamide], N-alkylated derivative of PTA 및 3, 5-didodecyloxybenzamidine로 구성된 군에서 선택되고,상기 양이온 고분자(cationic polymer)는 vinylpyrrolidone-N, N-dimethylaminoethyl methacrylate acid copolymer diethyl sulphate, polyisobutylene 및 poly(N-vinylcarbazole)로 구성된 군에서 선택되고,상기 pH 감응 고분자(pH sensitive polymers)는 polyacids, poly(acrylic acid), poly(methacrylic acid) 및 hydrolyzed polyacrylamide로 이루어진 군에서 선택되는 것을 특징으로 하는 핵산 복합체.
- 제1항에 있어서, 상기 핵산 복합체는 치료성 단백질, 치료성 화합물, 치료성 조성물, 암 화학치료제, 톡신, 세포독성 물질, 소염제, 관절염 치료제, 성장인자, 사이토킨, 케모카인, 하나 이상의 신호 전달 경로를 조절하는 화합물, 항체, 핵산, 핵산 유사체, 세포, 바이러스, 파지, 바이러스 입자, 파지 입자, 바이러스 캡시드, 파지 캡시드, 바이러스 유사입자, 리포솜, 미셀, 비드, 나노입자, 마이크로입자, 화학치료제, 조영제, 영상제, 표지(label), 표지화제 또는 그 배합물로 구성된 군에서 선택되는 어느 하나 이상을 추가로 더 포함하는 것을 특징으로 하는 피부 투과성 전달체.
- 제1항에 있어서, 상기 생활성 핵산은 DNA, RNA, LNA, PNA 및 변형된 펩티드 핵산으로 구성된 군에서 선택된 핵산 단량체로 이루어진 것을 특징으로 하는 피부 투과성 전달체.
- 제1항에 있어서, 상기 캐리어 펩티드 핵산은 상기 생활성 핵산과 염기서열 일부 혹은 전부가 상보적인 서열로 구성되는 것을 특징으로 하는 피부 투과성 전달체.
- 제1항에 있어서, 전체적으로 양전하를 가지는 것을 특징으로 하는 피부 투과성 전달체.
- 제1항에 있어서, 상기 캐리어 펩티드 핵산은 전체적으로 양전하를 가지도록 1개 이상의 gamma- 또는 alpha-backbone 변형 펩티드 핵산 단량체를 포함하는 것을 특징으로 하는 피부 투과성 전달체.
- 제11항에 있어서, 상기 gamma- 혹은 alpha-backbone 변형 펩티드 핵산 단량체는 양전하를 가지는 아미노산을 가지는 단량체가 음전하를 가지는 아미노산을 가지는 단량체에 비해 더 많이 포함되어 전체적인 캐리어 펩티드 핵산의 전하가 양성이 되는 것을 특징으로 하는 피부 투과성 전달체.
- 제1항에 있어서, 소수성 잔기(hydrophobic moiety), 친수성 잔기(hydrophilic moiety), 표적 항원 특이적 항체, 앱터머, 소광자, 형광 표지자 및 발광 표지자로 이루어진 군에서 선택된 하나 이상의 물질이 생활성 핵산 및/또는 캐리어 펩티드 핵산에 결합된 것을 특징으로 하는 피부 투과성 전달체.
- 제1항에 있어서, 상기 생활성 핵산 및 캐리어 펩티드 핵산은 각각의 핵산의 5'-방향성 및 3'-방향성에 따라 평행(parallel) 또는 역평행(antiparallel)으로 결합된 것을 특징으로 하는 피부 투과성 전달체.
- 제1항에 있어서, 상기 생활성 핵산 및 캐리어 펩티드 핵산의 결합력은 생활성 핵산과 생활성 핵산의 목적하는 유전자와의 결합력보다 낮은 것을 특징으로 하는 피부 투과성 전달체.
- 제15항에 있어서, 상기 생활성 핵산과 캐리어 펩티드 핵산은 평행 결합(Parallel binding) 또는 부분 특이결합(Partial specific binding)함으로써, 생활성 핵산 및 캐리어 펩티드 핵산의 결합력이 생활성 핵산과 생활성 핵산의 목적하는 유전자와의 결합력보다 낮은 것을 특징으로 하는 피부 투과성 전달체.
- 제1항 내지 제16항 중 어느 한 항에 따른 피부 투과성 전달체를 포함하는 질병의 진단용 조성물.
- 제1항 내지 제16항 중 어느 한 항에 따른 피부 투과성 전달체를 포함하는 질병의 예방 또는 치료용 조성물.
- 제18항에 있어서, 상기 질병은 피부질환, 암, 염증성 질환, 노인성 황반변성, 당뇨병성 망막병증, 희귀질환 및 중증 질환, 심혈관 질환, 또는 대사성 질환인 것을 특징으로 하는 조성물.
- 제19항에 있어서, 상기 피부 투과성 전달체 내에 포함된 생활성 핵산이 결합하는 표적 유전자는 IFI16, TGFβR2, ERBB3, TLR2, smad3 및 TIEG1으로 구성된 군에서 선택된 어느 하나 이상이고, 상기 질병은 피부질환인 것을 특징으로 하는 조성물.
- 제19항에 있어서, 상기 피부질환은 건선, 피부암, 아토피 피부염, 피부 손상, 색소침착 및 켈로이드성 질환으로 구성된 군에서 선택되는 어느 하나인 것을 특징으로 하는 조성물.
- 제18항에 따른 조성물을 포함하는 제형.
- 제22항에 있어서, 제형은 수성액, 겔, 연고, 크림, 로션, 페이스트, 도말제, 패치에서 선택되는 어느 하나인 것을 특징으로 하는 제형.
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CN201980028880.4A CN112135636A (zh) | 2018-02-08 | 2019-01-08 | 含有核酸复合物的皮肤渗透载体及其用途 |
EP19751328.6A EP3750561A4 (en) | 2018-02-08 | 2019-01-08 | SKIN PERMEATION VEHICLE CONTAINING A NUCLEIC ACID COMPLEX AND ITS USE |
US16/968,378 US12016930B2 (en) | 2018-02-08 | 2019-01-08 | Skin-permeating carrier containing nucleic acid complex and use thereof |
AU2019219473A AU2019219473B2 (en) | 2018-02-08 | 2019-01-08 | Skin-permeating carrier containing nucleic acid complex and use thereof |
CA3090765A CA3090765C (en) | 2018-02-08 | 2019-01-08 | Skin-permeating carrier containing nucleic acid complex and use thereof |
JP2020565245A JP2021520402A (ja) | 2018-02-08 | 2019-01-08 | 核酸複合体を含有する皮膚透過性伝達体及びその用途 |
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KR1020180015751A KR102484332B1 (ko) | 2018-02-08 | 2018-02-08 | 핵산 복합체를 함유하는 피부 투과성 전달체 및 이의 용도 |
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EP (1) | EP3750561A4 (ko) |
JP (1) | JP2021520402A (ko) |
KR (3) | KR102484332B1 (ko) |
CN (1) | CN112135636A (ko) |
AU (1) | AU2019219473B2 (ko) |
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JP2022552993A (ja) * | 2019-10-16 | 2022-12-21 | シーサン・セラピューティクス | 血液脳関門透過能を有するペプチド核酸複合体及びこれを含む組成物 |
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WO2020096234A1 (ko) * | 2018-11-07 | 2020-05-14 | 주식회사 시선테라퓨틱스 | 피부 투과성 핵산 복합체를 유효성분으로 함유하는 아토피 피부염의 예방 또는 치료용 조성물 |
JP7459298B2 (ja) * | 2020-01-06 | 2024-04-01 | シーサン・セラピューティクス | 細胞透過性核酸複合体を有効成分として含有する黄斑変性の予防又は治療用組成物 |
KR102630164B1 (ko) * | 2021-06-02 | 2024-01-29 | 주식회사 시선테라퓨틱스 | 펩티드 핵산 복합체를 유효성분으로 함유하는 비알콜성 지방간염 예방 또는 치료용 조성물 |
KR20230106326A (ko) * | 2022-01-06 | 2023-07-13 | 주식회사 시선테라퓨틱스 | 핵산 복합체를 포함하는 퇴행성 뇌질환의 예방 또는 치료용 조성물 |
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Publication number | Publication date |
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EP3750561A4 (en) | 2021-06-09 |
US20210177984A1 (en) | 2021-06-17 |
KR102484332B1 (ko) | 2023-01-04 |
AU2019219473B2 (en) | 2022-01-13 |
CA3090765C (en) | 2022-12-06 |
AU2019219473A1 (en) | 2020-09-10 |
EP3750561A1 (en) | 2020-12-16 |
JP2021520402A (ja) | 2021-08-19 |
KR20220104118A (ko) | 2022-07-26 |
US12016930B2 (en) | 2024-06-25 |
KR20210122731A (ko) | 2021-10-12 |
CA3090765A1 (en) | 2019-08-15 |
CN112135636A (zh) | 2020-12-25 |
KR20190096149A (ko) | 2019-08-19 |
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