KR102156323B1 - Composition for Preventing or Treating Psoriasis Comprising Skin Permeable Nucleic Acid Complex - Google Patents

Abstract

The present invention relates to a composition for preventing or treating psoriasis containing a skin-permeable nucleic acid complex as an active ingredient, and more specifically, a skin-permeable nucleic acid complex in which a bioactive nucleic acid targeting JAK1 and JAK2 and a carrier peptide nucleic acid are complementarily bound It relates to a pharmaceutical composition and skin external preparation for the prevention, improvement or treatment of psoriasis containing. The skin-permeable nucleic acid complex in which a bioactive nucleic acid targeting JAK1 and JAK2 according to the present invention is complementarily combined with a carrier peptide nucleic acid has high skin permeability and skin persistence, and is useful for the prevention, improvement or treatment of skin diseases such as psoriasis.

Description

Composition for Preventing or Treating Psoriasis Comprising Skin Permeable Nucleic Acid Complex containing skin permeable nucleic acid complex as an active ingredient

The present invention relates to a composition for preventing or treating psoriasis containing a skin-permeable nucleic acid complex as an active ingredient, and more specifically, a skin-permeable nucleic acid complex in which a bioactive nucleic acid targeting JAK1 and JAK2 and a carrier peptide nucleic acid are complementarily bound It relates to a pharmaceutical composition and skin external preparation for the prevention, improvement or treatment of psoriasis containing.

Psoriasis is a non-infectious, chronic inflammatory skin disease characterized by an increase in hyperproliferative keratinocytes and scales. Although the cause of the onset has not been clearly identified, chronic activation of inflammatory cell infiltration in the skin and epidermal keratinocytes. It is a skin disease caused by an immunological abnormality caused by a dysregulation of According to the research results, psoriasis is known to be one of the autoimmune diseases caused by the involvement of T lymphocytes, and it is reported that excessive activation of T lymphocytes is the main cause of the onset of psoriasis.

General treatment methods for psoriasis include topical treatment, systemic treatment, and phototherapy. Recently, immunobiological agents based on the etiology of psoriasis have been developed. In addition, in order to increase the effect and reduce the side effects, a combination therapy using them properly together is widely used. However, phototherapy has disadvantages that the skin ages quickly and the incidence of skin cancer is increased. Oral administration applied to the most severe psoriasis includes administration of methotrexate, oral retinoids, and cyclosporin. However, since cyclosporin can cause kidney function damage and blood pressure problems, careful observation is required. Methotrexate has the advantage of being effective in both psoriasis and psoriatic arthritis, but if used cumulatively, it may cause cirrhosis or fibrosis. Should be used. In addition, the use of oral retinoids has the potential to seriously impair a woman's childbirth, so it must be carefully controlled. As such, drugs that have been used to treat psoriasis have side effects or disadvantages.

However, topical treatments applied directly to the skin in the form of ointments, lotions, and gels are essential treatments for psoriasis patients, and are the first and most often used to control symptoms of psoriasis patients. In addition, the skin is an organ with the largest surface area in the human body, and it becomes a path through which drugs can be effectively delivered when appropriate methods are used. In order to overcome the defense mechanism against foreign substances in the skin barrier when delivering therapeutic drugs to the skin, studies on various skin penetration methods have been attempted, but the drug is not physically damaged or irritated, and the drug is used through the properties of chemical substances. It is believed that it is very difficult to deliver, and development of a material that has both skin-penetrating function and internal efficacy is required. The effectiveness of the external treatment material applied to the skin surface requires a lot of effort due to the technical difficulty of passing through the epidermis and the dermis.

On the other hand, unlike traditional drugs, nucleic acid drugs inhibit the expression of target-specific messenger RNA (mRNA), so it is possible to deal with research areas that were not possible to be treated with conventional drugs targeting 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.). Despite the excellent effects and various uses of gene expression regulation based on oligo nucleic acid, there are many obstacles to be overcome in order to develop a therapeutic agent based on nucleic acid. For this reason, the importance of developing a nucleic acid carrier based on a non-viral oligo nucleic acid having low cytotoxicity and low immunological activity is increasing.

In this regard, the present inventors believe that a nucleic acid complex in which a bioactive nucleic acid and a carrier peptide nucleic acid modified to have an overall positive charge are complementarily bound has surprisingly improved cell permeability, and using this It has been confirmed that the expression of the target gene can be very efficiently regulated, and a patent has been applied for a new construct that has low cytotoxicity and improved cell permeability and gene expression control ability of bioactive nucleic acids (PCT/KR2017/008636). ).

As a result of continuously conducting studies on the improvement of skin permeation and cell delivery functions of the constructs and therapeutic drugs, the present inventors believe that the carrier peptide nucleic acids modified to have a positive charge as a whole with Bioactive Nucleic Acid. Peptide Nucleic Acid) has the property of highly efficient passage through the skin, preferably the stratum corneum and/or epidermal layer, and thus it was confirmed that the nucleic acid complex has excellent skin permeability.

Accordingly, the present inventors have made intensive research efforts to develop a nucleic acid complex that can effectively treat psoriasis by improving skin permeability and skin persistence. As a result, a skin-permeable nucleic acid in which a bioactive nucleic acid targeting JAK1 and JAK2 and a carrier are complementarily combined. It was found that the complex exhibits an excellent effect in the prevention or treatment of psoriasis, and the present invention was completed.

An object of the present invention is to contain a skin-permeable nucleic acid complex in which a bioactive nucleic acid targeting JAK1 and JAK2, which are nucleic acid complexes having high skin permeability and skin persistence and excellent prevention or treatment effect of psoriasis, and a carrier are complementarily bound, as an active ingredient. It is to provide a pharmaceutical composition and an external preparation for skin for the prevention, improvement or treatment of psoriasis.

In order to achieve the above object, the present invention is a bioactive nucleic acid having a sequence capable of binding to JAK1 and JAK2 genes (Bioactive Nucleic Acid); And it provides a pharmaceutical composition for the prevention, improvement or treatment of psoriasis containing a skin-permeable nucleic acid complex complementarily bound to a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) as an active ingredient.

The present invention also provides an external preparation for skin comprising the composition.

The present invention also includes a bioactive nucleic acid having a sequence capable of binding to JAK1 and JAK2 genes; And it provides a method for preventing or treating psoriasis comprising administering a skin-permeable nucleic acid complex to which a carrier peptide nucleic acid is complementarily bound.

The present invention also includes a bioactive nucleic acid having a sequence capable of binding to JAK1 and JAK2 genes; And a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) is complementarily bound to a skin permeable nucleic acid complex to provide a use for the prevention or treatment of psoriasis.

The present invention also provides a bioactive nucleic acid (Bioactive Nucleic Acid) having a sequence capable of binding to JAK1 and JAK2 genes for the manufacture of a drug for the prevention or treatment of psoriasis; And a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) provides the use of a skin-penetrating nucleic acid complex complementarily bound.

The skin-permeable nucleic acid complex in which a bioactive nucleic acid targeting JAK1 and JAK2 according to the present invention is complementarily combined with a carrier peptide nucleic acid has high skin permeability and skin persistence, and is useful for the prevention, improvement or treatment of skin diseases such as psoriasis.

1 is a schematic representation of the effect according to the type of structure in which a bioactive peptide nucleic acid and a carrier peptide nucleic acid are bound in a psoriasis-like cell model, and bioactive peptide nucleic acids and carriers in a psoriasis-induced human keratin cell line induced by IFN-γ It shows the cell viability according to the type of structure to which the peptide nucleic acid is bound (1: SEQ ID NO: 1 and 3, 2: SEQ ID NO: 1 and 4, 3: SEQ ID NO: 1 and 5, 4: SEQ ID NO: 1 and 6; Table 2 Reference).
Figure 2 is a schematic representation of the effect according to the type of structure in which a bioactive peptide nucleic acid and a carrier peptide nucleic acid are bound in a psoriasis-like cell model, and bioactive peptide nucleic acid and carrier in a psoriasis-induced human keratin cell line induced by IFN-γ It shows the effect on the expression of target genes JAK1 and JAK2 and sub-step gene expression according to the type of structure to which the peptide nucleic acid is bound (1: SEQ ID NO: 1 and 3, 2: SEQ ID NO: 1 and 4, 3: SEQ ID NO: 1 And 5, 4: SEQ ID NOs: 1 and 6; see Table 2).
Figure 3a is a schematic representation of the effect according to the type of structure in which the bioactive peptide nucleic acid and the carrier peptide nucleic acid are bound in a psoriasis-like cell model, and bioactive peptide nucleic acid and carrier in psoriasis-induced human T lymphocyte induced by IFN-γ It shows the effect on the expression of the target gene JAK1 and sub-step gene expression according to the type of structure to which the peptide nucleic acid is bound (1: SEQ ID NO: 1 and 3, 2: SEQ ID NO: 1 and 4, 3: SEQ ID NO: 1 and 5 , 4: SEQ ID NOs: 1 and 6; see Table 2).
Figure 3b is a schematic representation of the effect according to the type of structure in which a bioactive peptide nucleic acid and a carrier peptide nucleic acid are bound in a psoriasis-like cell model, with IFN-γ, PMA (Phorbol-12-myristate-13-acetate) and Ionomycin. In the induced psoriasis-induced human T lymphocyte, the effect on the expression of the target gene JAK1 and the sub-step gene expression according to the type of structure in which the bioactive nucleic acid and the carrier peptide nucleic acid are bound is shown (1: SEQ ID NOs: 1 and 3, 2 : SEQ ID NO: 1 and 4, 3: SEQ ID NO: 1 and 5, 4: SEQ ID NO: 1 and 6; see Table 2).
Figure 4a shows the therapeutic effect of a nucleic acid complex targeting JAK1 and JAK2 in an animal model of psoriasis induction, and a photograph showing the decrease in the thickness of the ear, which is a psoriasis phenotype of the mouse, by the nucleic acid complex in C57BL/6 mice. .
4B shows the therapeutic effect of a nucleic acid complex using JAK1 and JAK2 as target genes in a psoriasis-inducing animal model. Concentration of IgE in serum by a nucleic acid complex in C57BL/6 mice induced psoriasis using 5% Imiquimod (PNA 2: SEQ ID NO: 1 and 4, PNA 4: SEQ ID NO: 1 and 6; see Table 3).
4C shows the therapeutic effect of a nucleic acid complex using JAK1 and JAK2 as target genes in a psoriasis-inducing animal model. IL-17A in serum by a nucleic acid complex in C57BL/6 mice induced psoriasis using 5% Imiquimod It shows a decrease in the concentration of (PNA 2: SEQ ID NO: 1 and 4, PNA 4: SEQ ID NO: 1 and 6; see Table 3).
4D shows the therapeutic effect of a nucleic acid complex using JAK1 and JAK2 as target genes in a psoriasis-inducing animal model. Target gene JAK1 by a nucleic acid complex in C57BL/6 mouse skin tissues inducing psoriasis using 5% Imiquimod And a decrease in the expression of JAK2 and substage genes (PNA 2: SEQ ID NOs: 1 and 4, PNA 4: SEQ ID NOs: 1 and 6; see Table 3).
4E shows the therapeutic effect of a nucleic acid complex using JAK1 and JAK2 as target genes in a psoriasis-inducing animal model. T helper type by nucleic acid complex in the spleen tissue of C57BL/6 mice induced psoriasis using 5% Imiquimod 17 and T helper type 1 marker of CCR6 expression reduction (PNA 2: SEQ ID NO: 1 and 4, PNA 4: SEQ ID NO: 1 and 6; see Table 3).
4F shows the therapeutic effect of a nucleic acid complex using JAK1 and JAK2 as target genes in a psoriasis-inducing animal model. Transdermal thickness reduction due to a nucleic acid complex in the skin tissue of C57BL/6 mice induced psoriasis using 5% Imiquimod (PNA 2: SEQ ID NO: 1 and 4, PNA 4: SEQ ID NO: 1 and 6; see Table 3).
4G shows the therapeutic effect of a nucleic acid complex using JAK1 and JAK2 as target genes in a psoriasis-inducing animal model, and decreases the inflammatory marker Ki67 by a nucleic acid complex in C57BL/6 mice that induce psoriasis using 5% Imiquimod. What to do and it is shown in numerical form (PNA 2: SEQ ID NO: 1 and 4, PNA 4: SEQ ID NO: 1 and 6; see Table 3).

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

In the present invention, it was confirmed that a nucleic acid complex in which a bioactive nucleic acid and a carrier peptide nucleic acid are complementarily bound has skin permeability and skin persistence, and in particular, a bioactive nucleic acid targeting JAK1 and JAK2 and a carrier peptide nucleic acid are complementarily bound. It was confirmed that it can be used in the treatment of skin diseases such as psoriasis by applying the skin-permeable nucleic acid complex to the skin surface.

Psoriasis is an inflammatory autoimmune disease. As the activity of immune T cells in the skin increases, secreted inflammatory substances stimulate keratinocytes of the skin, causing excessive proliferation and inflammation of keratinocytes. JAK1 and JAK2 are known to play a key role in mediating signaling between several cytokines, growth factors and immune cells, so selective inhibitory activity of JAK1 and JAK2 may be an important target of immunosuppressants.

The differentiation of CD4+ T helper type 1 and CD4+ T helper type 17 is increased due to the activation of JAK1 and JAK2 in the skin and blood of psoriasis patients, and the secretion and accumulation of inflammatory cytokines secreted into the skin thereby worsens psoriasis.

In one embodiment of the present invention, a skin-permeable nucleic acid complex in which a bioactive nucleic acid targeting JAK1 and JAK2 and a carrier peptide nucleic acid are complementarily combined is prepared, and T helper type 17 and T helper type 1 of CD4+ T lymphocytes are used. Differentiation inhibitory effect and psoriasis induction animal model confirmed the effect of psoriasis treatment.

Accordingly, the present invention provides, in one aspect, a bioactive nucleic acid having a sequence capable of binding to JAK1 and JAK2 genes (Bioactive Nucleic Acid); And it relates to a pharmaceutical composition for the prevention, improvement or treatment of psoriasis containing a skin-permeable nucleic acid complex complementarily bound to a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) as an active ingredient.

In the present invention, a nucleic acid complex in which a bioactive nucleic acid and a carrier peptide are complementarily bonded can be characterized by having a structure of the following structural formula (1).

Structural Formula (1)

[A ≡ C ⁽⁺⁾ ]

In the above 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 a negative charge or neutrality as a whole,

C ⁽⁺⁾ means that the carrier peptide nucleic acid has a positive charge as a whole,

The carrier peptide nucleic acid includes 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 the present invention may have anti-parallel binding or parallel binding.

In the present invention, the carrier peptide nucleic acid preferably contains one or more gamma- or alpha-backbone modified peptide nucleic acid monomers to have a positive charge throughout the carrier peptide nucleic acid, and the gamma- or alpha-backbone modified peptide nucleic acid As for the monomer, it is more preferable that a monomer having an amino acid having a positive charge is contained more than a monomer having an amino acid having a negative charge so that the charge of the entire carrier peptide nucleic acid is positive.

In the present invention, the bioactive nucleic acid and the carrier peptide nucleic acid are parallel binding or partial specific binding according to the 5'-direction and 3'-direction of each nucleic acid, It can be characterized in that the binding power (melting temperature, Tm) of the carrier peptide nucleic acid is lower than that of the bioactive nucleic acid and the desired gene, and also regulates the binding power between the bioactive nucleic acid and the carrier peptide nucleic acid. Through this, it may be characterized in that it is possible to control the timing of separation of the bioactive nucleic acid and the carrier peptide nucleic acid and the binding time of the bioactive nucleic acid and the desired gene of the bioactive nucleic acid.

In the present invention, the "living nucleic acid" refers to a nucleic acid having a complementary sequence capable of binding to a target gene of interest to reduce its expression, in particular, a complementary sequence capable of binding to the mRNA of the target gene of interest. , It means a nucleic acid involved in gene expression regulation, such as inhibiting the expression of the corresponding gene, and may be a nucleic acid having a sequence complementary to a target gene for which expression is to be reduced.

In particular, the "Bioactive Nucleic Acid" in the present invention is combined with a target gene and a nucleotide sequence including the target gene in vitro or in vivo , and the inherent function of the gene (for example, For example, it may be characterized by performing a function such as inhibiting (transcript expression or protein expression).

Therefore, the "Bioactive Nucleic Acid" in the present invention is preferably an antisense peptide nucleic acid of JAK1 (Janus kinase/Just another kinase 1) and JAK2 (Janus kinase/Just another kinase 2), which are target genes for psoriasis. And, more preferably, it is represented by the sequence of SEQ ID NO: 1 or 2, but is not limited thereto.

In the present invention, “carrier peptide nucleic acid” refers to a nucleic acid in which a bioactive nucleic acid and some or all of the bases complementarily bind to impart functionality, and the carrier peptide nucleic acid used in the present invention is a peptide nucleic acid (PNA: Peptide Nucleic Acid), as well as a modified nucleic acid similar thereto, may be used, and a peptide nucleic acid is preferred, but the meaning is not limited thereto.

In particular, in the present invention, the carrier peptide nucleic acid is preferably represented by a sequence selected from the group consisting of SEQ ID NOs: 3 to 7, but is not limited thereto.

In the present invention, the "skin-permeable nucleic acid complex" can penetrate a bioactive substance into the body and ultimately into cells through contact with the skin, and specifically, the stratum corneum and/or the epidermal layer, which is the outermost layer of the skin It has the ability to pass through, pass through the epidermal layer or the dermis layer, or pass through the dermal layer and pass into the body.

Depending on the net charge in the skin-permeable nucleic acid complex according to the present invention and/or the number of bioactive nucleic acids and/or carrier peptide nucleic acids in the nucleic acid complex, it remains in the stratum corneum, epidermal layer or dermal layer, Can be delivered to

Accordingly, in the present invention, the nucleic acid complex may be characterized in that it has skin persistence.

In particular, the "skin-permeable nucleic acid complex" according to the present invention has the property of transdermal delivery into the body through the skin, and then can be delivered into a target cell, and is used in any form containing the nucleic acid complex. Can be.

In the present invention, the skin-permeable nucleic acid complex is a bioactive nucleic acid represented by SEQ ID NO: 2; And a carrier peptide nucleic acid represented by any one sequence selected from the group consisting of SEQ ID NOs: 3 to 7, but is not limited thereto.

In addition, the binding power (melting temperature, melting temperature, Tm) of the bioactive nucleic acid and carrier peptide nucleic acid capable of binding to the JAK1 and JAK2 genes is lower than that of the bioactive nucleic acid and the desired JAK1 and JAK2 genes. It can be characterized.

The avidity of the bioactive nucleic acid and the carrier peptide nucleic acid is by parallel binding or partial specific binding according to the 5'-direction and 3'-direction of each nucleic acid, and thus the bioactive nucleic acid and carrier peptide nucleic acid The binding power (melting temperature, Tm) of the bioactive nucleic acid can be lower than the binding power of the bioactive nucleic acid and the desired gene.

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

Structural Formula (2)

[mA ≡ mC ⁽⁺⁾ ]

In the above structural formula (2),

'm' refers to a substance that helps endosome escape of bioactive nucleic acids and carrier peptide nucleic acids.

In the present invention, the "substance that helps the endosomes escape" may be characterized by helping to escape from the endosomes of the bioactive nucleic acid by increasing the osmotic pressure inside the endosome or destabilizing the membrane of the endosome. have. It means that bioactive nucleic acids move more efficiently and quickly to the nucleus or cytoplasm to meet and function with target genes (DW Pack, AS Hoffman, S. Pun, PS Stayton, “Design and development of polymers for gene delivery,” Nat. Rev. Drug. Discov., 4, 581-593 (2005)).

In the present invention, the substances that help escape the endosomes are peptides, lipid nanoparticles, polyplex nanoparticles, polymer nanospheres, inorganic nanoparticles, cationic lipids. It may be characterized in that any one or more selected from the group consisting of nanomaterials (cationic lipid-based nanoparticles), cationic polymers, and pH sensitive polymers.

In the present invention, a peptide (GLFDIIKKIAESF) may be linked to a bioactive nucleic acid as a substance that helps escape the endosomes, and Histisine (10) is linked to a carrier peptide nucleic acid through a linker medium. It can be, but is not limited thereto.

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

In the present invention, the conjugate nanomaterials (polyplex nanoparticles) may be characterized in that poly(amidoamine) or polyethylenimine (PEI).

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

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

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

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

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

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

The bioactive nucleic acid may be characterized in that it consists of a natural nucleic acid base and/or a modified nucleic acid monomer.

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

In the present invention, the bioactive nucleic acid and the carrier peptide nucleic acid are phosphodiester, 2'0-methyl (2' 0-methyl), 2'methoxy-ethyl, and phosphor. It may be characterized in that it further includes any one or more functional groups selected from the group consisting of amidate, methylphosphonate, and phosphorothioate.

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

In the present invention, each of the bioactive nucleic acid and the carrier peptide nucleic acid in the skin-permeable nucleic acid complex may be a complex characterized in that the electrical properties as a whole have a positive charge (positive), negative charge (negative), or a neutral charge.

In the expression of the above electrical properties, the meaning of “total” refers to the overall electrical properties of the entire bioactive nucleic acid or carrier peptide nucleic acid when viewed from the outside, not the electrical properties of individual bases. Even if some of the monomers in the sex nucleic acid are positive, if the number of negative monomers is greater, the bioactive nucleic acid will have a negative charge in terms of electrical properties “total”, and some bases and/or Even if the backbone is negative, if there are more positive bases and/or the number of backbones, the carrier peptide nucleic acid will have a positive charge in terms of electrical properties "total".

From this point of view, it is preferable that the nucleic acid complex of the present invention has a positive charge as a whole. In the nucleic acid complex, preferably, the bioactive nucleic acid has a negative or neutral characteristic when looking at electrical properties as a whole, and the carrier peptide nucleic acid may be characterized by having a positive charge characteristic when looking at an overall electrical property, It is not limited thereto.

In the present invention, a modified peptide nucleic acid monomer may be used to impart electrical properties between the bioactive nucleic acid and the carrier peptide nucleic acid, and the modified peptide nucleic acid monomer is a carrier peptide nucleic acid having a positive charge, and lysine (Lysine, Lys, K) , Arginine (Arginine, Arg, R), histidine (Histidine, His, H), diamino butyric acid (Diamino butyric acid, DAB), ornithine (Ornithine, Orn) and any one or more positive charges selected from the group consisting of amino acid analogs As a carrier peptide nucleic acid containing an amino acid of and having a negative charge, it may be characterized by containing a negatively charged amino acid of glutamic acid (Glu, E) or an amino acid analogue.

In the present invention, the carrier peptide nucleic acid may be characterized in that it contains 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 is lysine (Lysine, Lys, K), arginine (Arginine, Arg, R), histidine (Histidine, His, H), diamino butyric acid (Diamino butyric acid) so as to have electrical positivity. acid, DAB), ornithine (Orn), and amino acids having positive charges selected from the group consisting of amino acid analogs may be included in the backbone.

In the present invention, in addition to the backbone modification, a peptide nucleic acid monomer having a modified nucleobase may be used as the modification of the peptide nucleic acid monomer for imparting charge. Preferably, amine, triazole, and imidazole moeity may be included in the nucleobase to have electrical positivity, or carboxylic acid may be included in the base to have electrical negative.

In the present invention, 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 contains more positively charged monomers than the negatively charged monomers, so that the carrier peptide as a whole It is preferable that the charge of the nucleic acid is positive.

Preferably, the nucleic acid complex according to the present invention is characterized in that it has a positive charge as a whole.

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

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

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

The antiparallel binding and parallel binding are determined according to 5'-direction and 3'-direction in the binding method of DNA-DNA or DNA-PNA. Antiparallel binding is a general method of binding DNA-DNA or DNA-PNA, and when describing the nucleic acid complex according to the present invention as an example, the bioactive nucleic acid is from 5'to 3'direction, and the carrier peptide nucleic acid is from 3'to 5' It means a form that is combined with each other in a direction. The parallel bond is a form in which the binding force is somewhat lower than that of the antiparallel bond, and it refers to a form in which both the bioactive nucleic acid and the carrier peptide nucleic acid are bound to each other in a 5'to 3'direction or a 3'to 5'direction.

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

Examples of specific methods for lowering the binding strength (melting temperature, Tm) of the bioactive nucleic acid and the carrier peptide nucleic acid to the desired gene, particularly the mRNA of the bioactive nucleic acid and the bioactive nucleic acid, include the above 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.

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

In the present invention, the universal base is adenine (adenine), guanine (guanine), cytosine (cytosine), thymine (thymine), uracil (Uracil), such as natural bases and non-selective binding, complementary One or more selected from the group consisting of inosine PNA (inosine PNA), indole PNA (indole PNA), nitroindole PNA (nitroindole PNA), and absic may be used as a base having a binding power lower than that of binding power, and preferably It may be characterized by using inosine PNA.

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

In the present invention, the time point at which the bioactive peptide nucleic acid is bound to the target sequence in the presence of the target gene (the time point at which the bioactive nucleic acid is substituted with the target sequence, Target-specific separation and binding time) can be adjusted.

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

In the present invention, the particles of the nucleic acid complex may be characterized by having a size of 5 nm to 300 nm, preferably 10 nm to 80 nm, most preferably 15 nm to 70 nm.

In the present invention, the particle size of the nucleic acid complex may be controlled by adjusting the charge balance between the bioactive peptide nucleic acid and the carrier peptide nucleic acid. Specifically, when the positive charge of the carrier peptide nucleic acid increases, the size of the particles decreases, but when the positive charge of the carrier peptide nucleic acid exceeds a certain level, the size of the particles increases. In addition, as another important factor determining the particle size, the particle size is determined by an appropriate charge balance with the overall carrier peptide nucleic acid according to the charge of the bioactive peptide nucleic acid forming the complex.

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 has a net charge of charge balance of 0 to 5, preferably 0 to 3 negative charges.

In the present invention, the nucleic acid complex may be prepared by hybridizing a bioactive nucleic acid and a carrier peptide nucleic acid under appropriate conditions.

In the present invention, "hybridization" means that complementary single-stranded nucleic acids form double-stranded nucleic acids. Hybridization can occur when the complementarity between the two nucleic acid strands is perfect match or even in the presence of some mismatch bases. The degree of complementarity required for hybridization may vary according to hybridization conditions, and in particular, may be controlled by the bonding temperature.

“Target gene” of the present invention means a nucleic acid sequence (base sequence) to be activated, inhibited or labeled, and there is no difference from the term “target nucleic acid”, and are used interchangeably herein.

In the present invention, when the target nucleic acid (base sequence) containing the target gene is brought into contact (binding) with the complex in vitro or in vivo , the bioactive nucleic acid is separated from the carrier peptide nucleic acid. It becomes a biological activity.

In the present invention, diseases that can be prevented or treated using the nucleic acid complex may be determined according to a target gene to which the bioactive nucleic acid in the nucleic acid complex binds. In the present invention, it may be characterized in that JAK1 and JAK2 are target genes to which bioactive nucleic acids bind.

Therefore, it is preferable that the disease that can be prevented and treated by using the nucleic acid complex in the present invention is psoriasis.

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

The therapeutic composition of the present invention may be formulated in a form of skin delivery according to standard pharmaceutical practice. These formulations may contain additives such as carriers, excipients, adjuvants or diluents suitable for formulations in a form that is pharmaceutically acceptable, particularly applicable to the skin, in addition to the active ingredient.

Preferably, the composition according to the present invention may be formulated in the form of an aqueous solution, gel, ointment, cream, lotion, paste, smear or patch.

Most preferably, it may be formulated in the form of an aqueous solution, and the aqueous solution may be in the form of distilled water and a buffer solution.

The term "physiologically acceptable" refers to a property that does not impair the biological activity and physical properties of a compound.

The term “carrier” is defined as a compound that facilitates the addition of a nucleic acid complex into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic compounds into cells or tissues of an organism.

The term “diluent” is defined as a compound that is diluted in water that 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. The 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 alter the biological activity of a compound.

The substance containing the nucleic acid complex in the present invention can be administered to a patient as such or as a pharmaceutical composition mixed with other active ingredients, such as in combination therapy, or with a suitable carrier or excipient.

Pharmaceutical 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 refers to 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 the disease. Determination of a therapeutically effective amount is within the capabilities of one of ordinary skill in the art, particularly in light of the detailed disclosure provided herein.

The term "prevention" as used in the present invention means any action of preventing the onset of a disease or inhibiting its progression by administration (or application) of a therapeutic composition comprising the skin-permeable nucleic acid complex.

The term "improvement" of the present invention means any action of at least reducing the degree of a parameter related to the condition to be treated, for example, symptoms by administration (or application) of a therapeutic composition containing the skin-permeable nucleic acid complex. .

In addition, the term "treatment" used in the present invention refers to any action in which symptoms of a disease are improved or cured by administration (or application) of a therapeutic composition containing the skin-permeable nucleic acid complex.

In the present invention, the skin-permeable nucleic acid complex may be administered (or applied) using a carrier such as a liposome. The liposome may help target the complex to specific tissues, such as lymphoid tissue, or to selectively target to infected cells, and may also help to increase the half-life of the composition containing the complex. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, and lamellar layers. In such formulations, the complex to be delivered alone or to specific cells, together with a molecule that binds to a predominant receptor among lymphocytes, such as a monoclonal antibody that binds to the CD45 antigen, or with other therapeutic compositions, is a liposome. Incorporate as part of. Thus, liposomes filled or decorated with a predetermined complex of the present invention to deliver the nucleic acid complex composition can be directed to the site of the lymphocyte.

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. In general, lipids are selected in consideration of, for example, stability of liposomes in the bloodstream, acid lability, and size of liposomes. Various methods can be used to prepare liposomes. See, eg, Szoka, et al., Ann. Rev. Biophys. Bioeng., 9:467, 1980), and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369].

In another aspect, the present invention is a bioactive nucleic acid having a sequence capable of binding to the JAK1 and JAK2 genes (Bioactive Nucleic Acid); And a skin-permeable nucleic acid complex complementarily bound to a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) for the prevention, improvement, or treatment of psoriasis containing as an active ingredient.

Ingredients included in the composition for external application for skin of the present invention may include ingredients commonly used in the composition for external application for skin in addition to the active ingredient, for example, conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances. , And a carrier.

In order to formulate the active ingredient of the present invention, it can be easily formulated if carried out according to conventional methods, and surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffers, suspensions, and other commercially available auxiliary agents can be appropriately used.

The composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, It may be formulated as an oil, powder foundation, emulsion foundation, wax foundation, pack, massage cream and spray, but is not limited thereto. More specifically, it may be prepared in the form of a softening lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as carrier components. I can.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and crystallites Sex cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propane / May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a surfactant-containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether Sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethan-5-olamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester may be used.

In another aspect of the present invention, a bioactive nucleic acid having a sequence capable of binding to JAK1 and JAK2 genes (Bioactive Nucleic Acid); And it relates to a method for preventing or treating psoriasis comprising administering to an individual a skin-permeable nucleic acid complex to which a carrier peptide nucleic acid is complementarily bound.

The composition comprising the nucleic acid complex according to the present invention can be applied to the skin in an amount effective as a medicament to treat psoriasis or to suppress (or alleviate) symptoms of psoriasis. It may vary depending on various factors such as the type of psoriasis, the patient's age, weight, the characteristics and extent of symptoms, the type of current treatment, the number of treatments, the application form and route, and can be easily determined by experts in the field. The composition of the present invention may be applied together or sequentially with the pharmacological or physiological components described above, and may be applied in combination with an additional conventional therapeutic agent, and may be applied sequentially or simultaneously with the conventional therapeutic agent. These applications can be single or multiple applications.

In the present invention, "individual" refers to a mammal suffering from or at risk of a condition or disease that can be alleviated, suppressed or treated by administering (applying) the skin-permeable nucleic acid complex according to the present invention, and preferably refers to a human it means.

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

The toxicity and therapeutic efficiency of a composition comprising a skin-permeable nucleic acid complex as described herein are, for example, LD50 (lethal dose for 50% of the population), ED50 (dose with therapeutic effect for 50% of the population). , To determine the IC50 (dose with therapeutic inhibitory effect on 50% of the population), it can be estimated by standard pharmaceutical procedures in cell culture or laboratory animals. 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 a high therapeutic index are preferred. The data obtained from these cell culture assays can be used to estimate a range of doses for human use. The dosage or application of such compounds is preferably within a range of circulating concentrations including the ED50 (or IC50) with little or no toxicity.

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

The route of administration 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, intradermal, oral, intranasal, pulmonary, or rectal as desired. In addition, the composition can be administered by any device capable of moving the active substance to the target cell.

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

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

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

In another aspect of the present invention, a bioactive nucleic acid having a sequence capable of binding to JAK1 and JAK2 genes (Bioactive Nucleic Acid); And a skin-penetrating nucleic acid complex complementarily bound to a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) to use in the prevention or treatment of psoriasis.

In another aspect, the present invention is a bioactive nucleic acid (Bioactive Nucleic Acid) having a sequence capable of binding to the JAK1 and JAK2 genes for the manufacture of a drug for the prevention or treatment of psoriasis; And to the use of a skin-penetrating nucleic acid complex to which a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) is complementarily bound.

[ Example ]

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

Example One: Life Peptide nucleic acid ( Bioactive Peptide Nucleic Acid) and carrier Preparation of peptide nucleic acids, and complexes using them

JAK1 and 2 were used as target genes to test the effect of the nucleic acid complex of the present invention on psoriasis, and antisense peptide nucleic acid as a bioactive peptide nucleic acid (Bioactive Peptide Nucleic Acid) against JAK1 and JAK2 to confirm the psoriasis treatment effect (antisense PNA) was used.

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

The peptide nucleic acid-based bioactive nucleic acid used in this example binds the peptide GLFDIIKKIAESF to 5'to help the endosomes escape ability, and the nucleotide sequence, monomer modification and structure are shown in Table 1 below.

All of the peptide nucleic acids used in the present invention were synthesized in PANAGENE (Korea) through HPLC purification. All carrier peptide nucleic acids according to an embodiment of the present invention have peptides for helping endosomes escape ability bound to the 5'to 3'ends, and are composed of sequences described in SEQ ID NOs: 3 to 7 (Table 1) .

Sequence of bioactive nucleic acids and carrier peptide nucleic acids for verification of psoriasis treatment effect division Sequence number Base sequence Monomer modification Bioactive nucleic acid SEQ ID NO: 1 5'-GLFDIIKKIAESF-OC ^(-) ATG ⁽⁺⁾ GCAT ^(-) TG ⁽⁺⁾ CAGT ^(-) COK-3' -+-+- SEQ ID NO: 2 5'-GLFDIIKKIAESF-O-GT ^(-) CAA ⁽⁺⁾ TCC ^(-) GA ⁽⁺⁾ TTC ^(-) GG-OK-3' -+-+-
Carrier peptide
Nucleic acid SEQ ID NO: 3 5'-Histidine(10)-OG ⁽⁺⁾ TACCGTA ⁽⁺⁾ ACGTCA ⁽⁺⁾ GOK-3' +++ SEQ ID NO: 4 5'-KOG ⁽⁺⁾ ACTGCA ⁽⁺⁾ ATGCCAT ⁽⁺⁾ GO-Histidine (10)-3' +++ SEQ ID NO: 5 5'-Histidine(10)-O-GT ⁽⁺⁾ CA ⁽⁺⁾ GOK-3' ++ SEQ ID NO: 6 5'-KO-GA ⁽⁺⁾ CT ⁽⁺⁾ GO-Histidine(10)-3' ++ SEQ ID NO: 7 5'-Histidine(10)-O-CA ⁽⁺⁾ GTTAGG ⁽⁺⁾ CTAAG ⁽⁺⁾ CC-OK-3' +++

Table 1 shows the sequence information of the bioactive peptide nucleic acid and carrier peptide nucleic acid used as a control, and the bioactive peptide nucleic acid and carrier peptide nucleic acid used as a control, to confirm the effect of JAK1 and JAK2 as a targeting psoriasis therapeutic agent.

Modification of the monomer is to give the backbone of the peptide nucleic acid electrical properties with lysine (Lysine, Lys, K, ⁽⁺⁾ ) and glutamic acid (Glutamic acid, Glu, E, ^(-)) (Notation) was prepared to have a peptide backbone modified.

Each combination of bioactive nucleic acid and carrier peptide nucleic acid was hybridized under DMSO, resulting in a complex composed of bioactive nucleic acid and carrier peptide nucleic acid.

Example 2: Using skin-permeable nucleic acid complex psoriasis (Psoriasis) treatment effect analysis

According to Example 1, the effect of treating psoriasis was analyzed using a skin-permeable nucleic acid complex containing a bioactive peptide nucleic acid and a carrier peptide nucleic acid using JAK1 and JAK2 as target genes prepared in the structures shown in Table 2 below.

Example 2-1: cell culture

Human keratinocytes (HaCaT) obtained from CLS (CLS Cell Lines Service, Germany) were added to 10% (v/v) fetal bovine serum, penicillin 100 units/ml, strepto in DMEM culture medium (Dulbecco Modified Eagle Medium, Weljin, Korea). 100 µg/ml of mycin was added, and cultured under the conditions of 37°C and 5% (v/v) CO ₂ . To make a psoriasis-like cell model, IFN-γ was treated with 10 ng/mL and cultured for 24 hours.

Example 2-2: MTT ( MTT assay) to analyze cell viability in keratin cell lines

As the experimental conditions in Example 2-1, a human-derived keratin cell line was seeded in 96 wells at 6×10 ³ , cultured for 24 hours, and then a complex containing a bioactive nucleic acid prepared in the structure of Table 1 and a carrier peptide nucleic acid was used. To the treated cell line, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution was prepared in 1X PBS at a concentration of 5 mg/mL, treated with 20 μL per well, and incubated for 4 hours. After that, OD (optical density) was measured and analyzed with a spectrophotometer.

The nucleic acid complex combinations used in this example are shown in Table 2 below.

Combination of nucleic acid complexes for cell viability analysis in keratin cell lines division Nucleic acid complex One SEQ ID NO: 1 and 3 2 SEQ ID NO: 1 and 4 3 SEQ ID NO: 1 and 5 4 SEQ ID NO: 1 and 6

As a result, as shown in Fig. 1, it was confirmed that the cell viability decreased in a concentration-dependent manner by the nucleic acid complex in the IFN-γ-induced psoriasis-like cell model.

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

The human-derived keratin cell line was seeded in a 6-well plate at 1×10 ⁵ , cultured for 24 hours, and the experiment was conducted under the conditions of Example 2-1 to treat a complex containing a bioactive peptide nucleic acid and a carrier peptide nucleic acid. After incubation for 72 hours, 30 μL of RIPA buffer was added to each well to obtain protein lysate. Protein lysate is quantified using a BCA assay kit (Thermo Fisher, USA), and 30 μg of protein is separated by size through electrophoresis, and the protein is transferred to the PVDF membrane, and then the primary antibody, p-JAK1 (Cell signaling). Technology, USA), p-JAK2 (Cell signaling Technology, USA), p-stat1 (Cell signaling Technology, USA), p-stat3 (Cell signaling Technology, USA) at 1:1000 and left at 4℃ for one day I did. Washed using 1X T BS-T, and treated with a secondary antibody, Goat Anti-Rabbit (Cell signaling Technology, USA) 1:2000, and allowed to stand at room temperature for 1 hour. Supersignal ^TM West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA) was treated and the efficiency of suppressing the expression of target genes in keratin cell lines was analyzed using Image600 (Amersham, Germany) equipment.

In this example, changes in the expression of JAK1 and JAK2 and sub-step genes in a psoriasis-like cell model using IFN-γ were analyzed, and the nucleic acid complex combinations used are the same as in Table 2.

As a result, as shown in Fig. 2, the expression of the target gene and the expression of the sub-path gene in a concentration-dependent manner through the combination of SEQ ID NO. 1 and SEQ ID NO. It was confirmed that the most suppressed.

Example 3: Analysis of the inhibitory effect on T helper type 17 and T helper type 1 differentiation of CD4 positive T lymphocytes using skin-permeable nucleic acid complex

According to Example 1, the effect of inhibiting T helper cell differentiation was analyzed using a skin-permeable nucleic acid complex including a bioactive peptide nucleic acid and a carrier peptide nucleic acid using JAK1 and JAK2 as target genes prepared in the structures of Table 2 above.

Example 3-1: cell culture

Pure T cells (Jurkat, CD4+ naive T cells) obtained from ATCC (American Type Culture Collection, USA) were added to RPMI-1640 culture medium (ATCC, USA) at 10% (v/v) fetal bovine serum, penicillin 100 units/ml , Streptomycin 100 µg/ml was added, and culture was carried out under conditions of 37°C and 5% (v/v) CO ₂ . To differentiate into T helper type 17 and type 1, IFN-γ 10 ng/mL and PMA (Phorbol-12-myristate-13-acetate) 10 ng/ml + Ionomycin 1uM were treated and incubated for 24, 48, and 72 hours.

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

Pure T cells (Jurkat, CD4+ naive T cells) were seeded in a 6-well plate at 2×10 ⁵ and cultured for 24 hours, followed by experiments under the conditions of Example 3-1, including bioactive peptide nucleic acids and carrier peptide nucleic acids. After processing the complex and incubating for 24, 48, and 72 hours, 30 μL of RIPA buffer was added to each well to obtain protein lysate. Protein lysate is quantified using a BCA assay kit (Thermo Fisher, USA), and 30 μg of protein is separated by size through electrophoresis, and the protein is transferred to the PVDF membrane, and then the primary antibody, p-JAK1 (Cell signaling). Technology, USA), p-stat1 (Cell signaling Technology, USA), p-stat3 (Cell signaling Technology, USA) were treated at 1:1000 and left at 4°C for one day. Washed using 1X TBS-T, and treated with a secondary antibody, Goat Anti-Rabbit (Cell signaling Technology, USA) at 1:2000, and left at room temperature for 1 hour. Supersignal ^TM West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA) was treated and the efficiency of suppressing the expression of target genes in keratin cell lines was analyzed using Image600 (Amersham, Germany) equipment.

JAK1 and substage gene expression changes in T helper type 17 and T helper type 1 differentiation models using IFN-γ and IFN-γ + PMA + Inomoycin were analyzed, and the nucleic acid complex combinations used are the same as in Table 2.

As a result, as shown in Figures 3a to 3b, the combination of the nucleic acid complex of SEQ ID NO: 1 and SEQ ID NO: 4, and SEQ ID NO: 1 and SEQ ID NO: 6 most efficiently expresses JAK1 and JAK2 and lower-stage genes in a psoriasis-like cell model. Suppressed.

Example 4: 5% Imiquimod Used psoriasis In the induced animal model psoriasis Phenotypic effect analysis

The left ear of C57BL/6 mice was depilated, and Imiquimod powder (Sigma, USA) was made into a 5% Imiquimod cream formulation, and then applied at 25 mg once a day for 2 weeks to construct an animal model for psoriasis induced by Imiquimod. A total of 7 cream-formed nucleic acid complexes were treated once a day for a week, and the psoriasis animal model phenotype was analyzed by photographs, and the thickness of the left ear region was measured by DIGMATIC MICROMETER (Mitutoyo Corporation, Japan).

In this Example, the nucleic acid combinations whose effects were verified through Examples 2 and 3 were selected to analyze phenotypic and histological findings, JAK1 and JAK2 gene expression changes in a psoriasis-like animal model using Imiquimod, and the nucleic acid complex combination used was It is shown in Table 3 below.

Combination of nucleic acid complexes for analysis of therapeutic effects in animal models of psoriasis division Nucleic acid complex PNA 2 SEQ ID NO: 1 and 4 PNA 4 SEQ ID NO: 1 and 6

As a result, it was confirmed that the level of psoriasis lesions by Imiquimod treatment was alleviated in the nucleic acid complex group (FIG. 4A), and when the skin thickness according to the proliferation of epidermal cells was compared with the animal group inducing psoriasis, nucleic acid It was confirmed that the ear thickness was decreased in the group to which the complex was applied (FIG. 4A; Cream control is a group containing a cream formulation but not a nucleic acid complex, and positive control is a conventional treatment, Lidomex steroid ointment).

Example 4-1: in serum IgE And IL-17A concentration change analysis

In the animal experiment conducted under the conditions of Example 4, mouse blood was collected through orbital blood collection on the final day of the final experiment, left at room temperature for at least 2 hours, and serum was collected using a centrifuge (14,000 rpm, 15 min). The collected serum was measured for the concentration of IgE and IL-17A in the serum using an experimental method provided by the IgE ELISA kit (Koma Biotech, Korea) and the IL-17A ELISA kit (Koma Biotech, Korea).

As a result, as shown in FIGS. 4B and 4C, it was confirmed that the concentration of IgE and IL-17A decreased to a level similar to that of the negative control group in the group treated with the nucleic acid complex compared to the control group inducing psoriasis.

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

The experiment was conducted under the conditions of Example 4 to obtain protein lysate by adding 200 μL of RIPA buffer to each well of a portion of the biopsied mouse ear tissue. Protein lysate is quantified using a BCA assay kit (Thermo Fisher, USA), and 30 μg of protein is separated by size through electrophoresis, and the protein is transferred to the PVDF membrane, and then the primary antibody, p-JAK1 (Cell signaling). Technology, USA), p-JAK2 (Cell signaling Technology, USA), JAK1 (Cell signaling Technology, USA), and JAK2 (Cell signaling Technology, USA) were treated at 1:1000 and left at 4°C for one day. Washed using 1X T BS-T, and treated with a secondary antibody, Goat Anti-Rabbit (Cell signaling Technology, USA) at 1:2000, and allowed to stand at room temperature for 1 hour. Supersignal ^TM West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA) was treated and the efficiency of suppressing the expression of target genes in tissues such as mice was analyzed using Image600 (Amersham, Germany) equipment.

As a result, it was confirmed that the expression of JAK1 and JAK2 and phosphorylation JAK1 and JAK2 genes decreased in the group treated with the nucleic acid complex in the psoriasis-like animal model tissue as shown in FIG. 4D.

Example 4-3: Flow cytometry Analyzer ( FACS ) Through psoriasis T cell analysis in an induced animal model

In the animal experiment conducted under the conditions of Example 4, the mouse spleen was biopsied on the end of the final experiment, and the tissue was crushed with a Handy Tissue Grinder (BIOFACT, Korea). After collecting the crushed splenocytes 3x10 ⁷ in 0.5% bovine serum albumin, 120 μL of 2mM EDTA buffer, 30 μL of Biotin antibody from CD4+ T Cell Isolation Kit (MACS Miltenyi Biotec, USA) was treated for 5 minutes, and Anti-Biotin MicroBeads 60 μL was treated for 10 minutes. After inserting an LS Column (MACS Miltenyi Biotec, USA) into a magnetic MACS sepeparator (MACS Miltenyi Biotec, USA), CD4+ T cells were separated by clolumn flow of cells labeled with CD4+ antibody. Treat the separated T cells with 5 μL of CCR6-PE antibody (BD, USA) for 30 minutes, dissolve in 500 μL of BD Perm/Wash buffer (BD, USA), transfer to a flow cytometer dedicated tube, and FACS CantoⅡ (BD, USA). USA)

As a result, it was confirmed that CCR6 expression decreased in the group treated with the nucleic acid complex compared to the control group inducing psoriasis, as shown in FIG. 4e, and it was confirmed that it decreased even when compared with the numerical value (FIG. 4e ).

Example 4-4: H&E dyeing psoriasis Phenotypic analysis in induced animal models

The experiment was conducted under the conditions of Example 4, and on the final day of the final experiment, the mouse ear tissue was biopsied and fixed in 4% formalin solution for one day, the fixed tissue was embedded in paraffin, and the tissue was sectioned into 5 μm and mounted on a slide glass. The mounted tissue was stained with Hematoxylin:Eoin staining solution for a certain period of time, washed with 1X PBS, and analyzed under a microscope.

As a result, it was confirmed that the thickness of the transdermal tissue and the number of inflammatory cells decreased in the group treated with the nucleic acid complex compared to the control group inducing psoriasis as shown in FIG. 4F.

Example 4-5: by immunostaining psoriasis Inflammation in tissues in induced animal models Marker analysis

The experiment was conducted under the conditions of Example 4, and on the end of the final experiment, the mouse ear tissue was biopsied and fixed in 4% formalin solution for one day, and the fixed tissue was embedded in paraffin, sectioned at 5 μm, and mounted on a slide glass. The mounted tissue was blocked in 0.5% BSA solution for 1 hour, and treated with Ki67 primary antibody solution for one day. Subsequently, the primary antibody solution was removed, washed with 1X PBS, treated with the secondary antibody solution, treated at room temperature for 2 hours, and analyzed by DAB staining.

As a result, as shown in Fig. 4g, it was confirmed that Ki67, an inflammatory marker in the tissue, decreased in the nucleic acid complex group compared to the control group inducing psoriasis, and it was confirmed that it decreased even when compared with the numerical value (Fig. 4g).

As described above, specific parts of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> SEASUN THERAPEUTICS <120> Composition for Preventing or Treating Psoriasis Comprising Skin Permeable Nucleic Acid Complex <130> P18-B278 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> BNA1 <400> 1 catggcattg cagtc 15 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> BNA2 <400> 2 gtcaatccga ttcgg 15 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CPNA3 <400> 3 gtaccgtaac gtcag 15 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CPNA4 <400> 4 gactgcaatg ccatg 15 <210> 5 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> CPNA5 <400> 5 gtcag 5 <210> 6 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> CPNA6 <400> 6 gactg 5 <210> 7 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CPNA7 <400> 7 cagttaggct aagcc 15

Claims (17)

Bioactive Nucleic Acid capable of binding to JAK1 and JAK2 genes and comprising a sequence represented by SEQ ID NO: 1 or 2; And a skin-permeable nucleic acid complex to which a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) comprising a sequence selected from the group consisting of SEQ ID NOs: 3 to 7 is complementarily bound, as an active ingredient, for the prevention, improvement or treatment of psoriasis Composition.
delete delete The method of claim 1, wherein the nucleic acid complex is a bioactive nucleic acid represented by SEQ ID NO: 2; And A pharmaceutical composition comprising a carrier peptide nucleic acid represented by any one sequence selected from the group consisting of SEQ ID NOs: 3 to 7.
The method of claim 1, wherein the binding power (melting temperature, melting temperature, Tm) of the bioactive nucleic acid and carrier peptide nucleic acid capable of binding to the JAK1 and JAK2 genes is determined by the target JAK1 and JAK2 genes of the bioactive nucleic acid and the bioactive nucleic acid. Pharmaceutical composition, characterized in that lower than the binding force of.
The method of claim 1, wherein the bioactive nucleic acid or carrier peptide nucleic acid is characterized in that the peptide GLFDIIKKIAESF or Histidine (10) is additionally bound to the 5'-end or 3'-end of each nucleic acid to help escape the endosomes. Pharmaceutical composition.
delete delete The pharmaceutical composition of claim 1, wherein the bioactive nucleic acid has a negative charge or neutrality as a whole.
The pharmaceutical composition of claim 1, wherein the carrier peptide nucleic acid comprises one or more gamma- or alpha-backbone modified peptide nucleic acid monomers so as to have a positive charge as a whole.
The method of claim 10, wherein the gamma- or alpha-backbone modified peptide nucleic acid monomer contains more positively charged amino acid monomers than negatively charged amino acid monomers, so that the overall charge of the carrier peptide nucleic acid is positive. A pharmaceutical composition characterized in that.
The method of claim 11, wherein the positively charged amino acids are lysine (Lysine, Lys, K), arginine (Arginine, Arg, R), histidine (Histidine, His, H), diamino butyric acid (DAB), Ornithine (Ornithine, Orn) and a pharmaceutical composition, characterized in that at least one selected from the group consisting of amino acid analogs.
The pharmaceutical composition of claim 11, wherein the amino acid having a negative charge is glutamic acid (Glu, E), aspartic acid (Asp, D), or an amino acid analog.
The pharmaceutical composition according to claim 1, wherein the nucleic acid complex has an overall positive charge.
The pharmaceutical composition according to claim 1, wherein the nucleic acid complex is skin-permeable with skin persistence.
An external preparation for skin comprising the composition according to claim 1.
The external preparation for skin according to claim 16, which is any one selected from aqueous solutions, gels, ointments, creams, lotions, pastes, smears, and patches.

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