Double-stranded oligonucleotide structure containing androgen receptor specific sequence, and composition for preventing hair loss and promoting hair growth containing the same

Abstract

To provide a double stranded oligonucleotide construct that efficiently delivers an androgen-receptor-specific oligonucleotide into a cell, a nanoparticle, and a composition for preventing hair loss and promoting hair growth, comprising the oligonucleotide construct.SOLUTION: Disclosed are a double stranded oligonucleotide construct, configured such that a hydrophilic material and a hydrophobic material are conjugated through a simple covalent bond or a linker-mediated covalent bond to both ends of a double stranded oligonucleotide, and a nanoparticle capable of being produced by self-assembling the double stranded oligonucleotide construct in an aqueous solution through hydrophobic interactions.EFFECT: The double stranded oligonucleotide construct according to the present invention and a composition for preventing hair loss or promoting hair growth containing the same as an active ingredient can suppress the expression of an androgen receptor with high efficiency without side effects, and can thus exhibit excellent effects on preventing hair loss, particularly androgenetic alopecia, alopecia areata, and telogen effluvium, and promoting hair growth.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present invention relates to a double-stranded oligonucleotide structure containing an androgen receptor-specific sequence and a composition for preventing hair loss and regrowth comprising the same, and more particularly to a double-stranded oligonucleotide structure having a structure in which a hydrophilic substance and a hydrophobic substance are bonded to both ends of a double-stranded oligonucleotide by a simple covalent bond or a linker-mediated covalent bond in order to efficiently deliver nucleotides of an androgen receptor-specific sequence into cells, nanoparticles that can be produced by self-assembling the double-stranded oligonucleotide structure in an aqueous solution through hydrophobic interactions, and a composition for preventing hair loss and regrowth comprising the double-stranded oligonucleotide structure. [Background technology]

[0002] Hair plays an important role in protecting the body and in appearance, and the purpose of managing hair is to protect the scalp, maintain healthy hair condition, and improve appearance. Hair loss refers to the natural loss of hair that has stopped growing due to the growth cycle, and serious hair loss has generally been recognized as a genetic phenomenon that occurs mainly in men. However, recently, the importance of environmental factors such as work stress, environmental pollution, exposure to harmful environments, and incorrect eating habits has become more prominent, and alopecia has come to be recognized as a disease that means a state in which hair is not present in areas where it should be. Alopecia is classified into cicatricial alopecia, in which the hair follicle is destroyed and restored as fibrous tissue, resulting in permanent hair loss, and non-cicatricial alopecia, in which the tissue does not become fibrous and the hair follicle is preserved as it is. Non-cicatricial alopecia includes telogen effluvium, hereditary androgenetic alopecia, alopecia areata, and anagen alopecia.

[0003] Hair goes through a cycle of growth, degeneration, resting, and exogen stages over time, which is called the hair cycle. The lifespan of the growth stage is usually 2-8 years, accounting for about 90% of the total hair, and hair is generated by continuous division of hair matrix cells in the lower half of the hair orifice that contacts the hair papilla. After the growth stage, there is a period when growth stops for a while, which is called the exogenous stage. This is the period when hair production and growth stops and the hair transitions to the resting stage, during which changes occur in the hair roots, and the activity of hair matrix cells and pigment cells stops, keratin is not produced, and hair growth is interrupted. During the resting stage, the hair orifice shrinks, and hair falls out only when the exogenous stage is reached, and proteolytic enzymes are known to be involved in this period. Factors that are thought to be involved in hair growth include androgens, estrogen, thyroid hormones, steroids, prolactin, and growth hormones, among which androgens are known to be the most important regulator. The most common example of hormones being involved in hair loss is temporary hair loss after childbirth. During pregnancy, estrogen increases and inhibits the progression of the hair cycle from the growth phase to the resting phase, but after childbirth, estrogen rapidly decreases and the progression to the resting phase accelerates, resulting in telogen effluvium. In other words, there is hormone-dependent alopecia, but other causes of hair loss can be selected from genetic factors, male hormones, aging, blood circulation disorders, stress, superoxide radicals, etc., and the countermeasures can vary depending on the cause. For hair loss caused by male hormones, DHT inhibitors are used as treatments, and the basic mechanism of these inhibitors is to prevent testosterone from being converted to the highly active dihydrotestosterone (hereinafter referred to as DHT) by 5-alpha-reductase.On the other hand, DHT has more than five times the ability to bind to the androgen receptor (AR) compared to testosterone, so substances that slow down protein synthesis in hair follicles to prevent excessive production of DHT and block binding to the androgen receptor are used as therapeutic agents (Dallob AL et al., 1994. J. Clin. Endocrinol. Metab. 79, 703-709; Ellsworth, K and Harris G., 1995, Biochem. Biophys. Res. Commun. 215, 774-780; Kaufman KD., 2002. Mol and Cell Endocrinology. 198, 85-89).

[0004] In 1942, Hamilton clarified the relationship between hair loss and male hormones. In androgenetic alopecia (AGA), testosterone present in hair root cells is converted into DHT, a powerful metabolite, which binds to the androgen receptor (AR) in the hair follicle and inhibits the activity of adenyl cyclase, which activates intracellular metabolism, thereby lowering the concentration of cAMP in the cells and reducing glucose metabolism. As a result, energy supply is inhibited and protein synthesis is delayed, shortening the growth phase of the hair follicle. As this phenomenon is repeated, the ratio of hair follicles in the resting phase increases, and gradually thinner and shorter hairs grow. In other words, the occurrence of androgenetic alopecia is believed to be due to the activity of DHT receptors and 5-alpha reductase, which are hormone components associated with the overexpression of testosterone and androgen receptors present in hair root cells. It is known that testosterone is overproduced into DHT (Dihydrotestosterone) by 5-alpha reductase, and this metabolite stimulates the production of hair cycle inhibitors, shortening the anagen phase and suppressing the hair production ability of hair follicles (Kaufman KD., 2002. Mol and Cell Endocrinology. 198, 89-85; Naito et al., 2008. Br. J. Dermatol. 159, 300-305).

[0005] DHT is known to have five times stronger binding power with the androgen receptor (AR) than testosterone, and in androgen-specific cells and tissues, DHT is known to be more involved in androgen activity than testosterone. There are two subtypes of 5-α-reductase, which is responsible for this metabolic process, and their roles differ slightly depending on the tissue. Type 1 5-α-reductase is found in the sebaceous gland, and Type 2 5-α-reductase is found mainly in the genitourinary tract and hair follicles.

[0006] Finasteride and dutasteride are drugs that target 5-alpha reductase to suppress the excessive production of DHT. Finasteride only acts on Type 2 5-alpha reductase, while dutasteride acts on Type 1 and 2 5-alpha reductase, and is known to be effective against prostate-related diseases. In particular, the drug that has been approved by the FDA as a baldness treatment is Propecia, which contains finasteride as its main ingredient. The hair loss treatment drugs developed to date are mainly single compounds, including minoxidil to promote blood circulation, and finasteride and dutasteride as male hormone inhibitors. Recently, drugs against JAK inhibitors (ruxolitinib and tofacitinib) have been approved by the FDA. However, research continues to find substances that have better effects than these substances.

[0007] The androgen receptor is a 110KDa steroid receptor, and one of its important functions is the transcription of androgen-related genes. The androgen receptor plays an important role in androgen-related diseases such as prostate cancer, prostatic hyperplasia, male pattern baldness, muscle loss, and hirsutism, and for this reason has been used as a target for the treatment of diseases that only appear in men, such as prostate cancer and male pattern baldness. In the case of male hormones, collectively known as androgens, testosterone is produced in the pituitary gland, adrenal gland, testes, etc., enters the cells of the target organ, is reduced to dihydrotestosterone (DHT) by testosterone 5-alpha reductase, and then binds to the receptor and acts as an androgen. Therefore, as mentioned above, efforts are being made to develop treatments for this disease by suppressing the production of DHT by inhibiting the action of 5-alpha-reductase, which reduces testosterone to DHT, and by suppressing the action of androgens by inhibiting the binding of DHT produced from testosterone to its receptor.

[0008] The technology of suppressing gene expression is an important tool in the development of therapeutic agents and target validation for disease treatment. Among these technologies, the role of interfering RNA (RNA interference; hereafter referred to as 'RNAi') has been discovered, and it has been shown to act on sequence-specific mRNA in various mammalian cells (Silence of the transcripts: RNA interference in medicine. J Mol Med (2005) 83: 764-773). When a long double-stranded RNA is delivered to a cell, it is converted into small interfering RNA (hereinafter referred to as 'siRNA') processed into 21-23 double strands (base pairs, bp) by an endonuclease called Dicer. The siRNA binds to RISC (RNA-induced silencing complex), and the guide (antisense) strand recognizes and degrades the target mRNA, thereby inhibiting the expression of the target gene in a sequence-specific manner (NUCLEIC-ACID THERAPEUTICS: BASIC PRINCIPLES AND RECENT APPLICATIONS. Nature Reviews Drug Discovery. 2002.1, 503-514).

[0009] According to Bertrand's research team, siRNA for the same target gene has a superior effect of inhibiting mRNA expression in vitro and in vivo compared to antisense oligonucleotide (ASO), and the effect lasts longer (Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem. Biophys. Res. Commun. 2002. 296: 1000-1004). In addition, the mechanism of action of siRNA is to bind complementarily to the target mRNA and regulate the expression of the target gene sequence-specifically, so it has the advantage of being able to dramatically expand the range of applicable subjects compared to existing antibody-based drugs or small molecule drugs (Progress Towards in Vivo Use of siRNAs. MOLECULAR THERAPY. 200613(4): 664-670).

[0010] Despite the excellent efficacy and wide range of uses of siRNA, in order for siRNA to be developed as a therapeutic agent, it is necessary to improve the stability of siRNA in the body and the cell delivery efficiency so that siRNA can be effectively delivered to target cells (Harnessing in vivo siRNA delivery for drug discovery and therapeutic development. Drug Discov Today. 2006 Jan;11(1-2):67-73).

[0011] In order to solve the above problems, active research is being conducted on the modification of some nucleotides or backbones of siRNA to make them resistant to nucleases in order to improve stability in the body, or the use of delivery vehicles such as viral vectors, liposomes, or nanoparticles.

[0012] Delivery systems using viral vectors such as adenovirus and retrovirus have high transfection efficacy but high immunogenicity and oncogenicity. In contrast, non-viral delivery systems including nanoparticles have lower cell transduction efficiency than viral delivery systems, but have high in vivo stability, target-specific delivery, and improved delivery efficiency such as uptake and internalization of encapsulated RNAi oligonucleotides into cells or tissues, as well as little cytotoxicity and immune stimulation. Therefore, they are currently evaluated as a more powerful delivery method than viral delivery systems (Nonviral delivery of synthetic siRNA s in vivo. J Clin Invest. 2007 December 3; 117(12): 3623-3632).

[0013] Among the non-viral delivery systems, the method using a nanocarrier uses various polymers such as liposomes and cationic polymer complexes to form nanoparticles, and siRNA is carried on the nanoparticles, i.e., nanocarriers, and delivered to cells. Among the methods using nanocarriers, the most commonly used methods include polymeric nanoparticles, polymer micelles, and lipoplexes. Among these, the method using lipoplexes is composed of cationic lipids, and plays a role in inducing the destabilization effect of endosomes by interacting with anionic lipids in the endosomes of cells, thereby delivering the siRNA into cells (Proc. Natl. Acad. Sci. 15; 93 (21): 11493-8, 1996).

[0014] In order to improve the intracellular delivery efficiency of siRNA, a technology for ensuring siRNA stability and efficient cell membrane permeability has been developed using an siRNA conjugate in which a biocompatible polymer, a hydrophilic substance (e.g., polyethylene glycol (PEG)), is conjugated to siRNA by simple covalent bond or linker-mediated covalent bond (Korean Patent No. 883471). However, chemical modification of siRNA and conjugation with polyethylene glycol (PEG) alone (PEGylation) still have disadvantages such as low stability in vivo and insufficient delivery to target organs. To solve these disadvantages, a double-stranded oligonucleotide structure in which hydrophilic and hydrophobic substances are bound to oligonucleotides, particularly double-stranded oligonucleotides such as siRNA, has been developed, and the structure is capable of binding SAM iRNA to the siRNA by the hydrophobic interaction of the hydrophobic substance. TM It forms self-assembled nanoparticles named SAMiRNA (self assembled micelle inhibitory RNA) (Korea Patent No. 1224828). TM The technology has the advantage of being able to obtain homogenous nanoparticles that are very small in size compared to existing delivery technologies.

[0015] SAMiRNA TMAs a specific example of the technology, PEG (polyethylene glycol) or HEG (hexaethylenglycol) is used as a hydrophilic substance. PEG is a synthetic polymer and is usually used to increase the solubility of pharmaceuticals, especially proteins, and to adjust their pharmacokinetics. PEG is a polydisperse substance, and one batch of polymer is composed of a sum of different numbers of monomers, and the molecular weight shows a Gaussian curve shape, and the degree of homogeneity of the substance is expressed by the polydisperse value (Mw / Mn). That is, when PEG has a low molecular weight (3~5kDa), it shows a polydispersity index of about 1.01, and when it has a high molecular weight (20kDa), it shows a high polydispersity index of about 1.2, and the higher the molecular weight, the lower the homogeneity of the substance (FM Veronese. Peptide and protein PEGylation: a review of problems and solutions. Biomaterials (2001) 22: 405-417). Therefore, when PEG is bound to a drug, the polydispersity characteristics of PEG are reflected in the conjugate, and it is difficult to verify a single substance. Due to this disadvantage, there is a trend to produce substances with low polydispersity indexes by improving the synthesis and purification process of PEG, but there are problems due to the polydispersity characteristics of the substance, such as the inconvenience of it being difficult to confirm whether the binding was easily achieved, especially when PEG is bound to a substance with a low molecular weight. (Francesco M.Veronese and Gianfranco Pasut.PEGylation,successful approach to drug delivery.DRUG DISCOVERY TODAY(2005)10(21):1451-1458).

[0016] Therefore, recently, existing self-assembled nanoparticles, SAMiRNA TM As an improved form of the technique, SAMiRNA TMThe hydrophilic substance of the double-stranded nucleotide structure constituting the present invention is blocked into a basic unit containing 1 to 15 uniform monomers having a certain molecular weight and a linker as necessary, and the basic unit is used in an appropriate number as necessary to produce the existing SAMiRNA. TM A new form of vehicle technology has been developed that has smaller size and dramatically improved polydispersity compared to conventional vehicles.

[0017] Meanwhile, it has been reported (Grand View Research, Inc.) that the global market size related to hair loss is expected to reach $11.8 billion in 2024, and it is known that four out of seven American men and one out of five Chinese men are bald, with more than 90% of cases being caused by androgenetic alopecia. However, it has been confirmed that most hair loss treatments developed to date target DHT and 5-alpha reductase, and no treatments or hair growth products targeting the androgen receptor, which has a direct link with androgen, have been developed.

[0018] Therefore, the inventors have made extensive efforts to develop hair growth-related products that target the androgen receptor, which has a direct link with androgen. As a result, they have confirmed that a specific androgen receptor-specific sequence can effectively inhibit the expression of the androgen receptor, and that a double-stranded oligonucleotide structure containing the same and a composition containing the same exhibit excellent effects in preventing hair loss or promoting hair growth, thereby completing the present invention. Summary of the Invention

[0019] An object of the present invention is to provide a novel oligonucleotide sequence that is specific to the androgen receptor and can inhibit its expression with extremely high efficiency, and a double-stranded oligonucleotide structure for effectively delivering the sequence to hair root cells.

[0020] Another object of the present invention is to provide a nanoparticle comprising the double-stranded oligonucleotide structure.

[0021] It is still another object of the present invention to provide a pharmaceutical composition for preventing hair loss or regrowing hair, which comprises the novel oligonucleotide sequence or the double-stranded oligonucleotide structure as an active ingredient.

[0022] It is still another object of the present invention to provide a cosmetic composition for preventing hair loss or promoting hair growth, which contains the novel oligonucleotide sequence or the double-stranded oligonucleotide structure as an active ingredient.

[0023] In order to achieve the above object, the present invention provides a double-stranded oligonucleotide structure having the following structural formula (1):

[0024] AXRYB structural formula (1)

[0025] In the structural formula (1), A is a hydrophilic substance, B is a hydrophobic substance, X and Y each independently represent a simple covalent bond or a covalent bond mediated by a linker, and R represents an androgen receptor-specific oligonucleotide comprising a sense strand comprising any one sequence selected from the group consisting of SEQ ID NOs: 6, 58, 68, 99, 107, 109, 260, 270, 284, 298, 348, 358, 359, and 434, and an antisense strand comprising a sequence complementary thereto.

[0026] The present invention also provides a nanoparticle comprising the double-stranded oligonucleotide structure.

[0027] The present invention also provides a pharmaceutical composition for preventing hair loss or promoting hair growth, which comprises the double-stranded oligonucleotide structure or nanoparticle as an active ingredient.

[0028] The present invention also provides a cosmetic composition for preventing hair loss or promoting hair growth, which comprises the double-stranded oligonucleotide structure or nanoparticle as an active ingredient.

[0029] The present invention also provides a method for treating hair loss, comprising administering the structure, nanoparticles or pharmaceutical composition of the present invention to a subject in need of hair growth, or applying the structure, nanoparticles or pharmaceutical composition of the present invention to a site in need of hair growth.

[0030] The present invention also provides a method for preventing hair loss or promoting hair growth, comprising administering or applying the structure, nanoparticles, or cosmetic composition according to the present invention to a subject or a corresponding area in need of hair loss prevention or hair growth.

[0031] The present invention also provides a use of the double-stranded oligonucleotide structure for preventing hair loss or promoting hair growth.

[0032] The present invention also provides a use of the double-stranded oligonucleotide structure for the manufacture of a medicine or cosmetic for preventing hair loss or promoting hair growth. [Brief description of the drawings]

[0033] [Figure 1] The exon maps and isoform common regions of human androgen receptor mRNA NM_000044.3 (isoform 1, 10,661 bp) and NM_001011645.2 (isoform 2, 8112 bp) for designing human androgen receptor-specific oligonucleotide candidate sequences are shown.

[0034] [Diagram 2] This shows the process of selecting candidate sequences consisting of 19 bases by applying the 2-base sliding window algorithm to the isoform common region for designing candidate oligonucleotide sequences specific to the human androgen receptor.

[0035] [Diagram 3]1 shows nanoparticle size distribution of double-stranded oligonucleotides, including randomly selected androgen receptor-specific oligonucleotides.

[0036] [Figure 4] 1 shows the results of a primary SAMiRNA screen of 544 targeting the androgen receptor.

[0037] [Diagram 5] FIG. 4 shows the results of screening 14 sequences with the highest androgen receptor expression inhibitory effect, and shows the results of selecting SAMiRNA containing androgen receptor-specific oligonucleotides.

[0038] [Figure 6] The results of secondary screening of SAMiRNA containing androgen receptor-specific oligonucleotides selected in the primary screening are shown.

[0039] [Figure 7] The results are as follows: after processing SAMiRNA constructs for the 14 selected sequences and sequences known in the prior art, the protein expression level of the androgen receptor was confirmed.

[0040] [Figure 8] The results of FIG. 7 show that inhibition of protein expression was confirmed after treatment with SAMiRNA constructs for the two selected sequences and a sequence from a prior literature.

[0041] [Figure 9] The effect of SAMiRNA nanoparticles on intracellular delivery into hair root cells was confirmed using a confocal laser scanning microscope. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein and the experimental procedures described below are those well known and commonly used in the art.

[0043] In the present invention, in order to select oligonucleotides capable of targeting androgen receptor and inhibiting its expression, a 2-base sliding window algorithm was applied to the entire androgen receptor to select a list of candidate sequences, from which 468 candidate sequences with 15 bases or less of identity to other genes and RNA sequences were finally selected, and the degree of androgen receptor inhibition was tested using a total of 544 oligonucleotide sequences, including 76 siRNA sequences disclosed in a known prior art document (US Patent Publication US2007-0141009), and 14 particularly effective oligonucleotides were selected. In addition, the oligonucleotides were prepared as double-stranded oligonucleotide structures to increase intracellular delivery efficiency and improve hair loss prevention and hair growth effects.

[0044] Therefore, in one aspect, the present invention relates to an androgen receptor-specific double-stranded oligonucleotide comprising a sense strand containing any one of sequences selected from the group consisting of SEQ ID NOs: 6, 58, 68, 99, 107, 109, 260, 270, 284, 298, 348, 358, 359 and 434, and an antisense strand containing a sequence complementary thereto.

[0045] The double-stranded oligonucleotide in the present invention is a concept including all substances having a general RNAi (RNA interference) action, and the androgen receptor-specific sequence includes androgen receptor-specific shRNA, ASO, etc., which are obvious to those with ordinary knowledge in the technical field to which the present invention belongs. Conventional methods for delivering siRNA into target cells still have problems that it is delivered into cells through the cell membrane, and that it is easily degraded by degradative enzymes present in the body as it escapes from endosomes in the cell and moves to the cytoplasm. Another form of DNA-RNA hybrid, which is an antisense oligo, is joined to siRNA for degrading target mRNA, and is more stable in vivo than existing double-stranded oligo RNA, and the DNA portion has an aptamer base sequence that can bind to target proteins, so it is efficiently delivered into target cells, and has an siRNA base sequence that inhibits RNA from being expressed as a protein, so it binds to target mRNA in target cells to inhibit gene expression. Since such DNA-RNA hybrid particles are composed only of biological materials, they are non-toxic and highly resistant to DNase and RNase, which are nucleic acid-degrading enzymes present in the body, and therefore can be said to be a new RNAi technology.

[0046] Furthermore, as long as the specificity for the androgen receptor is maintained, it is clear to a person having ordinary skill in the art to which the present invention pertains that androgen receptor-specific siRNAs including sense strands and antisense strands containing a sequence in which one or more bases are substituted, deleted, or inserted in a sense strand containing any one of the sequences selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:468 or an antisense strand complementary thereto are also included in the scope of the present invention.

[0047] The sequences of SEQ ID NOs: 1 to 468 are sequences specific to the human androgen receptor, and are RNA sense strand sequences having a homology of 15 bases or less to other sites of the androgen receptor mRNA (see Table 2). Meanwhile, the sequences of SEQ ID NOs: 469 to 544 represent human androgen receptor-specific siRNA sequences known from an existing patent (US2007-0141009) (see Table 3).

[0048] In the present invention, as a result of comparing the intracellular activity with that of androgen receptor-specific oligonucleotide sequences disclosed in existing patents, an RNA sequence with excellent efficiency and low homology to other human mRNAs has been invented.The oligonucleotide according to the present invention is preferably an androgen receptor-specific oligonucleotide containing any one of the sequences selected from the group consisting of SEQ ID NO: 6, 58, 68, 99, 107, 109, 260, 270, 284, 298, 348, 358, 359 and 434 as a sense strand, and more preferably an androgen receptor-specific oligonucleotide containing the sequence of SEQ ID NO: 68 or 109 as a sense strand.

[0049] The sense strand or antisense strand of the oligonucleotide of the present invention preferably consists of 19 to 31 nucleotides, and includes a sense strand containing any one of the sequences selected from SEQ ID NO: 1 to SEQ ID NO: 468, and an antisense strand complementary thereto.

[0050] The androgen receptor-specific oligonucleotide provided by the present invention has a base sequence designed to be capable of binding complementarily to the mRNA encoding the gene of interest, and is therefore characterized in that it can effectively suppress the expression of the gene of interest. In addition, the oligonucleotide may contain an overhang, which is a structure containing one or more unpaired nucleotides, at the 3' end of the oligonucleotide.

[0051] In addition, the oligonucleotide may include various modifications for improving the in vivo stability, imparting resistance to nucleases, and reducing non-specific immune responses. The modification of the first or second oligonucleotide constituting the oligonucleotide may be a modification in which the -OH group at the 2' carbon position of the sugar structure in one or more nucleotides is replaced with -CH3 (methyl), -OCH3 (methoxy), -NH2, -F (fluorine), -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamide or -O-dimethylamidooxyethyl; a modification in which oxygen in the sugar structure in the nucleotide is replaced with sulfur; or a modification to a phosphorothioate, boranophosphate or methyl phosphonate bond in the nucleotide bond, which may be used in combination. The modified oligonucleotide may be a peptide nucleic acid (PNA), locked nucleic acid (LNA) or unlocked nucleic acid (UNA). Modifications to the nucleotide (nucleotide) form are also available (Ann. Rev. Med. 55, 61-652004; US 5,660,985; US 5,958,691; US ​​6,531,584; US 5,808,023; US 6,326,358; US 6,175,001; Bioorg. Med. Chem. Lett. 14: 1139-1143, 2003; RNA, 9: 1034-1048, 2003; Nucleic Acid Res. 31: 589-595, 2003; Nucleic Acids Research, 38(17) 5761-5773, 2010; Nucleic Acids Research, 39(5) 1823-1832, 2011).

[0052] The androgen receptor-specific oligonucleotides provided by the present invention not only inhibit the expression of the gene of interest, but also significantly inhibit the expression of the protein of interest.

[0053] In another aspect of the present invention, a conjugate is provided in which a hydrophilic substance and a hydrophobic substance are conjugated to both ends of a double-stranded oligonucleotide for efficient delivery and improved stability of an androgen receptor-specific double-stranded oligonucleotide in vivo.

[0054] As described above, in the case of a double-stranded oligonucleotide conjugate in which a hydrophilic substance and a hydrophobic substance are bound to a double-stranded oligonucleotide, self-assembled nanoparticles are formed by the hydrophobic interaction of the hydrophobic substances (Korea Patent Registration No. 1224828). Such nanoparticles have excellent delivery efficiency and stability in the body, as well as excellent particle size uniformity and easy quality control, which makes the manufacturing process as a drug simple.

[0055] Therefore, in another aspect, the present invention relates to a double-stranded oligonucleotide structure having the structure of the following structural formula (1):

[0056] AXRYB structural formula (1)

[0057] In the structural formula (1), A is a hydrophilic substance, B is a hydrophobic substance, X and Y each independently represent a simple covalent bond or a covalent bond mediated by a linker, and R represents an androgen receptor-specific oligonucleotide comprising a sense strand having any one sequence selected from the group consisting of SEQ ID NOs: 6, 58, 68, 99, 107, 109, 260, 270, 284, 298, 348, 358, 359, and 434, and an antisense strand having a sequence complementary thereto.

[0058] More preferably, the double-stranded oligonucleotide structure comprising the androgen receptor-specific oligonucleotide of the present invention has the structure of the following structural formula (2):

[0059] [ka]

[0060] In the structural formula (2), A, B, X, and Y are the same as those in the structural formula (1), S represents the sense strand of the androgen receptor-specific oligonucleotide, and AS represents the antisense strand of the androgen receptor-specific oligonucleotide.

[0061] More preferably, the double-stranded oligonucleotide structure comprising the androgen receptor-specific oligonucleotide has the structure of the following structural formula (3) or (4).

[0062] [ka]

[0063] [ka]

[0064] In the structural formula (3) and the structural formula (4), A, B, S, AS, X and Y are the same as defined in the structural formula (1), and 5' and 3' represent the 5' end and 3' end of the androgen receptor-specific oligonucleotide sense strand.

[0065] It will be apparent to those skilled in the art to which the present invention pertains that the double-stranded oligonucleotide constructs comprising the androgen receptor-specific oligonucleotides in the above structural formulas (1) to (4) may have one to three phosphate groups bound to the 5'-end of the antisense strand, and that shRNA may be used instead of RNA.

[0066] The hydrophilic substances in the structural formulas (1) to (4) are preferably polymeric substances having a molecular weight of 200 to 10,000, and more preferably 1,000 to 2,000. For example, the hydrophilic polymeric substance is preferably a nonionic hydrophilic polymer compound such as polyethylene glycol, polyvinylpyrrolidone, or polyoxazoline, but is not necessarily limited thereto.

[0067] In particular, the hydrophilic substance (A) in structural formulas (1) to (4) can be used in the form of a hydrophilic substance block as shown in the following structural formula (5) or structural formula (6). By using an appropriate number of such hydrophilic substance blocks (n in structural formula (5) or structural formula (6)) as necessary, problems due to polydispersity that may occur when using general synthetic polymer substances, etc., can be greatly improved.

[0068] (A' m -J) n Structural formula (5)

[0069] (J-A' m ) n Structural formula (6)

[0070] In the structural formula (5), A' is a hydrophilic substance monomer, J is a linker connecting m hydrophilic substance monomers or connecting m hydrophilic substance monomers and siRNA, m is an integer of 1 to 15, and n is an integer of 1 to 10. m -J) or (J-A' m The repeating units represented by the symbols "a" and "b" correspond to the basic units of the hydrophilic block.

[0071] When the hydrophilic substance block such as structural formula (5) or structural formula (6) is present, the double-stranded oligonucleotide structure including the androgen receptor-specific oligonucleotide of the present invention may have a structure such as structural formula (7) or structural formula (8) below.

[0072] (A' m -J) n -XRYB Structural formula (7)

[0073] (J-A' m ) n -XRYB Structural formula (8)

[0074] In the structural formula (7) and the structural formula (8), X, R, Y, and B are the same as defined in the structural formula (1), and A', J, m, and n are the same as defined in the structural formula (5) and the structural formula (6).

[0075] In the structural formula (5) and the structural formula (6), the hydrophilic substance monomer (A') may be any monomer of a nonionic hydrophilic polymer that satisfies the object of the present invention, and may preferably be a monomer selected from compounds (1) to (3) shown in Table 1, more preferably a monomer of compound (1), and in compound (1), G may preferably be selected from CH2, O, S, and NH.

[0076] In particular, among the hydrophilic monomers, the monomer represented by compound (1) has excellent biocompatibility, such as being capable of introducing various functional groups, having good affinity in vivo, and inducing a small immune response. In addition, it has the advantage of being able to increase the in vivo stability and delivery efficiency of the oligonucleotide contained in the structure represented by structural formula (7) or structural formula (8), and is therefore very suitable for producing the structure according to the present invention.

[0077] [Table 1]

[0078] It is particularly preferred that the hydrophilic substance in the structural formulae (5) to (8) has a total molecular weight within the range of 1,000 to 2,000. Therefore, for example, when hexaethylene glycol according to compound (1) is used in structural formulae (7) and (8), that is, a substance in which G is O and m is 6, since the molecular weight of the hexaethylene glycol spacer is 344, it is preferred that the number of repetitions (n) is 3 to 5. In particular, the present invention can be modified as necessary by adding (A' m -J) or (J-A' m), i.e., a suitable number of hydrophilic block units represented by n may be used. The hydrophilic monomer A and linker J contained in each hydrophilic block may be independently the same or different between each hydrophilic block. That is, when three hydrophilic blocks are used (n=3), a hydrophilic monomer based on compound (1) may be used in the first block, a hydrophilic monomer based on compound (2) may be used in the second block, and a hydrophilic monomer based on compound (3) may be used in the third block, and different hydrophilic monomers may be used for all hydrophilic blocks, or all hydrophilic blocks may use the same hydrophilic monomer selected from the hydrophilic monomers based on compounds (1) to (3). Similarly, the linker mediating the bond between the hydrophilic monomers may be the same for each hydrophilic block, or different linkers may be used for each hydrophilic block. In addition, the number m of hydrophilic substance monomers may be the same or different between the hydrophilic substance blocks. That is, different numbers of hydrophilic substance monomers may be used, such as three hydrophilic substance monomers linked together (m=3) in the first hydrophilic substance block, five hydrophilic substance monomers linked together (m=5) in the second hydrophilic substance block, and four hydrophilic substance monomers linked together (m=4) in the third hydrophilic substance block, or the same number of hydrophilic substance monomers may be used in all the hydrophilic substance blocks.

[0079] In the present invention, the linker (J) is a PO3 - It is preferable that the linker is selected from the group consisting of SO3 and CO2, but it is not limited thereto, and it will be apparent to one of ordinary skill in the art that any linker that meets the objectives of the present invention can be used depending on the monomer of the hydrophilic substance used, etc.

[0080] The hydrophobic substance (B) in the structural formulas (1) to (4), (7) and (8) plays a role in forming nanoparticles composed of oligonucleotide structures represented by the structural formulas (1) to (4), (7) and (8) through hydrophobic interaction. The hydrophobic substance preferably has a molecular weight of 250 to 1,000, and is preferably a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, C 12 ~C 50 Examples of the hydrophobic substances that can be used include unsaturated or saturated hydrocarbons, diacylphosphatidylcholine, fatty acids, phospholipids, and lipopolyamines, but are not limited thereto. It will be apparent to a person skilled in the art to which the present invention pertains that any hydrophobic substance that meets the objectives of the present invention can be used.

[0081] The steroid derivative may be a group consisting of cholesterol, cholestanol, cholic acid, cholesterol formate, cholestanyl formate and cholesterylamine, and the glyceride derivative may be selected from mono-, di- and tri-glycerides, etc., in which the fatty acid of the glyceride is selected from the group consisting of C 12 ~C 50 Preferred are unsaturated or saturated fatty acids of the formula:

[0082] In particular, among the hydrophobic substances, saturated or unsaturated hydrocarbons or cholesterol are preferred because they can be easily bound to the oligonucleotide structure of the present invention during synthesis. 24 Carbohydrates, especially those containing disulfide bonds, are most preferred.

[0083] The hydrophobic substance is attached to the distal end of the hydrophilic substance, and may be attached to either the sense strand or the antisense strand of the oligonucleotide.

[0084] In the structural formulae (1) to (4), (7) and (8) according to the present invention, the hydrophilic or hydrophobic substance and the androgen receptor-specific oligonucleotide are bound by a simple covalent bond or a linker-mediated covalent bond (X or Y). The linker mediating the covalent bond is not particularly limited as long as it covalently binds the hydrophilic or hydrophobic substance to the end of the androgen receptor-specific oligonucleotide and provides a bond that can be degraded in a specific environment as necessary. Therefore, the linker can be any compound that is bound to activate the androgen receptor-specific oligonucleotide and / or the hydrophilic substance (or the hydrophobic substance) during the preparation process of the double-stranded oligonucleotide structure according to the present invention. The covalent bond may be either a non-degradable bond or a degradable bond. In this case, the non-degradable bond may be an amide bond or a phosphorylated bond, and the degradable bond may be a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond or an enzymatically degradable bond, but is not limited thereto.

[0085] Furthermore, the androgen receptor-specific oligonucleotide represented by R (or S and AS) in structural formulas (1) to (4), (7), and (8) can be any sequence having the property of being able to specifically bind to the mRNA of the androgen receptor, and preferably, in the present invention, the oligonucleotide is composed of a sense strand containing any one sequence selected from the group consisting of SEQ ID NOs: 6, 58, 68, 99, 107, 109, 260, 270, 284, 298, 348, 358, 359, and 434, and an antisense strand containing a sequence complementary thereto.

[0086] In particular, the siRNAs represented by the structural formulas (1) to (4), (7) and (8) of the present invention may be androgen receptor-specific oligonucleotides each comprising a sense strand containing any one of the sequences selected from the group consisting of SEQ ID NOs: 6, 58, 68, 99, 107, 109, 260, 270, 284, 298, 348, 358, 359 and 434, and an antisense strand containing a sequence complementary thereto.

[0087] In the double-stranded oligonucleotide structure comprising the androgen receptor-specific oligonucleotide according to the present invention, an amine group or a polyhistidine group may be further introduced into the opposite end of the hydrophilic substance bound to the oligonucleotide in the structure.

[0088] This is to facilitate intracellular introduction and endosomal escape of a carrier of a double-stranded oligonucleotide structure including the androgen receptor-specific oligonucleotide according to the present invention, and it has already been reported that introduction of an amine group and a polyhistidine group can be used to facilitate intracellular introduction and endosomal escape of carriers such as quantum dots, dendrimers, and liposomes, and the effects thereof.

[0089] Specifically, it is known that the primary amine group modified at the end or outside of the carrier is positively charged at the pH level in vivo and forms a conjugate with the negatively charged gene through electrostatic interaction, and after intracellular entry, the internal tertiary amine, which has a buffering effect at the low pH level of the endosome, facilitates escape from the endosome, thereby protecting the carrier from lysosomal degradation (Gene transfer and expression inhibition using polymer-based hybrid materials. Polymer Sci. Technol., Vol. 23, No. 3, pp 254-259). Histidine, a non-essential amino acid, has an imidazoline residue (pKa3 6.04) in the -R residue, which increases the buffering capacity in endosomes and lysosomes. It is also known that histidine modification can be used to increase the efficiency of endosomal escape in non-viral gene carriers such as liposomes (Novel histidine-conjugated galactosylated cationic liposomes for efficient hepatocyte selective gene transfer in human hepatoma). HepG2 cells.J.Controlled Release 118,pp262-270).

[0090] The amine or polyhistidine groups may be linked to a hydrophile or hydrophile block via one or more linkers.

[0091] When an amine group or a polyhistidine group is introduced into the hydrophilic substance of the double-stranded oligonucleotide structure according to the structural formula (1) of the present invention, it may have a structure as shown in the structural formula (7).

[0092] P-J1-J2-AXRYB Structural formula (9)

[0093] In the structural formula (9), A, B, R, X, and Y are the same as those defined in the structural formula (1), P represents an amine group or a polyhistidine group; J1 and J2 are linkers, and J1 and J2 are independently a simple covalent bond, PO3 - , SO3, CO2, C 2-12 It is obvious to one of ordinary skill in the art that J1 and J2 may be any linker suitable for the purposes of the present invention, including but not limited to, alkyl, alkenyl, and alkynyl, depending on the hydrophilic substance used.

[0094] Preferably, when an amine group is introduced, J2 is a simple covalent bond or PO3 - Preferably, J1 is C6 alkyl, but is not limited thereto.

[0095] In addition, when a polyhistidine group is introduced, J2 in the structural formula (9) is a simple covalent bond or PO3 - J1 is preferably, but not limited to, compound (4).

[0096] [ka]

[0097] In addition, when the hydrophilic substance of the double-stranded oligonucleotide structure represented by structural formula (9) is a hydrophilic substance block represented by structural formula (5) or structural formula (6) to which an amine group or a polyhistidine group is introduced, it can have a structure such as structural formula (10) or structural formula (11).

[0098] P-J1-J2-(A' m -J) n -XRYB Structural formula (10)

[0099] P-J1-J2-(J-A' m ) n -XRYB Structural formula (11)

[0100] In the structural formula (10) and the structural formula (11), X, R, Y, B, A', J, m, and n are the same as those defined in the structural formula (5) or the structural formula (6), and P, J1, and J2 are the same as those defined in the structural formula (9).

[0101] In particular, in the structural formula (10) and the structural formula (11), it is preferable that the hydrophilic substance is bound to the 3' end of the androgen receptor-specific oligonucleotide sense strand, and in this case, the structural formulas (9) to (11) can have the following structural formulas (12) to (14).

[0102] [ka]

[0103] [ka]

[0104] [ka]

[0105] In the structural formulas (12) to (14), X, R, Y, B, A, A', J, m, n, P, J1, and J2 are the same as defined in the structural formulas (9) to (11), and 5' and 3' represent the 5' end and 3' end of the androgen receptor-specific oligonucleotide sense strand.

[0106] The amine group that can be introduced in the present invention may be a primary to tertiary amine group, and it is particularly preferable to use a primary amine group. The introduced amine group may exist as an amine salt. For example, the salt of the primary amine group can be prepared by reacting NH3 + It may be present in the form:

[0107] Moreover, the polyhistidine group that can be introduced in the present invention preferably contains 3 to 10 histidines, particularly preferably 5 to 8 histidines, and most preferably 6 histidines. Furthermore, in addition to histidines, one or more cysteines may also be contained.

[0108] Meanwhile, when the double-stranded oligonucleotide structure containing the androgen receptor-specific oligonucleotide according to the present invention and the nanoparticles formed therefrom are provided with a targeting moiety, they can be efficiently delivered to target cells, and therefore can be delivered to target cells even at a relatively low dose, exhibiting high target gene expression regulation function, and preventing non-specific delivery of the androgen receptor-specific oligonucleotide to other organs and cells.

[0109] Accordingly, the present invention provides a double-stranded oligonucleotide structure in which a ligand (L), particularly a ligand having a property of specifically binding to a receptor that promotes internalization into a target cell through receptor-mediated endocytosis (RME), is further bound to the structure represented by structural formula (1) to structural formula (4), structural formula (7), and structural formula (8). For example, the form in which a ligand is bound to the double-stranded oligonucleotide structure represented by structural formula (1) has a structure as represented by the following structural formula (15).

[0110] (L i -Z)-AXRYB Structural formula (15)

[0111] In the structural formula (15), A, B, X, and Y are the same as those defined in the structural formula (1), L represents a ligand having a property of specifically binding to a receptor that enhances internalization into a target cell through receptor-mediated endocytosis (RME), and i represents an integer of 1 to 5, preferably an integer of 1 to 3.

[0112] The ligand in structural formula (15) may be selected from, but is not limited to, a target receptor-specific antibody, aptamer, or peptide having RME properties that promote internalization in a target cell-specific manner; or a chemical substance such as folate (generally, folate and folic acid are used interchangeably, and folate in the present invention refers to folate in its natural state or in its activated state in the human body), hexoamines such as N-acetylgalactosamine (NAG), sugars or carbohydrates such as glucose and mannose.

[0113] Additionally, the hydrophilic substance A in the structural formula (15) may be used in the form of a hydrophilic substance block according to the structural formula (5) or (6).

[0114] In yet another aspect of the present invention, there is provided a nanoparticle comprising a double-stranded oligonucleotide structure comprising an androgen receptor-specific oligonucleotide.

[0115] As described above, the double-stranded oligonucleotide structure containing the androgen receptor-specific oligonucleotide is amphiphilic, containing both hydrophobic and hydrophilic substances, and the hydrophilic portion has affinity with water molecules present in the body through interactions such as hydrogen bonds, and faces outward, while the hydrophobic portion faces inward through hydrophobic interactions between them, forming thermodynamically stable nanoparticles. That is, the hydrophobic substance is located at the center of the nanoparticle, and the hydrophilic substance is located toward the outside of the androgen receptor-specific oligonucleotide, forming a nanoparticle in a form that protects the androgen receptor-specific oligonucleotide. The nanoparticles thus formed improve the intracellular delivery of the androgen receptor-specific oligonucleotide and the efficacy of the oligonucleotide.

[0116] The nanoparticles according to the present invention are characterized in that they may be composed only of double-stranded oligonucleotide structures containing oligonucleotides having the same sequence, or may be composed of double-stranded oligonucleotide structures containing oligonucleotides having different sequences. The oligonucleotides having different sequences in the present invention may be oligonucleotides specific to different target genes, for example, androgen receptors, and are interpreted as including cases where the oligonucleotides have the same target gene specificity but have different sequences.

[0117] Furthermore, the nanoparticles of the present invention may contain a double-stranded oligonucleotide structure containing an siRNA specific to another hair loss-related gene in addition to the androgen receptor-specific oligonucleotide.

[0118] In yet another aspect, the present invention relates to a pharmaceutical composition for preventing hair loss, in particular androgenic alopecia, or for promoting hair growth, which comprises, as an active ingredient, an androgen receptor-specific double-stranded oligonucleotide, a double-stranded oligonucleotide structure comprising the same, and / or nanoparticles comprising the double-stranded oligonucleotide structure.

[0119] The pharmaceutical composition may be characterized in that it is used in a dosage form selected from, but not limited to, an ointment, a paste, a gel, a jelly, a serum, an aerosol spray, a non-aerosol spray, a foam, a cream, a lotion, a solution or a suspension.

[0120] The composition according to the present invention is effective in preventing hair loss or inducing hair growth by inhibiting the binding of DHT, a metabolic product of testosterone, to the androgen receptor.

[0121] In the present invention, the composition of the present invention may further contain a double-stranded oligonucleotide specific to a hair loss disease-related gene other than the androgen receptor, other than the double-stranded oligonucleotide or its structure of the present invention, or a double-stranded oligonucleotide structure containing the same.

[0122] The composition of the present invention may be applied to hair loss associated with genes involved in up- or down-signaling of the androgen receptor, in particular, androgenetic alopecia, but is not limited thereto.

[0123] The composition of the present invention may further contain one or more pharma- ceutically acceptable carriers in addition to the active ingredient. The pharma-ceutically acceptable carrier must be compatible with the active ingredient of the present invention, and may be used as a mixture of one or more of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and other conventional additives such as antioxidants, buffers, bacteriostatic agents, etc., if necessary. In addition, the composition may be formulated into an injectable dosage form such as an aqueous solution, suspension, emulsion, etc., by further adding a diluent, dispersant, surfactant, binder, and lubricant. In particular, it is preferable to provide the composition in a lyophilized form. For the preparation of the lyophilized dosage form, a method generally known in the art to which the present invention belongs may be used, and a stabilizer for lyophilization may be added. In addition, the composition may be formulated according to each disease or ingredient by using a suitable method in the art or the method disclosed in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton PA).

[0124] The content of the active ingredient contained in the composition of the present invention and the administration method can be determined by a person skilled in the art based on the symptoms of an individual and the severity of alopecia. Also, the composition can be formulated in various forms such as powder, tablet, injection, ointment, etc., and can be provided in a unit dose or multi-dose container, such as a sealed ampule or bottle.

[0125] In yet another aspect, the present invention relates to a cosmetic composition for preventing hair loss, in particular androgenic alopecia, or for promoting hair growth, comprising as an active ingredient an androgen receptor-specific double-stranded oligonucleotide, a double-stranded oligonucleotide structure comprising the same, and / or nanoparticles comprising the double-stranded oligonucleotide structure.

[0126] The composition may be characterized in that it is used in a formulation selected from, but not limited to, hair tonic, hair conditioner, hair essence, hair lotion, hair nourishing lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nourishing cream, hair moisture cream, hair massage cream, hair wax, hair aerosol, hair pack, hair nourishing pack, hair soap, hair cleansing foam, hair oil, hair drying agent, hair preservative, hair dye, hair waving agent, hair bleach, hair gel, hair glaze, hair dressing, hair lacquer, hair moisturizer, hair mousse, or hair spray.

[0127] In yet another aspect, the present invention relates to a method for treating hair loss, comprising the step of administering the structure, nanoparticles or pharmaceutical composition according to the present invention to a subject in need of hair growth, or applying the structure, nanoparticles or pharmaceutical composition according to the present invention to a site in need of hair growth.

[0128] The present invention also relates to a method for preventing hair loss or promoting hair growth, comprising administering or applying the structure, nanoparticles or cosmetic composition according to the present invention to a subject or a corresponding area in need of hair loss prevention or hair growth.

[0129] The present invention also relates to the use of said double-stranded oligonucleotide structure for preventing hair loss or promoting hair growth.

[0130] The present invention also relates to the use of said double-stranded oligonucleotide structure for the manufacture of a medicine or cosmetic for preventing hair loss or promoting hair growth.

[0131] In the present invention, hair loss includes androgenetic alopecia, alopecia areata, and telogen effluvium.

[0132] The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are merely intended to more specifically describe the present invention, and that the scope of the present invention is not limited by these examples.

[0133] Example 1. Algorithm and candidate sequence selection for screening oligonucleotides targeting the androgen receptor siRNA based drug high-throughput screening is a method in which a 1-base or 2-base sliding window algorithm is applied to the entire mRNA to generate all possible candidate sequences, and then homology filtering is performed to remove unnecessary candidate sequences, and the degree of inhibition of gene expression in the corresponding gene is confirmed for all the finally selected oligonucleotides.

[0134] First, the design process for candidate oligonucleotide sequences for the androgen receptor was carried out by checking the exon maps of human androgen receptor mRNA NM_000044.3 (isoform 1, 10,661bp) and NM_001011645.2 (isoform 2, 8112bp) to extract isoform common regions, and applying a 2-base sliding window algorithm to the extracted isoform common regions to select 3,956 candidate sequences consisting of 19 bases.

[0135] The selected oligonucleotide candidate sequence list was subjected to BLAST e-value of 100 or less against human total reference seq RNA, and 468 candidate sequences with identity of 15 bases or less against other genes and RNA sequences were finally selected. At this time, an androgen receptor inhibition experiment was performed using a total of 544 oligonucleotide sequences, including 76 siRNA sequences mentioned in a previously known prior art (US Patent Publication US2007-0141009).

[0136] [Table 2] JPEG0007625033000010.jpg246151 JPEG0007625033000011.jpg246151 JPEG0007625033000012.jpg246151 JPEG0007625033000013.jpg246151 JPEG0007625033000014.jpg246151 JPEG0007625033000015.jpg246151 JPEG0007625033000016.jpg246151 JPEG0007625033000017.jpg246151 JPEG0007625033000018.jpg246151 JPEG0007625033000019.jpg246151 JPEG0007625033000020.jpg246151 JPEG0007625033000021.jpg246151 JPEG0007625033000022.jpg246151 JPEG0007625033000023.jpg75151

[0137] [Table 3] JPEG0007625033000025.jpg246151 JPEG0007625033000026.jpg90151

[0138] Example 2. Synthesis of double-stranded oligonucleotide constructs The double-stranded oligonucleotide structure (SAMiRNA) prepared in the present invention has the following structural formula:

[0139] [ka]

[0140] The synthesis process begins with a nucleoside-bound solid support (CPG), and repeats a cycle of deblocking, coupling, capping, and oxidation to obtain a single strand of RNA with the desired sequence. A RNA synthesizer (384 synthesizer, BIONEER, Korea) was used for the synthesis of the series of double-stranded oligo-RNA.

[0141] The sense strand of the double-stranded oligonucleotide construct is synthesized by using polyethylene glycol (PEG)-CPG as a support and β-cyanoethyl phosphoramidite to link the phosphodiester bonds that form the DNA backbone structure, synthesizing a double-stranded oligonucleotide-hydrophilic substance construct of the sense strand with polyethylene glycol bound to the 3' end, followed by synthesis of a C 24was attached to the 5' end. In the case of the antisense strand that is annealed with the sense strand, a method of linking phosphodiester bonds forming an RNA backbone structure using β-cyanoethyl phosphoramidite was used to prepare an antisense strand with a sequence complementary to the sense strand, and then a chemical phosphorylation reagent (CPR) was used to attach a phosphate group to the 5' end to prepare an antisense strand with a phosphate group attached to the 5' end.

[0142] After the synthesis was completed, the oligonucleotide single strand and oligonucleotide-polymer construct were separated from the CPG by treating with 28% (v / v) ammonia in a water bath at 60°C, and the protective residue was removed by a deprotection reaction. The oligonucleotide single strand and oligonucleotide-polymer construct from which the protective residue was removed were treated with N-methylpyrolidone, triethylamine, and triethylamine trihydrofluoride in a volume ratio of 10:3:4 in a 70°C oven to remove 2'. The oligonucleotide single strand, oligonucleotide-polymer construct, and oligonucleotide-polymer construct bound to a ligand were separated from the reaction product by high performance liquid chromatography (HPLC), and the molecular weights of the oligonucleotides were measured by a MALDI-TOF mass spectrometer (MALDI TOF-MS, SHIMADZU, Japan) to confirm whether they matched with the base sequence and oligonucleotide-polymer construct to be synthesized. Then, to prepare each double-stranded oligonucleotide construct, equal amounts of the sense strand and the antisense strand were mixed and placed in 1X annealing buffer (30 mM HEPES, 100 mM potassium acetate, 2 mM magnesium acetate, pH 7.0 or higher), and reacted in a 90°C thermostatic water bath for 3 minutes, and then further reacted at 37°C to prepare the desired SAMiRNA, monoSAMiRNA (n=1), monoSAMiRNA (n=2), monoSAMiRNA (n=3), and monoSAMiRNA (n=4), respectively. Annealing of the prepared double-stranded oligonucleotide constructs was confirmed by electrophoresis.

[0143] Example 3. Screening of SAMiRNA nanoparticles that target androgen receptor to induce RNAi 3.1 Preparation and particle size analysis of SAMiRNA nanoparticles To analyze the particle size of 544 types of SAMiRNA targeting the androgen receptor sequence synthesized in Example 2, the size and polydispersity index of the SAMiRNA were measured using a Zetasizer Nano ZS (Malvern, UK). The nanoparticle size and polydispersity index for randomly selected SAMiRNA are shown in Table 4 below, and a representative graph is shown in Figure 3.

[0144] [Table 4]

[0145] 3.2 Intracellular processing method of SAMiRNA nanoparticles To discover SAMiRNA that suppresses the expression of androgen receptor, we used LNCaP, a human prostate cancer cell line. The LNCaP cell line was cultured at 37°C and 5% CO2 in RPMI medium (Hyclone, US) containing 10% fetal bovine serum (Hyclone, US) and 1% penicillin-streptomycin (Hyclone, US). Using the same medium as above, the LNCaP cell line was cultured in a 12-well plate (Costar, US) at 4 × 10 4 The next day, SAMiRNA was diluted with 1X DPBS and treated with 50 nM SAMiRNA. SAMiRNA was treated once every 12 hours for a total of four times, and cells were cultured at 37℃ and 5% CO2.

[0146] 3.3 SAMiRNA Screening Using Androgen Receptor mRNA Expression Inhibition Efficacy Assay RNA extracted from cells treated with SAMiRNA in the same manner as in Example 3.2 was analyzed using AccuPower® RocketScript TMAfter synthesizing cDNA using Cycle RT Premix with oligo(dT)20, the relative expression rate of the androgen receptor gene was analyzed in comparison with the SAMiRNA control sample using multiplex qPCR with Taqman probe.

[0147] As a result, as shown in Figure 4, 9 sequences mentioned in an existing patent (US2007-0141009A) and 14 sequences in Table 2 were selected from 544 SAMiRNAs targeting the androgen receptor that showed 60% or more androgen receptor mRNA suppression ability (Figure 5), and the evaluation of the reproducibility of androgen receptor mRNA suppression ability for the 14 sequences is shown in Figure 6. Two SAMiRNAs that most effectively suppress androgen receptor gene expression were finally selected. The sequence information of the SAMiRNAs is shown in Table 5 below.

[0148] [Table 5]

[0149] 3.4 Evaluation of the efficacy of selected SAMiRNA in suppressing androgen receptor protein expression In order to confirm whether the 14 SAMiRNAs selected together with the sequences 68 and 109 selected in Example 3.3 effectively suppressed the expression of androgen receptor protein, Western blot (WB) analysis was performed. LNCaP cells were cultured at 1.2 × 10 5After dispensing cells / well, the cells were cultured at 37℃ and 5% CO2. The next day, transfection was performed at a concentration of 50nM using lipofectamine (Invitrogen, USA). After 48 hours of culture, the medium was removed and proteins were isolated using cell lysis buffer (Cell signaling technology, USA) containing a protease inhibitor cocktail (Sigma Aldrich, USA). After quantifying the amount of protein using a BCA assay kit (Thermo, USA), 20ug of protein was boiled at 95℃ for 10 minutes with Laemmli's 5x sample buffer. The denatured proteins were electrophoresed on SDS-polyacrylamide gel and then transferred to a PVDF membrane. The membrane was immersed in blocking solution (5% skim milk powder in TBS and 0.05% Tween 20) for 1 hour at room temperature, and then reacted with primary antibodies AR antibody (1:2000, Santa Cruz, USA) and GAPDH antibody (1:5000, Cell Signaling Technology, USA) in a refrigerator at 4°C overnight. After washing three times with TBST, it was reacted with horseradish peroxidase-labeled secondary antibody (Cell signaling technology) for 1 hour at room temperature, and protein bands were detected using the chemiluminescent reagent SuperSignal® Pico Chemiluminescent Substrate (Thermo, USA).

[0150] The ability of 14 types of SAMiRNA to suppress the expression of androgen receptor protein was confirmed as shown in FIG. 7, and SEQ ID NOs: 68 and 109 showed the highest suppressive ability in terms of protein expression.

[0151] 3.5 Evaluation of the efficacy of suppressing androgen receptor protein expression in human hair follicle dermal papilla cells (HFDPC) In order to confirm whether the sequences of SEQ ID NOs: 68 and 109 finally selected from Example 3.4 actually suppress the expression of androgen receptor protein in human hair root cells, the presence or absence of protein expression suppression was confirmed using human hair papilla dermal cells (HFDPC), which are hair root cells derived from humans (FIG. 8). It was confirmed that both sequences could suppress the expression of androgen receptor protein.

[0152] Example 4. Confirmation of intradermal delivery effect of SAMiRNA nanoparticles In order to confirm whether the finally selected SAMiRNA-AR#68 and SAMiRNA-AR#109, which were prepared using sequence numbers 68 and 109, were actually delivered to human hair roots, the gene delivery effect was confirmed using human hair.

[0153] On the day of the experiment, hair was harvested by removing the tip of the hair, cut into pieces about 1 cm from the root, and then incubated in 200 ul M199 medium (10% FBS + 1% penicillin) in a 96-well plate for 1 hour. Then, to observe the presence or absence of gene transfer, the hair was incubated for 24 hours in 200 ul M199 medium containing 2 uM or 10 uM SAMiRNA labeled with a fluorescent substance (FAM dye). After 24 hours of treatment with the substance, the hair was washed three times with DPBS, and finally the hair root was fixed for 20 minutes in PBS containing 3.7% formaldehyde and 2% FBS.

[0154] The hair roots that had been fixed were placed in a base mold containing OCT compound and placed on a pre-frozen stainless steel plate to completely freeze the OCT compound. The frozen tissue was stored at -70°C and placed at -20°C for about 30 minutes to facilitate tissue sectioning before cutting with a tissue slicer. The sections were placed on slides at a thickness of 10 μm and dried for 1 hour. Once dried, they were mounted using a mounting medium containing DAPI. Fluorescence was observed using a confocal laser scan microscope (LSM5 LIVE CONFIGURATION VARIOTWO VRGB), confirming that SAMiRNA was delivered to the hair root cells of the hair tissue (Figure 9).

[0155] As described above, certain parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents. [Industrial Applicability]

[0156] The double-stranded oligonucleotide structure containing the androgen receptor-specific oligonucleotide according to the present invention and the composition for preventing hair loss or promoting hair growth containing the same as an active ingredient can highly efficiently suppress the expression of the androgen receptor without side effects, and can exhibit excellent effects in preventing hair loss, particularly androgenetic alopecia, alopecia areata, and telogen effluvium, and promoting hair growth.

Claims

1. A double-stranded oligonucleotide structure having the structure of the following structural formula (1): AX-RY-B Structural formula (1) In the structural formula (1), A is a hydrophilic substance, and the hydrophilic substance is any one selected from the group consisting of polyethylene glycol (PEG), polyvinylpyrrolidone, polyoxazoline, and a structure represented by the following structural formula (5) or structural formula (6): (A' ｍ -J) ｎ Structural formula (5) (J-A' ｍ ) ｎ Structural formula (6) In the formula (5) and the formula (6), A' is a hydrophilic monomer, J is a linker connecting m hydrophilic monomers or connecting m hydrophilic monomers and an oligonucleotide, m is an integer of 1 to 15, and n is an integer of 1 to 10; The hydrophilic substance monomer A' is any one compound selected from the compounds (1) to (3), and the linker (J) is PO ３ － , S.O. ３ and CO ２ Selected from the group consisting of: 【Chemistry 1】 B is a steroid derivative, a glyceride derivative, a glycerol ether, a polypropylene glycol, C １２ ~C ５０ is a hydrophobic substance selected from the group consisting of unsaturated or saturated hydrocarbons, diacylphosphatidylcholine, fatty acids, phospholipids, and lipopolyamines; X and Y each independently represent a simple covalent bond or a linker-mediated covalent bond, and R represents an androgen receptor-specific DNA-RNA hybrid oligonucleotide comprising a DNA sense strand having a sequence selected from the group consisting of SEQ ID NO: 68 or 109 and an RNA antisense strand having a complementary sequence thereto.

2. The double-stranded oligonucleotide structure according to claim 1 , having the structure of the following structural formula (2): 【Chemistry 2】 In the structural formula (2), S represents the sense strand of the oligonucleotide according to claim 1, AS represents the antisense strand, and A, B, X and Y are the same as those defined in claim 1.

3. The double-stranded oligonucleotide structure according to claim 2, having the structure represented by the following structural formula (3) or structural formula (4). 【Chemistry 3】 In the structural formula (3) and the structural formula (4), A, B, X, Y, S and AS are the same as defined in claim 2, and 5' and 3' mean the 5' end and 3' end of the oligonucleotide sense strand.

4. 2. The double-stranded oligonucleotide structure according to claim 1, wherein the steroid derivative is selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesterol formate, cholestanyl formate and cholesterylamine.

5. 2. The double-stranded oligonucleotide structure according to claim 1, wherein the glyceride derivative is selected from mono-, di- and tri-glycerides.

6. 2. The double-stranded oligonucleotide structure of claim 1, wherein the covalent bonds represented by X and Y are non-degradable or degradable bonds.

7. 7. The double-stranded oligonucleotide structure according to claim 6, wherein the non-degradable bond is an amide bond or a phosphate bond.

8. 7. The double-stranded oligonucleotide structure according to claim 6, wherein the degradable bond is a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond or an enzymatically degradable bond.

9. The double-stranded oligonucleotide structure according to claim 1, further comprising a ligand that specifically binds to a receptor that promotes internalization into a target cell through receptor-mediated endocytosis (RME) and is attached to the hydrophilic substance.

10. The double-stranded oligonucleotide structure according to claim 9, wherein the ligand is selected from the group consisting of a target receptor-specific antibody, an aptamer, a peptide, folate, N-acetylgalactosamine (NAG), glucose, and mannose.

11. The double-stranded oligonucleotide structure according to claim 1, further comprising an amine group or a polyhistidine group introduced into the hydrophilic substance at the opposite end thereof bound to the oligonucleotide.

12. The double-stranded oligonucleotide structure according to claim 11, wherein the amine group or polyhistidine group is linked to a hydrophilic substance or hydrophilic block via one or more linkers.

13. The double-stranded oligonucleotide structure according to claim 11, wherein the amine group is any one selected from the group consisting of primary, secondary, and tertiary amine groups.

14. The double-stranded oligonucleotide structure according to claim 11, wherein the polyhistidine group contains 3 to 10 histidines.

15. A nanoparticle comprising the double-stranded oligonucleotide structure according to any one of claims 1 to 14.

16. 16. The nanoparticle of claim 15, comprising a mixture of double-stranded oligonucleotide structures comprising oligonucleotides with different sequences.

17. A pharmaceutical composition for preventing hair loss or promoting hair growth, comprising the double-stranded oligonucleotide structure according to any one of claims 1 to 14 as an active ingredient.

18. A pharmaceutical composition for preventing hair loss or promoting hair growth, comprising the nanoparticles according to claim 15 as an active ingredient.

19. The pharmaceutical composition according to claim 17, characterized in that the pharmaceutical composition is used in a dosage form selected from the group consisting of an ointment, a paste, a gel, a jelly, a serum, an aerosol spray, a non-aerosol spray, a foam, a cream, a lotion, a solution or a suspension.

20. The pharmaceutical composition according to claim 18, characterized in that the pharmaceutical composition is used in a dosage form selected from the group consisting of an ointment, a paste, a gel, a jelly, a serum, an aerosol spray, a non-aerosol spray, a foam, a cream, a lotion, a solution or a suspension.

21. A cosmetic composition for preventing hair loss or promoting hair growth, comprising the double-stranded oligonucleotide structure according to any one of claims 1 to 14 as an active ingredient.

22. A cosmetic composition for preventing hair loss or promoting hair growth, comprising the nanoparticles according to claim 15 as an active ingredient.

23. The cosmetic composition according to claim 21, characterized in that the composition is used in a formulation selected from the group consisting of hair tonic, hair conditioner, hair essence, hair lotion, hair nourishing lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nourishing cream, hair moisture cream, hair massage cream, hair wax, hair aerosol, hair pack, hair nourishing pack, hair soap, hair cleansing foam, hair oil, hair drying agent, hair preservative, hair dye, hair waving agent, hair bleach, hair gel, hair glaze, hair dressing, hair lacquer, hair moisturizer, hair mousse, and hair spray.

24. The cosmetic composition according to claim 22, wherein the composition is used in a formulation selected from the group consisting of hair tonic, hair conditioner, hair essence, hair lotion, hair nourishing lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nourishing cream, hair moisture cream, hair massage cream, hair wax, hair aerosol, hair pack, hair nourishing pack, hair soap, hair cleansing foam, hair oil, hair drying agent, hair preservative, hair dye, hair waving agent, hair bleach, hair gel, hair glaze, hair dressing, hair lacquer, hair moisturizer, hair mousse, and hair spray.

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