DNA-RNA heteroduplex targeting androgen receptor

Abstract

The present disclosure provides a DNA-RNA heteroduplex that targets and inhibits expression of an androgen receptor, and a composition thereof. The DNA-RNA heteroduplex and the composition can be used to inhibit symptoms associated with androgen receptor expression. In particular, methods of using the DNA-RNA heteroduplex and the compositions for hair improvement, increased hair growth, or reduced alopecia are provided.

Description

1. Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 492,794, filed March 29, 2023, which is hereby incorporated by reference in its entirety. 2. Sequence List

[0002] This application contains a sequence list that has been electronically filed in XML format and is hereby incorporated by reference in its entirety. The XML copy created in XX is named 41346-54988-SEQLIST.xml and has a size of XX bytes. Background Technology

[0003] Androgenetic alopecia (AGA) (commonly known as male pattern baldness (MPHL) in men and female pattern baldness (FPHL) in women) is the most common type of progressive hair loss. Androgens (i.e., male hormones) are one of the causes of AGA. Although the relationship between FPHL and androgens is not fully understood, FPHL is characterized by follicle miniaturization and thinning hair, similar to MPHL. The pathogenesis of both diseases is not fully understood, but their incidence has increased in recent years.

[0004] Androgens and AR signaling play important roles in regulating the hair cycle and skin pathogenesis, including in AGA. Endogenous androgens include testosterone and dihydrotestosterone (DHT). DHT is a more potent androgen synthesized from testosterone via 5-alpha reductase and exhibits approximately 10 times higher binding affinity to the androgen receptor (AR). Patients with AGA have elevated DHT and AR levels, and DHT-AR signaling has been reported to be closely related to the pathogenesis of AGA. Finasteride and dutasteride, developed as 5-alpha reductase inhibitors, have been approved by the FDA and are being used as primary treatments for AGA. However, these drugs have several side effects, such as decreased libido due to reduced DHT.

[0005] Recently, siRNA nanoparticles (SAMiRNA) targeting AR mRNA regions have been developed to reduce AR mRNA and protein levels in DPCs and hair follicles, as described in PCT / KR2019 / 015723, the entire patent of which is incorporated herein by reference. Their effects on hair growth have been tested in clinical studies, where continuous administration of SAMiRNA for 24 weeks showed similar efficacy to finasteride without side effects. See Yun, SI., Lee, SK., Goh, EA. et al. Weekly treatment with SAMiRNA targeting AR mRNA ameliorated androgenetic alopecia. Sci Rep 12, 1607 (2022).

[0006] These studies suggest that SAMiRNA may be a potential treatment for AGA. There is a need to develop SAMiRNA formulations to deliver and store SAMiRNA safely and efficiently over long periods. Furthermore, for the widespread use and commercialization of formulations, it is necessary to improve the safety, quality, and efficacy of the drug compound (i.e., SAMiRNA) and its timing of use. Summary of the Invention

[0007] This disclosure provides a DNA-RNA heteroduplex comprising: a DNA strand having the sequence of SEQ ID NO: 1 or 3; an RNA strand having the sequence of SEQ ID NO: 2 or 4, or a modified RNA strand comprising one or more modified sequences of SEQ ID NO: 2 or 4; a stearyl hexyl disulfide (C6-SS-C18) covalently attached to the 5' end of the DNA strand; and hexaethylene glycol (HEG) or polyethylene glycol (PEG) covalently attached to the 3' end of the DNA strand, wherein the DNA strand and the RNA strand or the modified RNA strand form a DNA-RNA hybrid.

[0008] In some embodiments, the DNA strand has the sequence of SEQ ID NO: 1, and the RNA strand has the sequence of SEQ ID NO: 2. In some embodiments, the DNA strand has the sequence of SEQ ID NO: 3, and the RNA strand has the sequence of SEQ ID NO: 4.

[0009] In some embodiments, the DNA-RNA heteroduplex comprises HEG, wherein the HEG is hexaethylene glycol-(-PO3-hexaethylene glycol)3.

[0010] In some embodiments, the DNA-RNA heteroduplex comprises the modified RNA strand, wherein the modified RNA strand comprises a sequence having one or more phosphate thioester modified SEQ ID NO: 2 or 4.

[0011] In some embodiments, the modified RNA strand comprises the sequence of SEQ ID NO: 2, and each nucleotide in the UCU at the 3' end of SEQ ID NO: 2 is a phosphate thioester nucleotide.

[0012] This disclosure also provides a composition for hair improvement comprising the DNA-RNA heteroduplex disclosed herein. In some embodiments, the composition further comprises one or more moisturizing agents selected from betaine and butylene glycol. In some embodiments, the composition further comprises one or more hair conditioning or soothing ingredients selected from panthenol, aminopropanol, biotin, niacinamide, and menthol. In some embodiments, the composition further comprises one or more buffering ingredients selected from citric acid and sodium citrate.

[0013] In some embodiments, 0.1%-2% by weight of the composition is the DNA-RNA heteroduplex. In some embodiments, 0.25%-1% by weight of the composition is the DNA-RNA heteroduplex. In some embodiments, 0.4%-0.6% by weight of the composition is the DNA-RNA heteroduplex. In some embodiments, 0.4%, 0.45%, 0.5%, 0.55%, or 0.6% by weight of the composition is the DNA-RNA heteroduplex. In some embodiments, 1%, 1.25%, 1.5%, 1.75%, or 2% by weight of the composition is the DNA-RNA heteroduplex.

[0014] In some embodiments, the composition at 1-20 mg / ml is the DNA-RNA heteroduplex. In some embodiments, the composition at 2.5-10 mg / ml is the DNA-RNA heteroduplex. In some embodiments, the composition at 4-6 mg / ml is the DNA-RNA heteroduplex. In some embodiments, the composition at 4 mg / ml, 4.5 mg / ml, 5 mg / ml, 5.5 mg / ml, or 6 mg / ml is the DNA-RNA heteroduplex. In some embodiments, the composition at 10 mg / ml, 12.5 mg / ml, 1.5 mg / ml, 1.75 mg / ml, or 2 mg / ml is the DNA-RNA heteroduplex.

[0015] In some embodiments, the composition comprises denatured ethanol, D-panthenol, betaine, biotin, nicotinamide, citric acid, menthol crystals, trisodium citrate dihydrate, and 1,3-butanediol. In some embodiments, the composition comprises 30%-35% phosphate-buffered saline, 0.4%-0.6% of the DNA-RNA heteroduplex, 45%-50% water, 14%-15% ethanol, 0.19%-0.2% panthenol, 1% nicotinamide, 0.02% citric acid, 0.05% menthol, 0.03% sodium citrate, 3% butanediol, and less than 0.01% aminopropanol. In some embodiments, the composition comprises 32.8% phosphate-buffered saline, 0.5%-0.6% of the DNA-RNA heteroduplex, 46.3% water, 14%-15% ethanol, 0.19%-0.2% panthenol, 1% nicotinamide, 0.02% citric acid, 0.05% menthol, 0.03% sodium citrate, 3% butylene glycol, and less than 0.01% aminopropanol.

[0016] In some embodiments, the composition is in the form of an ointment, paste, gel, jelly, serum, aerosol spray, non-aerosol spray, foam, cream, lotion, solution, or suspension formulation.

[0017] In some embodiments, the composition has a pH between 5 and 7.5. In some embodiments, the composition has a pH between 5.5 and 6.5, 6 and 6.5, or 7 and 7.5. In some embodiments, the composition has a pH of 6.29 or 7.02.

[0018] In some implementations, the DNA-RNA heteroduplex forms nanoparticles.

[0019] In some embodiments, the nanoparticles have an average diameter between 50 nm and 200 nm. In some embodiments, the nanoparticles have an average diameter between 50 nm and 150 nm, between 75 nm and 125 nm, between 80 nm and 120 nm, between 90 nm and 110 nm, or between 100 nm and 110 nm.

[0020] This disclosure also provides a method for inhibiting androgen receptor expression, the method comprising administering to a subject the DNA-RNA heteroduplex or composition disclosed herein.

[0021] In some embodiments, the DNA-RNA heteroduplex or the composition is applied topically. In some embodiments, the DNA-RNA heteroduplex or the composition is applied for hair improvement in the subject. In some embodiments, the DNA-RNA heteroduplex or the composition is applied to the scalp of the subject.

[0022] In some embodiments, the DNA-RNA heteroduplex or the composition is applied once a week, once every two weeks, or once every four weeks. In some embodiments, the DNA-RNA heteroduplex or the composition is applied for at least four months or at least six months. In some embodiments, the DNA-RNA heteroduplex or the composition is applied once a week for at least four months. In some embodiments, the DNA-RNA heteroduplex or the composition is applied once a week for four months.

[0023] In some embodiments, the method includes applying the DNA-RNA heteroduplex or the composition weekly for four months, and then applying the DNA-RNA heteroduplex or the composition less than once a week after the four months. In some embodiments, the method includes applying the DNA-RNA heteroduplex or the composition weekly for four months, and then applying the DNA-RNA heteroduplex or the composition every two weeks, every three weeks, or every four weeks after the four months.

[0024] In some embodiments, the method includes applying the DNA-RNA heteroduplex or the composition once a week for four months, and then applying the composition once a month, once every two months, once every three months, once every four months, once every five months, once every six months, or less after the four months.

[0025] In some embodiments, the method includes applying the DNA-RNA heteroduplex or the composition once a week for four months, and then discontinuing the application of the composition after said four months.

[0026] In some embodiments, the subject has alopecia. In some embodiments, the subject has moderate androgenetic alopecia. In some embodiments, the subject has alopecia with a Norwood scale rating of at least III or more severe. In some embodiments, the subject has alopecia with a Ludwig scale rating of at least I-3 or more severe.

[0027] In some embodiments, the composition administered to the subject comprises 1-20 mg / ml of the DNA-RNA heteroduplex. In some embodiments, the composition administered to the subject comprises 2.5-10 mg / ml of the DNA-RNA heteroduplex. In some embodiments, the composition administered to the subject comprises 4 mg / ml, 4.5 mg / ml, 5 mg / ml, 5.5 mg / ml, or 6 mg / ml of the DNA-RNA heteroduplex. In some embodiments, the composition administered to the subject comprises 4.5 mg / ml, 5 mg / ml, or 5.5 mg / ml of the DNA-RNA heteroduplex.

[0028] In some embodiments, the method includes administering 1-100 mg of the DNA-RNA heteroduplex once weekly. In some embodiments, the method includes administering 1-30 mg of the DNA-RNA heteroduplex once weekly. In some embodiments, the method includes administering 5-10 mg of the DNA strand or the RNA strand once weekly.

[0029] In some embodiments, the method includes administering 1-10 mg of the DNA strand or the RNA strand once weekly. In some embodiments, the method includes per scalp area (cm²) 2 ) Administer 1-100 mg of the aforementioned DNA-RNA heteroduplex once weekly. In some embodiments, the method includes per scalp area (cm²) 2 ) Administer 1-50 mg of the aforementioned DNA-RNA heteroduplex once weekly. In some embodiments, the method includes per scalp area (cm²) 2 ) Administer 5-20 mg of the aforementioned DNA-RNA heteroduplex once weekly. In some embodiments, the method includes per scalp area (cm²) 2 ) Administer 8-10 mg of the aforementioned DNA-RNA heteroduplex once weekly. In some embodiments, the method includes per scalp area (cm²) 2 0.5-5 ml of the DNA-RNA heteroduplex is applied once weekly. In some embodiments, the method includes applying per scalp area (cm²) 2 ) Apply 1-2 ml of the DNA-RNA heteroduplex once a week. In some embodiments, the method includes applying per scalp area (cm²) 2 Apply 1-1.5 ml of the DNA-RNA heteroduplex once a week. Attached Figure Description

[0030] These and other features, aspects, and advantages of the invention will be better understood with reference to the following description and accompanying drawings, in which:

[0031] Figures 1A-1C. Screening of effective SAMiRNAs targeting human AR. (Figure 1A) Schematic diagram of SAMiRNA nanoparticles. (Figures 1B and 1C) LNCap cells were treated with PBS or 14 SAMiRNA candidates for 48 h. Quantitative polymerase chain reaction (qPCR) assays were performed on total RNA extracts to evaluate AR knockdown efficacy, and AR expression was normalized relative to the expression of the ribosomal protein stalk subunit P0 (RPLP0) gene (Figure 1B). Immunoblot analysis was performed on whole-cell lysates to compare AR and GAPDH protein levels. The intensity of AR and GAPDH bands was quantified using ImageJ software, and AR protein expression was normalized relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) values ​​(Figure 1C, bottom inset). Statistical significance was assessed using a two-tailed Student's t-test, ***p < 0.001.

[0032] Figures 2A and 2B illustrate the AR silencing effect of each SAMiRNA candidate in LNCap cells, as analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Fourteen SAMiRNA candidates were selected based on their knockdown efficiency (>50% AR silencing efficacy).

[0033] Figures 3A-3D. Efficacy of SAMiRNA-AR68 in reducing AR expression in HFDP cells. (Figures 3A and 3B) Human follicular dermal papilla (HFPD) cells were treated with PBS, SAMiRNA-AR68, or SAMiRNA-AR109 for 48 h. Total RNA extracts were analyzed by RT-qPCR, and AR expression was normalized relative to RPLPO gene expression. (Figure 3A). Immunoblot analysis of whole-cell lysates was performed (Figure 3B, left inset). The intensity of AR and GAPDH bands was quantified using ImageJ software, and AR protein expression was normalized relative to GAPDH values ​​(Figure 3B, right inset). (Figure 3C) HFDP cells were treated with PBS, SAMiRNA control, or an indicator dose of SAMiRNA-AR68 for 48 h. Total RNA extracts were analyzed by qPCR, and AR expression was normalized relative to RPLPO gene expression. (Figure 3D) HFDP cells were treated with PBS, SAMiRNA control, or 10 μM SAMiRNA-AR68 for 48 h. Whole-cell lysates were analyzed by enzyme-linked immunosorbent assay (ELISA) to measure AR protein. Statistical significance was assessed using a two-tailed Student's t-test, with **p < 0.01 and ***p < 0.001.

[0034] Figures 4A-4C. AR silencing effect of SAMiRNA-AR68 in human hair follicles. (Figure 4A) Plucking human hair follicles were treated in culture medium with PBS or 10 μM FAM-labeled SAMiRNA-AR68 for 24 h. Immunofluorescence (IF) analysis was performed on the hair follicles, and they were counterstained with 4',6-diamidinyl-2-phenylindole (DAPI). Scale bar = 100 μm. (Figure 4B) Plucking human hair follicles were treated with PBS or 10 μM SAMiRNA-AR68 for 48 h. qPCR was performed on the total RNA extract from the hair bulb, and AR expression was normalized relative to RPLPO gene expression. (Figure 4C) Plucking human hair follicles were treated with PBS or 10 μM SAMiRNA-AR68 for 48 h. IF analysis of plucked scalp hair follicle sections incubated with anti-AR antibody and counterstained with DAPI. Scale bar = 100 μm. AR protein levels were measured by mean fluorescence intensity using ZEN software. Data are presented as mean ± SD normalized to DAPI intensity (n = 8 hairs / group). Statistical significance was assessed by a two-tailed Student's t-test, *p < 0.05 and **p < 0.01.

[0035] Figures 5A and 5B. Safety of SAMiRNA-AR68. (Figure 5A) HFDP and HaCaT cells were treated with PBS or an indicated dose of SAMiRNA-AR68 for 72 h. WST-1 assay was performed to measure cell viability. (Figure 5B) Human PBMCs were treated with 20 μg / ml concanavalin A (ConA) as a positive control, PBS, or an indicated dose of SAMiRNA-AR68 for 6 h. qPCR assays of total RNA extracts were performed to evaluate the expression of pro-inflammatory cytokines (including IL-1β, IL-6, INF-γ, and TNF-α), normalized relative to the expression of the ribosomal protein L13A (RPL13A) gene.

[0036] Figure 6A shows the average size of AR68 nanoparticles measured in the formulation after 6 months of storage. The average size is approximately 99.2 ± 5.1 nm (22ºC, 55% ± 5% RH) and 105.0 ± 2.5 nm (40ºC, 75% ± 5% RH). Figure 6B shows the AR knockdown measured by RT-qPCR using AR68 samples stored under indicated conditions for 6 months.

[0037] Figure 7 is a flowchart summarizing the Clinical Trial I study described in Chapter 6.5.

[0038] Figures 8A and 8B. Images of hair loss changes after 24 weeks of continuous administration of 0.5 mg / ml SAMiRNA-AR68 and placebo. (Figure 8A) Representative photographs of the frontal hairline and crown at baseline and at 8, 16, and 24 weeks after treatment with 0.5 mg / ml AR68 and placebo three times weekly. Phototrichogram analysis was performed at baseline and at 16 and 24 weeks. (Figure 8B) Representative graphs of the percentage increase in total hair count at 16 and 24 weeks compared to baseline.

[0039] Figure 9 is a flowchart summarizing the Clinical Trial II study described in Chapter 6.6.

[0040] Figures 10A and 10B. Images of hair loss changes after 24 weeks of continuous administration of 5 mg / ml SAMiRNA-AR68 and placebo products. (Figure 10A) Representative photographs of the frontal hairline and crown at baseline and at 8, 16, and 24 weeks after weekly treatment with 5 mg / ml AR68 and placebo. Hair morphological analysis was performed at baseline and at 16 and 24 weeks. (Figure 10B) Representative graphs of the percentage increase in total hair count at 16 and 24 weeks compared to baseline.

[0041] Figure 11 shows an exemplary device for packaging and delivering SAMiRNA preparations. It is a piston-type bottle with a silicone adapter for massage.

[0042] Figure 12 is a graph summarizing the demographic characteristics of the subjects tested in the Clinical Trial III study described in Section 6.8. The pie chart (left) shows the extent of skin defects in the subjects, and the bar chart (right) shows their age distribution.

[0043] Figure 13A shows the percentage (%) of hair in the anagen phase calculated in the placebo group and the treatment group (weekly, twice a month, once a month) before SAMiRNA-AR68 administration (start) and after 6 months (6 months) of SAMiRNA-AR68 treatment. Figure 13B shows the change in the percentage of hair in the anagen phase after 6 months of SAMiRNA-AR68 treatment compared to the percentage before treatment.

[0044] Figure 14A shows the mean change (in percentage) in anagen hair in male subjects treated with placebo or SAMiRNA-AR68 for 6 months. Figure 14B shows the mean change (in percentage) in anagen hair in female subjects treated with placebo or SAMiRNA-AR68 for 6 months.

[0045] Figure 15 shows the number of hairs lost (counts) in the placebo group and the treatment group (weekly, twice a month, once a month) before (start) administration of SAMiRNA-AR68 and after 4 or 6 months of treatment with SAMiRNA-AR68.

[0046] Figure 16 shows the mean change (in percentage) in hair loss among subjects treated with placebo or SAMiRNA-AR68 for 4 months.

[0047] Figure 17A shows the mean change (in percentage) in hair loss in male subjects treated with placebo or SAMiRNA-AR68 for 4 months. Figure 17B shows the mean change (in percentage) in hair loss in female subjects treated with placebo or SAMiRNA-AR68 for 4 months.

[0048] Figure 18 shows the mean change (in percentage) in hair loss in subjects treated with placebo or SAMiRNA-AR68 for 4 or 6 months weekly (4 times / month), bi-weekly (2 times / month), or monthly (1 time / month).

[0049] Figures 19A and 19B show the results of treatment with placebo or SAMiRNA-AR68 at different dosing frequencies (weekly (4 times / month), bi-weekly (2 times / month), and monthly (1 time / month)) at 1 / cm² before or 4 or 6 months after treatment. 2 (Fig. 19A) or % (Fig. 19B) the density of the terminal hair.

[0050] Figure 20 shows the results of different dosing frequency groups (weekly (4 times / month), every two weeks (2 times / month), and monthly (1 time / month)) before or 4 or 6 months after treatment with placebo or SAMiRNA-AR68, at a rate of 1 / cm. 2 The density of the hairs measured.

[0051] Figure 21 shows hair thickness in mean µm measured in different dosing frequency groups (weekly (4 times / month), bi-weekly (2 times / month), and monthly (1 time / month)) before or 4 or 6 months after treatment with placebo or SAMiRNA-AR68.

[0052] Figure 22 provides representative photographs of the hairline and crown of the head before or after treatment with SAMiRNA-AR68 at different dosing frequencies (weekly (4 times / month), bi-weekly (2 times / month), and monthly (1 time / month)).

[0053] Figure 23 shows the inhibitory effect of SAMiRNA-AR68 or its variants with or without phosphate thioester modification on AR mRNA expression in human follicular dermal papilla cells, as analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

[0054] Figure 24 shows the half-maximal inhibitory concentration (IC50) of SAMiRNA-AR68 or its variants on AR mRNA expression.

[0055] Figure 25A shows a fluorescence image of mouse skin treated with SAMiRNA-SRN-021 labeled with fluorescein (Cy-5). It illustrates the subcutaneous delivery of SAMiRNA-SRN-021 via appendages (intercellular spaces). Figure 25B shows the inhibition (approximately 50% knockdown) of AR mRNA expression in skin tissue treated with SAMiRNA-SRN-021.

[0056] Figure 26A shows the detection of SAMiRNA by RT-qPCR amplification using stem-loop primers, as indicated by Ct (cycle threshold). There was no significant difference between the control group and the group receiving topical SAMiRNA-SRN-021. Figure 26B shows AR mRNA expression measured in the kidneys of mice treated with placebo or SAMiRNA-SRN-021. There was no significant difference between the control group and the group receiving topical SAMiRNA-SRN-021. Detailed Implementation 6.1. Definition

[0057] The term "hair improvement" is used to refer to, but is not limited to, prevention of hair loss and / or increase of hair growth. As used herein, hair improvement can refer to improvements in hair health, strength, and / or thickness. 6.2. DNA-RNA heteroduplex

[0058] In one aspect, this disclosure provides a DNA-RNA heteroduplex that can be used to inhibit androgen receptor (AR) mRNA expression. In some embodiments, the DNA-RNA heteroduplex is used for hair improvement. In some embodiments, the DNA-RNA heteroduplex is used to treat diseases associated with androgen receptor (AR) expression.

[0059] A DNA-RNA heteroduplex is a double-stranded molecule consisting of a DNA strand and an RNA strand, or modifications thereof, wherein the DNA strand and the RNA strand, or modifications thereof, form a double-stranded molecule, i.e., a DNA-RNA hybrid. In various embodiments, the DNA strand contains a sequence specific to an androgen receptor-specific oligonucleotide (e.g., AR mRNA). For example, the DNA strand may contain sequences selected from SEQ ID NO: 1, 3, 5-17. In some embodiments, the DNA strand has the sequence of SEQ ID NO: 1. In some embodiments, the DNA strand has the sequence of SEQ ID NO: 3. The RNA strand may be completely or partially complementary to the DNA strand.

[0060] In certain embodiments, this disclosure provides a DNA-RNA heteroduplex comprising a DNA strand having the sequence of SEQ ID NO: 1 and an RNA strand having the sequence of SEQ ID NO: 2, or a modification thereof. In some embodiments, the DNA-RNA heteroduplex comprises a DNA strand having the sequence of SEQ ID NO: 1 and an RNA strand having the sequence of SEQ ID NO: 2, or a modification thereof.

[0061] In some implementations, the DNA-RNA heteroduplex comprises a DNA strand of SEQ ID NO: 1 and an RNA strand of SEQ ID NO: 2.

[0062] In some embodiments, the DNA-RNA heteroduplex comprises a DNA strand of SEQ ID NO: 1 and a modified RNA strand comprising a sequence of SEQ ID NO: 2 having one or more modifications.

[0063] In certain embodiments, this disclosure provides a DNA-RNA heteroduplex comprising a DNA strand having the sequence of SEQ ID NO: 3 and an RNA strand having the sequence of SEQ ID NO: 4, or a modification thereof. In some embodiments, the DNA-RNA heteroduplex comprises a DNA strand having the sequence of SEQ ID NO: 3 and an RNA strand having the sequence of SEQ ID NO: 4, or a modification thereof.

[0064] In some implementations, the DNA-RNA heteroduplex comprises a DNA strand of SEQ ID NO: 3 and an RNA strand of SEQ ID NO: 4.

[0065] In some embodiments, the DNA-RNA heteroduplex comprises a DNA strand of SEQ ID NO: 3 and a modified RNA strand comprising a sequence of SEQ ID NO: 4 having one or more modifications.

[0066] In some embodiments, one or more modifications are post-translational modifications. In some embodiments, one or more modifications are chemical modifications of nucleotides. In some embodiments, one or more modifications are phosphate thioester substitutions.

[0067] In some embodiments, the chemical modification is one or more selected from, but not limited to, the following: a modification in which the OH group at the 2' carbon position of the sugar structure in one or more nucleotides is substituted by any one of -CH3 (methyl), OCH3 (methoxy), amine (-NH2), fluorine (-F), -O-2-methoxyethyl, -O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamido, and -O-dimethylamidooxyethyl; a modification in which the oxygen in the sugar structure of the nucleotide is substituted by sulfur; a bond modification between nucleotides in which the bond is selected from any one of phosphate thioester bonds, boranophosphophate bonds, and methylphosphoester bonds; a modification in which the bond is PNA (peptide nucleic acid), LNA (locked nucleic acid), or UNA (unlocked nucleic acid); and a modification in which the bond is a DNA-RNA hybrid (Ann. Rev. Med. 55, 61-65). 2004; US Patent Nos. 5,660,985; 5,958,691; 6,531,584; 5,808,023; 6,326,358; 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) 5761-773, 2010; Nucleic Acids Research, 39(5):1823-1832, 2011).

[0068] In some embodiments, the modified RNA chain comprises one or more phosphate-thioester nucleotides. In this case, the phosphate-thioester (PS) bond replaces the non-bridging oxygen in the phosphate backbone of the RNA chain with a sulfur atom. In some embodiments, the modified RNA chain comprises one, two, three, four, five, six, seven, eight, nine, or more phosphate-thioester nucleotides.

[0069] In some embodiments, the modified RNA strand contains one, two, three, or more phosphate-thioester nucleotides at the 3' end of the sequence CAAACUCU in SEQ ID NO: 2. In some embodiments, the modified RNA strand contains one, two, three, or more phosphate-thioester nucleotides at the 3' end of the sequence AAACUCU in SEQ ID NO: 2. In some embodiments, the modified RNA strand contains one, two, three, or more phosphate-thioester nucleotides at the 3' end of the sequence AACUCU in SEQ ID NO: 2. In some embodiments, the modified RNA strand contains one, two, three, or more phosphate-thioester nucleotides at the 3' end of the sequence ACUCU in SEQ ID NO: 2. In some embodiments, the modified RNA strand contains one, two, three, or more phosphate-thioester nucleotides at the 3' end of the sequence CUCU in SEQ ID NO: 2. In some embodiments, the modified RNA strand contains one, two, or three phosphate-thioester nucleotides at the 3' end of the sequence UCU in SEQ ID NO: 2. In some implementations, each nucleotide in the UCU at the 3' end of SEQ IDNO: 2 is a phosphate thioester nucleotide.

[0070] In some embodiments, the DNA-RNA heteroduplex further comprises a hydrophobic compound covalently attached to the DNA strand. In some embodiments, the hydrophobic compound is attached to the 5' end of the DNA strand. In some embodiments, the hydrophobic compound is attached to the 3' end of the DNA strand.

[0071] In some embodiments, the hydrophobic compound has a molecular weight of 250 to 1,000 and may be selected from, but is not limited to, steroid derivatives, glycerol ester derivatives, glycerol ethers, polypropylene glycol, C12-C50 unsaturated or saturated hydrocarbons, diacylphosphatidylcholine, fatty acids, phospholipids, lipopolyamines, lipids, tocopherols, and tocotrienols. Steroid derivatives may be selected from cholesterol, cholesterol, cholic acid, cholesterol carbamate, cholesterol alkyl carbamate, and cholesterol amine, and glycerol ester derivatives may be selected from monoglycerides, diglycerides, and triglycerides. In some embodiments, the fatty acid of the glycerol ester is C12-C50. 12 -C 50 Unsaturated or saturated fatty acids. In some implementations, C24 hydrocarbons are used, particularly hydrophobic hydrocarbons containing disulfide bonds.

[0072] In some embodiments, the DNA-RNA heteroduplex further comprises a stearylhexyl disulfide (C6-SSC) covalently attached to the 5' end of the DNA strand. 18In some embodiments, the DNA-RNA heteroduplex further comprises a stearylhexyl disulfide (C6-SSC) covalently attached to the 3' end of the DNA strand. 18 ).

[0073] In some embodiments, the DNA-RNA heteroduplex further comprises a hydrophilic compound covalently attached to the DNA strand. In some embodiments, the hydrophilic compound is attached to the 5' end of the DNA strand. In some embodiments, the hydrophilic compound is attached to the 3' end of the DNA strand.

[0074] In some embodiments, the hydrophilic compound is polyethylene glycol (PEG), hexaethylene glycol (HEG), polyvinylpyrrolidone, or polyoxazoline, but this disclosure is not limited thereto.

[0075] In some embodiments, the DNA-RNA heteroduplex further comprises hexaethylene glycol (HEG) covalently attached to the 3' end of the DNA strand. In some embodiments, the DNA-RNA heteroduplex further comprises hexaethylene glycol (HEG) covalently attached to the 5' end of the DNA strand. In some embodiments, the DNA-RNA heteroduplex further comprises hexaethylene glycol-(-PO3-hexaethylene glycol) covalently attached to the 3' end of the DNA strand. In some embodiments, the DNA-RNA heteroduplex further comprises hexaethylene glycol-(-PO3-hexaethylene glycol)2 covalently attached to the 5' end of the DNA strand. In some embodiments, the DNA-RNA heteroduplex further comprises hexaethylene glycol-(-PO3-hexaethylene glycol)2 covalently attached to the 3' end of the DNA strand. In some embodiments, the DNA-RNA heteroduplex further comprises hexaethylene glycol-(-PO3-hexaethylene glycol)2 covalently attached to the 5' end of the DNA strand. In some embodiments, the DNA-RNA heteroduplex further comprises hexaethylene glycol-(-PO3-hexaethylene glycol)3 covalently attached to the 3' end of the DNA strand. In some embodiments, the DNA-RNA heteroduplex further comprises hexaethylene glycol-(-PO3-hexaethylene glycol)3 covalently attached to the 5' end of the DNA strand.

[0076] In some embodiments, the hydrophobic and hydrophilic compounds attach to the RNA or DNA strand without a linker. In other embodiments, the hydrophobic and hydrophilic compounds attach to the RNA or DNA strand using a linker to form a linker-mediated covalent bond.

[0077] In some embodiments, the bond between the hydrophobic or hydrophilic compound and the RNA or DNA strand is a non-degradable bond or a degradable bond. In some embodiments, the non-degradable bond is an amide bond or a phosphate ester bond. In some embodiments, the degradable bond is selected from any one of disulfide bonds, acid-degradable bonds, ester bonds, acid anhydride bonds, biodegradable bonds, and enzyme-degradable bonds.

[0078] Methods for producing DNA-RNA heteroduplexes are described in PCT / KR2019 / 015723 and are generally described in U.S. Application No. 17 / 057,852, which are incorporated herein by reference in their entirety.

[0079] In some embodiments, the DNA-RNA heteroduplex is SAMiRNA-AR68 or a variant thereof. In some embodiments, the DNA-RNA heteroduplex is SAMiRNA-SRN-021.

[0080] In one aspect, this disclosure provides a DNA strand having the sequence SEQ ID NO: 1. In some embodiments, the DNA strand can be used to target AR mRNA without forming a DNA-RNA hybrid. In some embodiments, the DNA strand is used to inhibit AR mRNA expression. In some embodiments, the 5' end of the DNA strand is covalently attached to stearylhexyl disulfide (C6-SSC). 18 In some implementations, the 3' end of the DNA strand is covalently attached to hexaethylene glycol (HEG) or polyethylene glycol (PEG).

[0081] In one aspect, this disclosure provides an RNA strand having the sequence SEQ ID NO: 2. In some embodiments, the RNA strand can be used to target AR mRNA without forming a DNA-RNA hybrid. In some embodiments, the RNA strand is used to suppress the expression of AR mRNA.

[0082] In some embodiments, the compositions, kits, or methods of use disclosed herein use DNA strands or RNA strands instead of DNA-RNA heteroduplexes. 6.3. Composition of DNA-RNA heteroduplexes

[0083] In another aspect, this disclosure provides a composition comprising the DNA-RNA heteroduplex disclosed herein.

[0084] In some embodiments, 0.1%-2% by weight of the composition is a DNA-RNA heteroduplex. In some embodiments, 0.25%-1.5% by weight of the composition is a DNA-RNA heteroduplex. In some embodiments, 0.25%-1.25% by weight of the composition is a DNA-RNA heteroduplex. In some embodiments, 0.25%-1% by weight of the composition is a DNA-RNA heteroduplex. In some embodiments, 0.4%-0.6% by weight of the composition is a DNA-RNA heteroduplex. In some embodiments, 0.4%, 0.45%, 0.5%, 0.55%, or 0.6% by weight of the composition is a DNA-RNA heteroduplex. In some embodiments, 1%, 1.25%, 1.5%, 1.75%, or 2% by weight of the composition is a DNA-RNA heteroduplex.

[0085] In some embodiments, the composition at 1-20 mg / ml is a DNA-RNA heteroduplex. In some embodiments, the composition at 2.5-10 mg / ml is a DNA-RNA heteroduplex. In some embodiments, the composition at 4-6 mg / ml is a DNA-RNA heteroduplex. In some embodiments, the composition at 4 mg / ml, 4.5 mg / ml, 5 mg / ml, 5.5 mg / ml, or 6 mg / ml is a DNA-RNA heteroduplex. In some embodiments, the composition at 10 mg / ml, 12.5 mg / ml, 1.5 mg / ml, 1.75 mg / ml, or 2 mg / ml is a DNA-RNA heteroduplex.

[0086] In some embodiments, the composition further comprises a moisturizer, a hair conditioning or soothing ingredient, and / or one or more buffering ingredients. In some embodiments, the composition comprises one or more moisturizers selected from betaine and butylene glycol. In some embodiments, the composition comprises one or more hair conditioning or soothing ingredients selected from panthenol, aminopropanol, biotin, niacinamide, and menthol. In some embodiments, the composition comprises one or more buffering ingredients selected from citric acid and sodium citrate.

[0087] In some embodiments, the composition comprises denatured ethanol, D-panthenol, betaine, biotin, nicotinamide, citric acid, menthol crystals, trisodium citrate dihydrate, and 1,3-butanediol. In some embodiments, the composition comprises 30%-35% phosphate-buffered saline, 0.4%-0.6% DNA-RNA heteroduplex, 45%-50% water, 14%-15% ethanol, 0.19%-0.2% panthenol, 1% nicotinamide, 0.02% citric acid, 0.05% menthol, 0.03% sodium citrate, 3% butanediol, and less than 0.01% aminopropanol. In some embodiments, the composition comprises 30%-35% phosphate-buffered saline, 0.4%-0.6% DNA-RNA heteroduplex, 45%-50% water, 14%-15% ethanol, 0.19%-0.2% panthenol, 1% nicotinamide, 0.02% citric acid, 0.05% menthol, 0.03% sodium citrate, and 3% butanediol. In some embodiments, the composition comprises 32.8% phosphate-buffered saline, 0.5%-0.6% DNA-RNA heteroduplex, 46.3% water, 14%-15% ethanol, 0.19%-0.2% panthenol, 1% nicotinamide, 0.02% citric acid, 0.05% menthol, 0.03% sodium citrate, 3% butanediol, and less than 0.01% aminopropanol.

[0088] In some embodiments, the composition is in the form of ointment, paste, gel, jelly, serum, aerosol spray, non-aerosol spray, foam, cream, lotion, solution, or suspension formulation.

[0089] In some embodiments, the composition has a pH between 5 and 7. In some embodiments, the composition has a pH between 5.5 and 6.5 or between 6 and 6.5. In some embodiments, the composition has a pH of 6. In some embodiments, the composition has a pH of 6.29. In some embodiments, the composition has a pH of 6.50. In some embodiments, the composition has a pH of 7. In some embodiments, the composition has a pH of 7.02.

[0090] In some embodiments, the DNA-RNA heteroduplex in the composition is transmitted through a hydrophobic material (e.g., stearylhexyl disulfide (C6-SSC)). 18 The hydrophobic interactions between the DNA and RNA molecules form self-assembled micellar repressive RNA (SAMiRNA) nanoparticle-type siRNA. In some embodiments, the DNA-RNA heteroduplexes in the composition do not form nanoparticle-type siRNA. In some embodiments, some, but not all, of the DNA-RNA heteroduplexes in the composition form SAMiRNA.

[0091] In some embodiments, DNA-RNA heteroduplexes form nanoparticles. In some embodiments, the nanoparticles have an average diameter between 50 nm and 200 nm. In some embodiments, the nanoparticles have an average diameter between 50 nm and 150 nm, 75 nm and 125 nm, 80 nm and 120 nm, 90 nm and 110 nm, or 100 nm and 110 nm.

[0092] In some embodiments, the composition is administered directly to the subject. In some embodiments, the composition is diluted before administration to the subject. 6.4. Reagent Kit

[0093] In one aspect, this disclosure provides a kit for hair improvement. In some embodiments, the kit comprises a container and a composition of the DNA-RNA heteroduplex disclosed herein. In some embodiments, the container contains a unit dose of the DNA-RNA heteroduplex composition. In some embodiments, the container contains multiple unit doses of the DNA-RNA heteroduplex composition.

[0094] In some embodiments, the container includes a dispenser for delivering the composition. In some embodiments, the container includes a pump dispenser for delivering the composition. In some embodiments, the container includes a pump dispenser for delivering the composition. In some embodiments, the container includes a piston for delivering a unit dose of the composition. In some embodiments, the container is configured to deliver a unit dose of the composition at a time.

[0095] In some embodiments, the container contains a tool for massage. In some embodiments, the massage tool is made of silicone. In some embodiments, the tool is configured to massage the scalp. 6.5. Instructions for Use

[0096] In one aspect, this disclosure provides a method of using a DNA-RNA heteroduplex or a composition thereof. It can be used to inhibit the expression of AR mRNA. In some embodiments, the method includes the step of applying a DNA-RNA heteroduplex or a composition thereof to locally or systemically inhibit AR mRNA expression. In some embodiments, the DNA-RNA heteroduplex or a composition thereof is applied topically to the skin. In some embodiments, the DNA-RNA heteroduplex or a composition thereof is applied topically.

[0097] In some embodiments, a DNA-RNA heteroduplex or a combination thereof is administered to a subject suffering from symptoms associated with AR mRNA expression. In some embodiments, a DNA-RNA heteroduplex or a combination thereof is administered to a subject to treat a disease associated with AR mRNA expression. In some embodiments, the subject is a human or an animal.

[0098] In some embodiments, this disclosure provides a method for improving hair growth, the method comprising applying the composition disclosed herein to the scalp of a subject. In some embodiments, the scalp is massaged after application of the composition. In some embodiments, the scalp is massaged for one, two, three, four, five, six, seven, eight, nine, or ten minutes.

[0099] The composition may be applied once a week, once every two weeks, or once every four weeks. In some embodiments, the composition may be applied once a month, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once every twelve months.

[0100] In some embodiments, the composition is applied once weekly for at least four months. In some embodiments, the composition is applied once weekly for at least five months. In some embodiments, the composition is applied once weekly for at least six months. In some embodiments, the composition is applied once weekly for at least seven months. In some embodiments, the composition is applied once weekly for at least eight months. In some embodiments, the composition is applied once weekly for at least nine months. In some embodiments, the composition is applied once weekly for at least ten months.

[0101] In some embodiments, the composition is applied for two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve months. In some embodiments, the composition is applied for more than one year.

[0102] In some embodiments, the composition is applied once weekly for four months. In some embodiments, the composition is applied once weekly for five months. In some embodiments, the composition is applied once weekly for six months. In some embodiments, the composition is applied once weekly for seven months.

[0103] In some embodiments, the composition is used at a lower frequency after its maximum effect has been achieved.

[0104] In some embodiments, the composition is applied once weekly for four months, and then less than once weekly thereafter. In some embodiments, the composition is applied once weekly for four months, and then once every two weeks, three weeks, or four weeks thereafter.

[0105] In some embodiments, the composition is applied once a week for four months, and then less than once a week thereafter. In some embodiments, the composition is applied once a week for four months, and then applied once a month, once every two months, once every three months, once every four months, once every five months, once every six months, or less thereafter.

[0106] In some implementations, the composition is applied once a week for four months, and then application is discontinued after four months.

[0107] In some implementations, the subject has hair loss. In some implementations, the subject has moderate androgenetic alopecia. In some implementations, the subject has hair loss with a Norwood scale rating of at least III or higher. In some implementations, the subject has hair loss with a Ludwig scale rating of at least I-3 or higher.

[0108] In some implementations, the subjects are male. In some implementations, the subjects are female.

[0109] In some embodiments, the composition administered to the subject comprises 1-50 mg / ml of a DNA-RNA heteroduplex. In some embodiments, the composition administered to the subject comprises 1-40 mg / ml of a DNA-RNA heteroduplex. In some embodiments, the composition administered to the subject comprises 1-30 mg / ml of a DNA-RNA heteroduplex. In some embodiments, the composition administered to the subject comprises 1-20 mg / ml of a DNA-RNA heteroduplex. In some embodiments, the composition administered to the subject comprises 2.5-10 mg / ml of a DNA-RNA heteroduplex. In some embodiments, the composition administered to the subject comprises 4 mg / ml, 4.5 mg / ml, 5 mg / ml, 5.5 mg / ml, or 6 mg / ml of a DNA-RNA heteroduplex. In some embodiments, the composition administered to the subject comprises 4.5 mg / ml, 5 mg / ml, or 5.5 mg / ml of a DNA-RNA heteroduplex.

[0110] In some embodiments, the method includes administering 0.1-10 ml of DNA-RNA heteroduplex more than once per week. In some embodiments, the method includes administering 0.1-7.5 ml of DNA-RNA heteroduplex more than once per week. In some embodiments, the method includes administering 0.5-5 ml of DNA-RNA heteroduplex more than once per week. In some embodiments, the method includes administering 0.5-3 ml of DNA-RNA heteroduplex more than once per week. In some embodiments, the method includes administering 0.5-2 ml of DNA-RNA heteroduplex more than once per week. In some embodiments, the method includes administering 0.5-1.5 ml of DNA-RNA heteroduplex more than once per week. In some embodiments, the method includes administering 1-1.5 ml of DNA-RNA heteroduplex more than once per week.

[0111] In some embodiments, the method includes administering 1-100 mg of DNA-RNA heteroduplex more than once a week. In some embodiments, the method includes administering 1-100 mg of DNA-RNA heteroduplex once a week. In some embodiments, the method includes administering 1-100 mg of DNA-RNA heteroduplex less than once a week.

[0112] In some embodiments, the method includes administering 1-100 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes administering 1-50 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes administering 1-40 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes administering 1-30 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes administering 1-20 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes administering 1-15 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes administering 1-10 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes administering 1-5 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes administering 2-10 mg of DNA-RNA heteroduplex once weekly. In some implementations, the method includes administering 2.5–7.5 mg of DNA-RNA heteroduplex once a week.

[0113] In some embodiments, the method includes administering 1-100 mg of DNA or RNA once weekly. In some embodiments, the method includes administering 1-50 mg of DNA or RNA once weekly. In some embodiments, the method includes administering 1-25 mg of DNA or RNA once weekly. In some embodiments, the method includes administering 1-15 mg of DNA or RNA once weekly.

[0114] In some implementations, the method includes per scalp area (cm²) 2 ) Apply 1-50 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes per scalp area (cm²) 2 ) Administer 1-40 mg of DNA-RNA heteroduplex once a week.

[0115] In some implementations, the method includes per scalp area (cm²) 2 ) Administer 1-200 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes per scalp area (cm²) 2 ) Administer 1-150 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes per scalp area (cm²) 2 ) Administer 1-100 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes per scalp area (cm²) 2 ) Apply 1-50 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes per scalp area (cm²) 2 ) Administer 1-30 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes per scalp area (cm²) 2 ) Administer 5-20 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes per scalp area (cm²) 2 ) Administer 6-15 mg of DNA-RNA heteroduplex once weekly. In some embodiments, the method includes per scalp area (cm²) 2 ) Administer 8-10 mg of DNA-RNA heteroduplex once a week.

[0116] In some implementations, the method includes per scalp area (cm²) 2 Apply 0.5-5 ml of DNA-RNA heteroduplex once a week. In some embodiments, the method includes applying per scalp area (cm²) 2) Apply 1-2 ml of DNA-RNA heteroduplex once a week. In some embodiments, the method includes applying per scalp area (cm²) 2 Apply 1-1.5 ml of DNA-RNA heteroduplex once a week. 7. Example 7.1. Screening for effective SAMiRNAs for AR silencing

[0117] As shown in Figure 1A, SAMiRNA nanoparticles consist of a hydrophilic polymer, polyethylene glycol (PEG), and a hydrophobic hydrocarbon conjugate at each end of an unmodified DNA / RNA heteroduplex. For effective SAMiRNAs specifically targeting AR, SAMiRNAs containing 19 base pairs of DNA / RNA heteroduplexes were designed using a sliding window algorithm, with selection based on specificity for AR mRNA. A total of 547 SAMiRNA candidate conjugated sense and antisense strands were synthesized, purified, and annealed to form nanoparticles. The AR silencing effect of each SAMiRNA candidate in LNCap cells was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Fourteen SAMiRNA candidates were selected based on their knockdown efficiency (>50% AR silencing efficacy) (Figures 1B and 2). Furthermore, we validated the reduction in AR protein levels in LNCap cells treated with the fourteen SAMiRNA candidates (Figure 1C). AR68 and AR109 were found to be the most effective SAMiRNAs. AR68 has SEQ ID NO: 3 as a meaningful chain, and AR109 has SEQ ID NO: 5 as a meaningful chain.

[0118] To confirm its efficacy in human hair cells, human follicular dermal papilla (HFDP) cells were treated with AR68 and AR109. AR68 and AR109 significantly reduced AR mRNA and protein levels (Figures 3A and 3B). As shown in Figure 2B, in HFDP cells, AR68 reduced AR protein levels more effectively than AR109, confirming that AR68 is a more effective siRNA sequence for AR silencing. Treatment of HFDP cells with AR68 reduced AR mRNA expression in a dose-dependent manner (Figure 3C), and the inhibitory effect on AR protein was confirmed by enzyme-linked immunosorbent assay (ELISA) (Figure 3D). 7.2. SAMiRNA-AR68 reduced AR mRNA and protein levels in human hair follicles.

[0119] To evaluate the delivery of SAMiRNA nanoparticles to human hair follicles, fluorescein (FAM)-labeled AR68 was applied to plucked hairs, and the samples were visualized by confocal microscopy. Plucking hairs typically lack dermal papillae compared to microscopically dissected hair follicles. We plucked multiple hairs and selected those with hair bulbs. FAM-labeled AR68 was added to the plucked hairs with dermal papillae. As expected, FAM-labeled AR68 was efficiently delivered to the outer root sheath (ORS) and the dermal papillae of the hair bulb (Fig. 4A). AR silencing in the hair follicle was confirmed by RT-qPCR analysis (Fig. 4B). Immunofluorescence analysis of AR68-treated plucked hairs showed a significant reduction in AR protein expression (Fig. 4C). AR protein is highly expressed in the dermal papillae and transactivates AR by binding to DHT in dermal papilla cells, inducing apoptosis that promotes AGA progression. Therefore, we demonstrated that 10 µM AR68 nanoparticles can be delivered to and silence AR in the dermal papilla cells of hair follicles. Clinical studies on the treatment of AGA investigated the efficacy of higher concentrations of AR68. 7.3. Safety of SAMiRNA-AR68 nanoparticles

[0120] One of the limitations in developing RNAi therapeutics for clinical applications is cytotoxicity and systemic toxicity, including innate immune stimulation. To evaluate the cytotoxicity of AR68, cell viability was measured in HFDP and HaCaT cells at an effective concentration (10 μM). Neither HFDP nor HaCaT cells showed cytotoxicity up to 20 μM AR68 (Fig. 5A). The study investigated whether AR68 induces nonspecific innate immune stimulation in human PBMCs. Treatment with AR68 at concentrations up to 10 μM was performed, and pro-inflammatory cytokines associated with TLR3 signaling were analyzed after 6 h. Compared to an unstimulated negative control, AR68 did not induce pro-inflammatory cytokines in PBMCs, including interleukin (IL)-1β, IL-6, interferon-γ (INF-γ), and tumor necrosis factor-α (TNF-α); as a positive control, concanavalin A significantly induced pro-inflammatory cytokines (Fig. 5B). In summary, these results demonstrate that 10 µM AR68 does not induce cytotoxicity or innate immune stimulation, and these results guide the determination of the effective dose of AR68 for clinical use. 7.4. Stability of SAMiRNA-AR68 nanoparticles

[0121] The storage stability of the SAMiRNA solution was tested at different temperatures. The long-term stability of the AR68 nanoparticles was evaluated by monitoring the nanoparticle size of SAMiRNA using qNano Gold. The average size of the AR68 nanoparticles was approximately 99.2 ± 5.1 nm (22ºC, 55% ± 5% RH) and 105.0 ± 2.5 nm (40ºC, 75% ± 5% RH). The AR68 formulation remained stable for 6 months (Figure 6A). Furthermore, AR knockdown was tested by RT-qPCR using AR68 samples stored for 6 months under indicated conditions. As expected, it showed similar AR mRNA inhibitory activity in HFDPC (Figure 6B). 7.5. Clinical Study I: Treatment with low-dose SAMiRNA-AR68 (0.5 mg / ml) three times weekly General characteristics of the subjects

[0122] A total of 48 male and female participants diagnosed with moderate androgenetic alopecia were recruited and randomly assigned to either the low-dose AR68 treatment group (test group) (n = 24) or the placebo group (n = 24). In the low-dose AR68 treatment group, 2 out of 24 participants withdrew (withdrew their consent); therefore, 22 participants completed the study. The mean age was 42.4 ± 8.10 years, and the male-to-female ratio was 8:14. In the placebo group, 1 out of 24 participants withdrew (withdrew their consent), and 23 completed the study. The mean age in the placebo group was 42.4 ± 6.69 years, and the male-to-female ratio was 6:17. Based on group homogeneity analysis performed to confirm the effectiveness of randomization, there were no statistically significant differences between the two groups (Figure 7 and Table 1). Table 1. Demographic characteristics of subjects in Clinical Study I. Analysis of hair density and total hair count

[0123] Photographic evaluation revealed an increase in hair density in the low-dose AR68 treatment group compared to baseline (Figure 8A and Table 2a). As shown by the analysis of hair follicle imaging, the total hair count increased at 24 weeks after AR68 treatment compared to baseline (from 133.14 hairs / cm²). 2 Up to 135.41 hairs / cm 2 (p < 0.01) (Table 2b). Compared with baseline, the mean change and rate of total hair count in the AR68 0.5 mg / ml treatment group increased by 2.273 ± 3.089 and 1.870% at 24 weeks, respectively (Figure 8B and Table 3). Table 2. Descriptive statistical analysis of hair density (a) and total hair count (b) between time points in Clinical Study I. Table 3. Statistical analysis of Δ total hair count (week-to-baseline) between the SAMiRNA-AR68 0.5 mg / ml treatment group and the placebo group. Subject self-assessment questionnaire

[0124] There was no significant difference between the AR68 0.5 mg / ml treatment group and the placebo group. Security assessment

[0125] In the safety assessment, no adverse events were observed in any of the 45 subjects during the clinical study period. 7.6. Clinical Study II: Treatment with a high-dose SAMiRNA-AR68 (5 mg / ml) once weekly General characteristics of the subjects

[0126] To improve efficacy and user convenience, we designed and conducted a clinical study of once-weekly high-dose (5 mg / ml) treatment. A total of 60 male and female subjects diagnosed with moderate androgenetic alopecia participated and were randomly assigned to either the AR68 5 mg / ml treatment group (n = 30) or the placebo group (n = 30). During the study period, six subjects withdrew (withdrawal of consent or loss to follow-up), ten subjects withdrew at the investigator's discretion (perming, dyeing, etc.), and one subject withdrew due to adverse reactions; therefore, 43 subjects completed the clinical study. In the AR68 treatment group, 8 out of 30 subjects withdrew, and 22 completed the study. The mean age was 44.77 ± 10.88 years, and the male-to-female ratio was 9:13. In the placebo group, 9 out of 30 subjects withdrew, and 21 completed the study; the mean age was 46.48 ± 7.93 years, and the male-to-female ratio was 10:11. There were no statistically significant differences between the two groups, confirming randomization (Figure 9 and Table 4). Table 4. Demographic characteristics of subjects in Clinical Study II. Total hair count analysis and photographic evaluation

[0127] Compared with baseline (pre-assessment), the AR68 5 mg / ml treatment group showed a significant increase in hair density as measured by photographic assessment at 8 weeks (0.091 ± 0.294), 16 weeks (0.159 ± 0.29; p < 0.05), and 24 weeks. A significant improvement was observed at week 24 (0.250 ± 0.551; p < 0.05) (Figure 10A and Table 5a). Hair morphological analysis showed an increase in density at 16 weeks (from 182.182 hairs / cm²) compared with baseline. 2 Up to 189.727 hairs / cm 2 (p < 0.001) and at 24 weeks (from 182.182 hairs / cm²). 2 Up to 189.909 hairs / cm 2 (p < 0.001), the total hair count in the AR68 treatment group was significantly higher (Table 4b). Compared with baseline, the mean change and rate of total hair count in the AR68 5 mg / ml treatment group increased by 7.545 ± 7.896 and 4.264% at week 16 (p < 0.001), and by 7.727 ± 8.659 and 4.421% at week 24 (p < 0.001) (Figure 10B and Table 6). Table 5. Descriptive statistical analysis of hair density (a) and total hair count (b) between time points in Clinical Study II. Table 6. Statistical analysis of Δ total hair count (week-to-baseline) between the SAMiRNA-AR68 5 mg / ml treatment group and the placebo group. Subject self-assessment questionnaire

[0128] Regarding the efficacy of the product, participants in the AR68 5 mg / ml treatment group gave positive feedback in the participant self-assessment questionnaire. Participants reported "satisfaction with the sample used" at weeks 8 (72.73%), 16 (81.82%), and 24 (59.09%). At weeks 8 (36.36%), 16 (63.64%), and 24 (50.00%), participants reported "feeling thicker hair in the crown area," and hair loss decreased at weeks 8 (45.45%), 16 (54.55%), and 24 (72.73%). Security assessment

[0129] In the safety assessment, one subject in the AR68 5 mg / ml treatment group experienced erythema, edema, and itching at the test site, but the symptoms subsided after diagnosis and treatment by a dermatologist. The dermatologist's diagnosis determined that there was little correlation between the symptoms and the test substance. 7.7. Methods Cell culture and reagents

[0130] Human prostate cancer LNCaP cells and human keratinocyte HaCaT cells were purchased from the American Type Culture Collection (ATCC), and human follicular dermal papillary cells (HFDPCs) were purchased from PromoCell (C-12071). Human peripheral blood mononuclear cells (PBMCs) were obtained from Cellular Technology Limited (CTL-UP1). LNCaP cells were cultured in RPMI medium (HyClone) supplemented with 1% penicillin-streptomycin (HyClone) and 10% fetal bovine serum (FBS, HyClone). HaCaT cells were cultured in DMEM (HyClone) supplemented with 1% penicillin-streptomycin and 10% FBS. HFDPCs were maintained in follicular dermal papillary cell growth medium (PromoCell). Hairs were plucked from the posterior scalp using forceps, and hairs with visible hair bulbs were selected by microscopy. Hair follicles were cultured in DMEM / F12 (Gibco) supplemented with the following: 1% penicillin-streptomycin, 10% FBS, 10 µg / mL insulin-transferrin-selenium-X supplement (Gibco), 2.5 µg / mL amphotericin B (Gibco), 1% GlutaMAX (Gibco), 20 ng / mL fibroblast growth factor (FGF, PeproTech), 20 ng / mL epidermal growth factor (EGF, Sigma), and 10 ng / mL hydrocortisone (Tokyo Chemical Industry). The plucked human hair follicles were treated with 10 μM SAMiRNA-AR68 for 48 h, followed by qPCR and immunofluorescence analysis. The synthesis and quality control of SAMiRNA nanoparticles have been previously described. Measurement of SAMiRNA nanoparticle size

[0131] To determine the long-term stability of SAMiRNA, nanoparticle size was monitored using qNano Gold (Izon Science) according to standard operating procedures. Briefly, 35 μl of SAMiRNA-AR68 was analyzed using an NP80 nanopore (Izon Science) with parameters of 47 mm stretch, 140 nA current, and 10 mbar using the qNano Gold instrument. Calibration particles (CPC100, Izon Science) were determined under the same conditions prior to the experimental samples. Finally, particle counts (≥ 50 events per count) were determined using qNano software (Izon Control Suite version 3.3) provided by Izon Science. Reverse transcription and quantitative polymerase chain reaction (RT-qPCR)

[0132] Perform RT-qPCR according to MIQE guidelines. Use AccuPrep according to the manufacturer's instructions. ® Total RNA was extracted from cells using a universal RNA extraction kit (K-3140, Bioneer). To extract total RNA from hair follicles, SAMiRNA-AR68-treated plucked hair follicles were washed with PBS, resuspended in TRIzol® reagent (Invitrogen), homogenized using a Biomasher II® disposable micro-tissue homogenizer (Polyscience), and purified using the AccuPrep® Universal RNA Extraction Kit (K-3140, Bioneer). Accupower was used according to the manufacturer's instructions. ® RocketScript TM Total RNA (1 μg) was reverse transcribed in 20 μl of Cycle RT premix (dT20) in a reaction (K-2201, Bioneer). AccuPower was used for qPCR analysis. ® 2X GreenStar TM10 μl of 10-fold diluted cDNA was amplified using the qPCR master mix (K-6253, Bioneer). The following primer sets were used: AR, forward primer 5'-TTGTACACGTGGTCAAGTGG-3' (SEQ ID NO: 18) and reverse primer 5'-TGGAGTTGACATTGGTGAAGG-3' (SEQ ID NO: 19); RPLPO, forward primer 5'-TGCCATTGCCCCATGTGAAG-3' (SEQ ID NO: 20) and reverse primer 5'-AGCTGCACATCACTCAGGATT-3' (SEQ ID NO: 21); IL-1B, forward primer 5'-CTGAGCTCGCCAGTGAAAT-3' (SEQ ID NO: 22) and reverse primer 5'-CTGTAGTGGTGGTCGGAGA-3' (SEQ ID NO: 23); IL-6, forward primer 5'-AGATGCAATAACCACCCCTG-3' (SEQ ID NO: 19) 24) and reverse primer 5'-TGCGCAGAATGAGATGAGTT-3' (SEQ ID NO: 25); TNF, forward primer 5'-CTGTAGCCCATGTTGTAGCA-3' (SEQ ID NO: 26) and reverse primer 5'-GGTTATCTCTCAGCTCCACG-3' (SEQ ID NO: 27); IFNG, forward primer 5'-GAATGTCCAACGCAAAGCAA-3' (SEQ ID NO: 28) and reverse primer 5'-ACCTCGAAACAGCATCTGAC-3' (SEQ ID NO: 29); RPL13A, forward primer 5'-TGCCATTGCCCCATGTGAAG-3' (SEQ ID NO: 20) and reverse primer 5'-AGCTGCACATCACTCAGGATT-3' (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22) (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 25) (SEQ ID NO: 26) (SEQ ID NO: 27) (SEQ ID NO: 28) (SEQ ID NO: 29) (SEQ ID NO: 20 ...0) (SEQ ID NO: 20) (SEQ ID NO: 20) (SEQ ID NO: 20) (SEQ ID NO: 20) (SEQ ID NO: 20) (SEQ ID NO: 20) (SEQ ID NO: 20) (SEQ ID NO: 20) (SEQ ID NO: 20) (SEQ ID NO 21); IL-1B, forward primer 5'-GTGTTTGACGGCATCCCACC-3' (SEQ ID NO: 30) and reverse primer 5'-TAGGCTTCAGACGCACGACC-3' (SEQ ID NO: 31). PCR amplification was performed in 50 μl of reaction as follows: one cycle at 95ºC for 10 min; followed by 40 cycles at 95ºC for 5 s, 58ºC for 25 s, and 72ºC for 30 s; and a final extension step at 72ºC for 5 min. All experiments were performed in triplicate. The Δ-ΔCt method was used to determine the relative fold change, and all data were normalized relative to the internal control gene. Immunoblotting analysis

[0133] LNCaP and HFDP cells were collected and lysed using cell lysis buffer containing a protease inhibitor mixture (Thermo Fisher Scientific) (Cell Signaling Technology). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Bio-Rad). After blocking the membranes with 5% skim milk in Tris-buffered saline (TBS) for at least 1 h, they were incubated overnight with indicated antibodies, washed three times with TBS, and then incubated for 2 h with horseradish peroxidase-conjugated secondary antibodies. Immunoblotting was performed using the following antibodies: anti-AR (ab133273, Abcam), anti-GAPDH (2118, Cell Signaling Technology), and horseradish peroxidase-conjugated anti-rabbit (7074, Cell Signaling Technology). The intensity of the immunoblot bands for AR and GAPDH was quantified using ImageJ software, and AR protein expression levels were normalized relative to GAPDH values. Enzyme-linked immunosorbent assay (ELISA)

[0134] Perform an ELISA to detect AR protein using the Human AR ELISA Kit (LS-F4505, LSBio) according to the manufacturer's instructions. Briefly, collect HFDP cells (5 × 10⁻⁶ cells). 4 Cells were lysed using cell lysis buffer (CellSignaling Technology), and then 100 μl of cell lysate was added to each well at 37ºC for 1 h. After incubation, a biotin-conjugated detection antibody (detection reagent A) bound to the captured antigen was added. Then, a biotin-bound avidin-horseradish peroxidase conjugate (detection reagent B) was added. The TMB substrate reacted with HRP enzyme, resulting in color development. The color development reaction was terminated with sulfuric acid solution (stop solution), and the optical density (OD) of each well was measured at 450 nm. The OD of the sample was calculated using an OD standard curve generated using known standard AR concentrations (0.313–20 ng / ml) to determine its AR concentration. Incubation and washing steps were performed according to the supplier's manual. The absorbance at 450 nm was monitored using a FLUOstar Omega microplate reader (BMG Labtech). Cell viability assay

[0135] To perform cell viability assays, 4 × 10⁻⁶ cells were used. 3HaCaT and HFDP cells were seeded in 96-well plates at a cell / well density and then treated with indicated conditions for 96 h. After incubation, 10 μl of WST (water-soluble tetrazolium salt) reagent (EZ-Cytox, DoGen) was added to each well at 37ºC for 30 min. Absorbance at 450 nm was monitored using a FLUOstar Omega microplate reader (BMG Labtech). Immunofluorescence (IF)

[0136] Hair follicles were plucked from healthy donors and collected from the posterior scalp. After incubation, the follicles were washed three times for 5 min each with 0.05% Tween-20 (Sigma) in PBS and permeabilized for 1 min with 0.1% Triton X-100 (Sigma) in PBS. The follicles were fixed in 4% formaldehyde (Sigma) in PBS for 20 min, placed on a freezing tray, and covered with an OCT compound (Tissue-Tek). The mold was slowly placed in liquid nitrogen until the entire tissue block was completely frozen. The frozen tissue block was sectioned at 10 µm using a cryostat (Leica Biosystems). The follicle sections were incubated overnight at 4ºC with an anti-AR antibody (ab133273, Abcam). The sections were then incubated at room temperature for 1 h with an anti-rabbit Fluor 568 (A11011, Invitrogen) secondary antibody. After at least three washes, sections were counterstained with DAPI (D9542, Sigma-Aldrich) and mounted in Immu-Mount solution (Thermo Fisher Scientific). All incubations were performed in a dark, humid environment. Fluorescence signals were visualized using a confocal microscope (LSM880, Carl Zeiss) at excitation wavelengths of 568 nm (Alexa Fluor 568) and 405 nm (DAPI). At least three fields of view were analyzed for each section. ZEN software (Zeiss) was used to analyze images of the desired areas to measure intensity pixel-by-pixel and automatically calculate the average intensity. Statistical analysis

[0137] All data are expressed as mean ± standard deviation (SD), and the sample size is indicated in each legend. Statistical significance of differences was assessed using a two-tailed Student's t-test. Results shown represent at least three independent experiments. Significance is expressed as *p < 0.05, **p < 0.01, and ***p < 0.001. Clinical Study I: Treatment with low-dose SAMiRNA-AR68 (0.5 mg / ml) three times weekly

[0138] Clinical Study I was a double-blind, randomized, placebo-controlled study conducted in accordance with the guidelines of the Korean Ministry of Food and Drug Safety (MFDS) regarding studies on the efficacy of cosmetics in alleviating hair loss (July 2018). The study was approved by the Ethics Review Committee of the Seowon Skin Research Center (Local Committee) and commenced on September 10, 2020 (IRB No. 1040820-202009-HR004-02), and all experimental protocols were followed in accordance with relevant guidelines and regulations. Prior to participation, all participants provided written informed consent for the publication of identifiable information / images in an online open-access publication. A summary of the study is available at CRIS registry number KCT0005501: https: / / cris.nih.go.kr / cris / search / detailSearch.do / 20301. Participants were selected based on inclusion and exclusion criteria and consisted of 48 Korean men and women (aged 22 to 53 years). Based on the Basic and Specific Types (BASP) classification, male subjects were diagnosed with a basic type exceeding the M1, C1, and U1 range and a specific type exceeding the V1 and F1 range. 49 Female participants were diagnosed according to the BASP and Ludwig classifications, with scores exceeding F1 and L1, respectively. Exclusion criteria included participation in a previous study within the past 6 months, prior surgical correction of scalp alopecia, use of topical minoxidil or finasteride within the past 6 months, and other scalp skin conditions, including severe seborrheic dermatitis, psoriasis, lichen planus, or other scalp infections. Changes in hairstyle and hair dyeing were not permitted during the study period. Clinical Study II: Weekly high-dose SAMiRNA-AR68 (5 mg / ml) treatment

[0139] The second clinical study was conducted as a double-blind, randomized, placebo-controlled trial in accordance with the Korean MFDS guidelines for studies on the efficacy of cosmetics in alleviating hair loss (July 2018). A summary of the study details is available at the following link: https: / / cris.nih.go.kr / cris / search / detailSearch.do / 20367 (CRIS registry number KCT0005618). Clinical Study II was approved by the Ethics Review Committee of the Ellead Skin Research Center (Local Committee) and commenced on September 7, 2020 (IRB number 200804T001), with all experimental protocols conducted in accordance with relevant guidelines and regulations. Prior to participation, all participants provided written informed consent for the publication of identifiable information / images in an online open-access publication. Participants were selected based on inclusion and exclusion criteria and consisted of 60 Korean men and women (aged 22 to 54 years). The high-dose AR68 formulation (5 mg / ml) was the same as that used in Clinical Study I, and the product was applied once weekly after shampooing in a randomized, blinded manner. Hair density and total hair count were measured and analyzed using the same protocol as described for Clinical Study I. Total hair count was measured using the Folliscope® 5.0 hair imaging system (LeadM). Statistical analysis was also performed using the same procedures (i.e., SPSS statistics version 26.0 (IBM)) as in Clinical Study I. SAMiRNA-AR68 formulations and treatments

[0140] SAMiRNA-AR68 was formulated as an aqueous hair conditioner containing ethanol (15%, v / v), nicotinamide (1%, w / v), betaine (1%, w / v), biotin (0.02%, w / v), and a buffer solution. The AR68 hair conditioner was packaged in a piston-type bottle with a silicone adapter for massage (Figure 11). A placebo was prepared in the same manner as the formulation, except without AR68, and packaged in the same bottle. In Clinical Study I, the randomly selected product (AR68, 0.5 mg / ml) was used three times a week after shampooing, with scalp massaged for 5 minutes after application. In Clinical Study II, the randomly selected product (AR68, 5 mg / ml) was used once a week after shampooing, with scalp massaged for 5 minutes after application. Product use and adherence were monitored for each subject at each scheduled visit (weeks 8, 16, and 24) by reviewing subject diaries and weighing returned test products. Measurement of hair density and total hair count

[0141] All participants had their heads placed in a hair photography device (Canfield Scientific) and photographs taken using an EOSRebel T6i digital camera (Canon) at a fixed distance, angle, and lighting. Overall evaluation was performed by comparing clinical images at baseline with those taken at 8, 16, and 24 weeks after treatment with the test product. Qualitative assessment of clinical images was conducted using a 7-point scale (-3, significant decrease; -2, moderate decrease; -1, slight decrease; 0, no change; +1, slight increase; +2, moderate increase; and +3, significant increase).

[0142] To evaluate total hair count, an evaluation area (1 cm) was selected in the area of ​​hair loss (frontal hairline or crown). 2 After applying the red dot tattoo, trim the hair to approximately 2 mm. Use Folliscope at baseline and at 16 and 24 weeks post-treatment. ® 2.8 Hair imaging system (14x magnification, LeadM) was used to assess the total hair count. Total hair count (number / cm²) 2 This is calculated as the number of hairs in a region. Subject self-assessment questionnaire

[0143] Self-assessment questionnaires regarding efficacy and safety were collected from participants at 8, 16, and 24 weeks after product use. Security assessment

[0144] The dermatologist observes and assesses the occurrence of objective irritants (such as edema, itching, and burning) in the subjects. Detailed information, including the start and end dates of occurrence, severity, treatment, and causal relationship with the test product, is recorded in a form. If any adverse reactions occur, the dermatologist further assesses the adverse reactions according to standard operating procedures for adverse reactions. Statistical analysis

[0145] Data is presented as the mean and the rate of change between the baseline value and the value obtained at each time point. SPSS is used. ® All statistical analyses were performed using a software package (IBM). For photographic assessment of hair density, the Wilcoxon signed-rank test and the Mann-Whitney U test were used to perform statistical analysis on variables comparing time points or groups (p < 0.05). To analyze total hair counts, paired t-tests were used to calculate the significance of differences between time points, where p < 0.05 was considered significant. 7.8. Clinical Study III: A single-center, randomized, double-blind, placebo-controlled clinical trial of high-dose SAMiRNA-AR68 (5 mg / ml) (with a total timeframe of 6 months, including an interim analysis after 4 months). General characteristics of the subjects

[0146] Based on the inclusion and exclusion criteria, the study included 40 female subjects aged 23-72 years and 80 male subjects.

[0147] Selection criteria: - 18 years and older - Women and men's health volunteers - Skin type: Any - Hair loss (alopecia), defined as at least grade III or higher on the Norwood scale, or at least grade I-3 or higher on the Ludwig scale. - Written informed consent from the subject or legal guardian can be obtained.

[0148] Exclusion criteria: - Severe or chronic skin inflammation - Serious medical or chronic illness - Taking medications that may interfere with skin reactions (corticosteroids, antihistamines, topical immunomodulators, etc.) - Apply products and care products containing active ingredients 7-10 days before the start of the test. - Any history of severe allergic reactions or serious side effects from cosmetic preparations - Sunbathing or using a solarium during the research period - Known neoplastic diseases - Pregnancy and breastfeeding

[0149] The demographic characteristics of the participants are summarized in Figure 12. A total of 115 male and female participants were randomly assigned to either the SRN-021 treatment group (test group) or the placebo group, as provided in Table 7 below. The test group was further divided into three subgroups—subjects in the first subgroup (28 participants) received SRN-021 (5 mg / ml) weekly, subjects in the second subgroup (28 participants) received SRN-021 (5 mg / ml) every two weeks, and subjects in the third subgroup (29 participants) received SRN-021 (5 mg / ml) monthly. The test product conditioner was applied to areas of the scalp where hair was less dense. Analysis of hair density and total hair count

[0150] The TrichoScan HD system (DermoScan GmbH, Germany) with its dedicated camera and software (version 4.0.10.102) was used to evaluate hair density (terminal and vellus hairs) and the state of individual hairs involved in the anagen or telogen phase in all analyses. Furthermore, hair thickness was determined using the TrichoScan system and, in addition to being expressed as the average of all analyzed hairs, as the median value. The scalp area measured by the TrichoScan HD system in a single session was fixed at 0.59 cm², corresponding to a circle with a diameter of 0.87 cm. The analysis areas on the scalp underwent the following preprocessing:

[0151] On each subject's scalp, a 2 cm diameter area was located in the transition zone between the balding area and the normal, multi-haired scalp using a dot mask. Hair was passed through the holes in the mask and roughly trimmed with a pair of scissors. The trimmed hair was then shaved to a uniform length of 0.8 mm using a Moser TrichoScan Edition razor. For this purpose, the razor was moved at a 90° angle relative to the scalp without pressure to obtain a 2 cm diameter shaved area.

[0152] Three days after shaving, the hair dye (Goldwell Topchic 2N) was applied to the shortened hair using a wooden spatula. Specifically, an equal amount of developer (oxidizing paste) was added to the hair dye (1:1 mixture). The hair dye and developer were thoroughly mixed until they reached a paste-like consistency. The dye mixture was applied to the shaved area of ​​the subject's scalp and left there for 15 minutes. After this incubation period, the dye mixture was roughly removed with a swab, and the shaved area was very carefully cleaned with an alcohol tincture (e.g., Kodan spray) and a soft swab.

[0153] The camera of the TrichoScan HD system was used to record image files at the planned analysis time. The scalp was thoroughly wetted (Kodan spray), and then images of the prepared area were recorded. Note that images without air bubbles and without surrounding hair were recorded. Using this procedure, the terminal hair and vellus hair density [1 / cm²], as well as the percentage of hair in the anagen (growth) phase and the percentage of hair in the telogen (resting) phase, were quantified using TrichoScan HD software. Additionally, the percentages of terminal hair and vellus hair [%] and hair thickness [μm] were determined. In this manner, an average of 8 records (hair maps) were prepared for each subject at each analysis time.

[0154] The amount of hair shed was determined as follows: After shampooing, subjects vigorously combed their wet hair with a provided standardized comb while placing their heads above a white DIN A4 sheet of paper. This caused hair to fall onto the paper. The number of shed hairs was counted by a qualified research nurse. This procedure was performed at the scheduled analysis time.

[0155] In addition to hair loss, the following standards were measured over a total period of 6 months (including an intermediate analysis after 4 months): hair in the growth phase (%), terminal hair density [1 / cm²], vellus hair density [1 / cm²], and hair thickness [µm]. Skin examination results

[0156] Skin examination was performed according to clinical skin evaluation criteria. All subjects exhibited healthy skin in the test areas before, during, and after the application period. No pathological skin lesions were found. Under no circumstances was there any interruption or reduction of product application at the discretion of a dermatologist. No treatment by a dermatologist was required. The product SAMiRNA-AR68 was very well tolerated and did not induce any skin-related changes in any subjects. Total hair count analysis and photographic evaluation

[0157] Trichoscan analysis was performed on a prepared area of ​​each subject's scalp at the indicated time. The camera head was positioned at eight different locations within the prepared scalp area, and the records were evaluated using Trichoscan HD software to determine the percentage of hair in the anagen phase. The mean of all repeated records for each subject was analyzed.

[0158] Before treatment (start) and after 6 months of treatment (6 months), the percentage (%) of hair in the anagen phase was calculated in each placebo group or treatment group (weekly, twice a month, once a month), as provided in Figure 13A. It shows that SAMiRNA-AR68 increased the percentage of hair in the anagen phase in all treatment groups after 6 months of administration. Furthermore, the change in the percentage of hair in the anagen phase after 6 months of treatment with SAMiRNA-AR68 was calculated and is presented in Figure 13B. Compared to placebo, weekly treatment with SAMiRNA-AR68 increased the percentage of hair in the anagen phase by 5%.

[0159] Further analysis was conducted on the changes in the percentage of hair in the anagen phase in men (Figure 14A) and women (Figure 14B). In men, SAMiRNA-AR68 increased the percentage of hair in the anagen phase (change in percentage) by more than 4.6% compared to placebo. In women, SAMiRNA-AR68 increased the percentage of hair in the anagen phase (change in percentage) by more than 5.6% compared to placebo.

[0160] The efficacy of SAMiRNA-AR68 was further tested by measuring the reduction in hair loss through counting the hairs that fell out after shampooing and combing with a standardized comb. The results are shown in Figure 15. The results show a significant reduction in the number of hairs lost after treatment with SAMiRNA-AR68.

[0161] Subjects treated weekly with SAMiRNA-AR68 showed a reduction in hair loss of approximately 30% after 4 months. As shown in Figure 16, at four months, SAMiRNA-AR68 resulted in an average reduction of 26% in overall hair loss compared to the placebo group, with an efficacy rate of 80%.

[0162] This effect was also compared between men and women. In men (Figure 17A), at four months, SAMiRNA-AR68 reduced hair loss by 35% compared to placebo. In women (Figure 17B), at four months, CosmeRNA reduced hair loss by 16% compared to placebo. This effect was more significant in men than in women.

[0163] Changes in hair loss were compared between different dosing frequencies (weekly (4 times / month), bi-weekly (2 times / month), and monthly (1 time / month)). Mean changes in each group were measured 4 months and 6 months after the start of SAMiRNA-AR68 treatment. As shown in Figure 18, weekly or bi-weekly (2 times / month) administration up to 4 months of treatment resulted in a net hair increase of 12–13%. Monthly administration up to 6 months achieved comparable net hair increase. In the monthly SAMiRNA-AR68 treatment group, there was a significant difference in effect between 4 and 6 months. However, in the weekly or bi-weekly SAMiRNA-AR68 treatment group, there was no significant difference between 4 and 6 months. (Figure 18 and Table 8)

[0164] Trichoscan technology was used to study terminal hair density as another way to test the efficacy of SAMiRNA-AR68. Figures 19A and 19B show the density of terminal hair at different dosing frequencies (weekly (4 times / month), bi-weekly (2 times / month), and monthly (1 time / month)) before or 4 or 6 months after SAMiRNA-AR68 treatment, with a density of 1 / cm. 2 (Figure 19A) or % (Figure 19B) measurement of terminal hair density. Data showed that the density of terminal hair in the treatment group tended to increase compared to the placebo group.

[0165] The hair density and hair bundle thickness in each group were also measured, and the results are shown in Figures 20 and 21, respectively.

[0166] The hair-improving effect of SAMiRNA-AR68 was significant, with visible improvements observed after 6 months of use (Figure 22). No adverse skin reactions were observed throughout the study. 7.9. Stability of SAMiRNA-AR68 formulation

[0167] SAMiRNA-AR68 was formulated as an aqueous hair conditioner with the following composition (Table 9). The formulation was used in Clinical Study III to assess and evaluate its stability. The formulation was a colorless or pale yellow transparent liquid. The pH was 5.12 and ranged from pH 4.10 to 6.10. The difference in retention time between the main peak obtained from the test solution of phosphate-buffered saline (and) stearylhexyl disulfide sh-DNA-2 PEG-45 / sh-RNA-1 (SAMiRNA-AR68) and the main peak obtained from the standard solution was within ± 2.5%. The particle size (mean) was 109 nm and within the specification requirement of < 200 nm.

[0168] To evaluate chemical and physical stability, stability tests were conducted using the formulation. Results (pH, weight loss, and total bacterial count) showed that the product was stable after 3 months of storage (40ºC, 75% RH). Empirically, "a period of one month storing a sample at 40ºC corresponds to an actual shelf life of 10–12 months at room temperature. This suggests that, for a minimum expected shelf life of 30 months, a period of 3 months at 40ºC is typically, or necessarily, necessary." (From: Praxishandbuch Kosmetische Mittel 19 10 29, II.7 Mikrobiologie kosmetischer Mittel, S. 12). 7.10. DNA-RNA heteroduplexes modified with phosphate thioester

[0169] Several variants of SAMiRNA-AR68 (#3480, #3492, #3493, #3494, #3495) were generated by altering the DNA / RNA strand sequence. Furthermore, the variants were further modified using phosphate-thioester nucleotides during DNA / RNA strand synthesis. The knockdown efficacy of each variant with and without phosphate-thioester modification in human follicular dermal papilla cells was tested. Human follicular dermal papilla (HFPD) cells were treated with SAMiRNA for 72 h. Total RNA extracts were analyzed by RT-qPCR, and ARmRNA expression levels were normalized relative to RPLPO gene expression levels. The results are presented in Figure 23. Numerical data are also presented in a table (Figure 23).

[0170] Overall, the results showed that SAMiRNAs modified with phosphate thioesters exhibited stronger inhibitory effects on AR mRNA expression. Furthermore, the assays allowed for the identification of AR-68 variants (e.g., #3494 and #3495) that showed stronger inhibitory effects on AR mRNA expression than other variants. The half-maximal inhibitory concentrations (IC50) of the variants were further tested and analyzed. The data presented in Figure 24 show that #3494 (SRN-021) had a significantly greater inhibitory effect on AR expression (approximately 30-fold) compared to SAMiRNA AR-68. The IC50 of #3494 (SRN-021) was 40 nM, while that of SAMiRNA-AR68 was 1163 nM.

[0171] Variant #3494 (SRN-021) and SAMiRNA-AR68 have several differences summarized in Table 10 below. SRN-021 contains a modified RNA strand (UUGGAGCCAUCCAAAC*U*C*U, SEQ ID NO: 32) with a phosphate-thioester modified sequence of SEQ ID NO: 2, wherein each nucleotide in UCU at the 3' end of SEQ ID NO: 2 is a phosphate-thioester nucleotide. Additionally, SAMiRNA-SRN-021 differs from SAMiRNA-AR68 in having hexaethylene glycol (HEG) covalently attached to the 3' end of the DNA strand. 7.11. SAMiRNA-SRN-021 reduces AR mRNA and protein levels in mouse skin.

[0172] To evaluate the delivery of SAMiRNA nanoparticles and the AR-inhibitory effect of SAMiRNA-SRN-021, fluorescein (Cy5)-labeled SAMiRNA-SRN-021 was administered to the skin of mice. The fluorescence image in Figure 25A shows that SAMiRNA-SRN-021 penetrates through appendages (intercellular spaces) to deliver RNAi. This indicates that SAMiRNA-SRN-021 provides efficient subcutaneous delivery of RNAi.

[0173] In addition, SAMiRNA-SRN-021 effectively knocked down AR mRNA expression in the skin. The in vivo knockdown achieved via topical delivery was approximately 50%. (Figure 25B)

[0174] SAMiRNA-SRN-021, applied topically to the skin, remained localized and was not delivered systemically. Serum from mice treated with SAMiRNA-SRN-021 was collected, and the presence of SAMiRNA was tested by RT-qPCR amplification using stem-loop primers. Serum at 10-fold serial dilutions was detected at a range of 7.99 to 23.39 Ct (cycle threshold). In the assay, 23.39 Ct is the limit of detection (LOD) of the stem-loop RT-PCR primer set for SAMiRNA-SRN-021. No SAMiRNA was detected in serum, as shown in Figure 26A. Additionally, AR mRNA expression in the kidneys was measured to test for any regulation of AR expression in distal organs. No changes in AR mRNA expression were detected in the kidneys (Figure 26B), indicating no systemic exposure to SAMiRNA-SRN-021 following topical application. 8. Sequence List 9. Equivalent schemes and incorporation by reference

[0175] Although the invention has been specifically shown and described with reference to preferred embodiments and various alternative embodiments, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

[0176] All references, authorized patents and patent applications cited in the text of this specification are incorporated herein by reference in their entirety for all purposes.

Claims

1. A DNA-RNA heteroduplex, said DNA-RNA heteroduplex comprising: a. A DNA strand having the sequence SEQ ID NO: 1 or 3; b. An RNA strand having a sequence of SEQ ID NO: 2 or 4, or a modified RNA strand containing one or more modified sequences of SEQ ID NO: 2 or 4; c. Stearylhexyl disulfide (C6-SSC) covalently attached to the 5' end of the DNA strand 18 );and d. Hexaethylene glycol (HEG) or polyethylene glycol (PEG) covalently attached to the 3' end of the DNA strand. The DNA strand and the RNA strand or the modified RNA strand form a DNA-RNA hybrid.

2. The DNA-RNA heteroduplex according to claim 1, wherein the DNA strand has the sequence of SEQ ID NO: 1 and the RNA strand has the sequence of SEQ ID NO:

2.

3. The DNA-RNA heteroduplex according to claim 2, wherein the DNA strand has the sequence of SEQ ID NO: 3 and the RNA strand has the sequence of SEQ ID NO:

4.

4. The DNA-RNA heteroduplex according to any one of claims 1-3, wherein the DNA-RNA heteroduplex comprises HEG, wherein the HEG is hexaethylene glycol-(-PO3-hexaethylene glycol)3.

5. The DNA-RNA heteroduplex according to any one of claims 1-4, wherein the DNA-RNA heteroduplex comprises the modified RNA strand, wherein the modified RNA strand comprises a sequence having one or more phosphate thioester modified SEQ ID NO: 2 or 4.

6. The DNA-RNA heteroduplex according to claim 5, wherein the modified RNA strand comprises the sequence of SEQ ID NO: 2, and each nucleotide in the UCU at the 3' end of SEQ ID NO: 2 is a phosphate thioester nucleotide.

7. A composition for hair improvement, said composition comprising a DNA-RNA heteroduplex according to any one of claims 1-6.

8. The composition according to claim 7, wherein the composition further comprises one or more humectants selected from betaine and butylene glycol.

9. The composition according to claim 7 or 8, wherein the composition further comprises one or more hair conditioning or soothing ingredients selected from panthenol, aminopropanol, biotin, nicotinamide and menthol.

10. The composition according to any one of claims 7-9, wherein the composition further comprises one or more buffering components selected from citric acid and sodium citrate.

11. The composition according to any one of claims 7-10, wherein 0.1%-2% by weight of the composition is the DNA-RNA heteroduplex.

12. The composition according to claim 11, wherein 0.25%-1% by weight of the composition is the DNA-RNA heteroduplex.

13. The composition according to claim 12, wherein 0.4%-0.6% by weight of the composition is the DNA-RNA heteroduplex.

14. The composition of claim 13, wherein 0.4%, 0.45%, 0.5%, 0.55% or 0.6% of the composition by weight is the DNA-RNA heteroduplex.

15. The composition of claim 11, wherein 1%, 1.25%, 1.5%, 1.75%, or 2% of the composition by weight is the DNA-RNA heteroduplex.

16. The composition according to any one of claims 7-10, wherein 1-20 mg / ml of the composition is the DNA-RNA heteroduplex.

17. The composition according to claim 16, wherein 2.5-10 mg / ml of the composition is the DNA-RNA heteroduplex.

18. The composition of claim 17, wherein 4-6 mg / ml of the composition is the DNA-RNA heteroduplex.

19. The composition of claim 18, wherein the composition at 4 mg / ml, 4.5 mg / ml, 5 mg / ml, 5.5 mg / ml or 6 mg / ml is the DNA-RNA heteroduplex.

20. The composition of claim 16, wherein the composition at 10 mg / ml, 12.5 mg / ml, 1.5 mg / ml, 1.75 mg / ml or 2 mg / ml is the DNA-RNA heteroduplex.

21. The composition according to any one of claims 7-20, wherein the composition comprises denatured ethanol, D-panthenol, betaine, biotin, nicotinamide, citric acid, menthol crystals, trisodium citrate dihydrate, and 1,3-butanediol.

22. The composition according to any one of claims 7-21, wherein the composition comprises 30%-35% phosphate-buffered saline, 0.4%-0.6% of the DNA-RNA heteroduplex, 45%-50% water, 14%-15% ethanol, 0.19%-0.2% panthenol, 1% nicotinamide, 0.02% citric acid, 0.05% menthol, 0.03% sodium citrate, 3% butylene glycol, and less than 0.01% aminopropanol.

23. The composition according to claim 22, wherein the composition comprises 32.8% phosphate-buffered saline, 0.5%-0.6% of the DNA-RNA heteroduplex, 46.3% water, 14%-15% ethanol, 0.19%-0.2% panthenol, 1% nicotinamide, 0.02% citric acid, 0.05% menthol, 0.03% sodium citrate, 3% butylene glycol, and less than 0.01% aminopropanol.

24. The composition according to any one of claims 7-23, wherein the composition is in the form of an ointment, paste, gel, jelly, serum, aerosol spray, non-aerosol spray, foam, cream, lotion, solution or suspension formulation.

25. The composition according to any one of claims 7-24, wherein the composition has a pH between 5 and 7.

5.

26. The composition according to claim 25, wherein the composition has a pH between 5.5 and 6.5, between 6 and 6.5, or between 7 and 7.

5.

27. The composition according to claim 25, wherein the composition has a pH of 6.29 or 7.

02.

28. The composition according to any one of claims 1-27, wherein the DNA-RNA heteroduplex forms nanoparticles.

29. The composition of claim 28, wherein the nanoparticles have an average diameter between 50 nm and 200 nm.

30. The composition of claim 29, wherein the nanoparticles have an average diameter between 50 nm and 150 nm, between 75 nm and 125 nm, between 80 nm and 120 nm, between 90 nm and 110 nm, or between 100 nm and 110 nm.

31. A method for inhibiting androgen receptor expression, the method comprising administering to a subject a DNA-RNA heteroduplex according to any one of claims 1-6 or a composition according to any one of claims 7-30.

32. The method of claim 31, wherein the DNA-RNA heteroduplex or the composition is applied topically.

33. The method of claim 31 or 32, wherein the DNA-RNA heteroduplex or the composition is administered to the subject for hair improvement.

34. The method of claim 33, wherein the DNA-RNA heteroduplex or the composition is applied to the scalp of the subject.

35. The method according to any one of claims 31-34, wherein the DNA-RNA heteroduplex or the composition is applied once a week, once every two weeks, or once every four weeks.

36. The method of claim 35, wherein the DNA-RNA heteroduplex or the composition is administered for at least four months or at least six months.

37. The method of claim 35, wherein the DNA-RNA heteroduplex or the composition is applied weekly for at least four months.

38. The method of claim 35, wherein the DNA-RNA heteroduplex or the composition is applied weekly for four months.

39. The method according to any one of claims 31-38, the method comprising applying the DNA-RNA heteroduplex or the composition once a week for four months, and after said four months applying the DNA-RNA heteroduplex or the composition less than once a week.

40. The method of claim 39, wherein the method comprises applying the DNA-RNA heteroduplex or the composition once a week for four months, and after the four months applying the DNA-RNA heteroduplex or the composition once every two weeks, once every three weeks, or once every four weeks.

41. The method of claim 39, wherein the method comprises applying the DNA-RNA heteroduplex or the composition once a week for four months, and after the four months applying the composition once a month, once every two months, once every three months, once every four months, once every five months, once every six months, or less.

42. The method of claim 39, wherein the method comprises applying the DNA-RNA heteroduplex or the composition once a week for four months, and discontinuing the application of the composition after said four months.

43. The method according to any one of claims 31-42, wherein the subject suffers from hair loss.

44. The method according to any one of claims 31-42, wherein the subject suffers from moderate androgenetic alopecia.

45. The method according to any one of claims 31-44, wherein the subject suffers from at least Norwood scale grade III or more severe hair loss.

46. ​​The method according to any one of claims 31-45, wherein the subject suffers from hair loss of at least Ludwig scale grade I-3 or more.

47. The method according to any one of claims 31-46, wherein the composition administered to the subject comprises 1-20 mg / ml of the DNA-RNA heteroduplex.

48. The method of claim 47, wherein the composition administered to the subject comprises 2.5-10 mg / ml of the DNA-RNA heteroduplex.

49. The method of claim 48, wherein the composition administered to the subject comprises 4 mg / ml, 4.5 mg / ml, 5 mg / ml, 5.5 mg / ml, or 6 mg / ml of the DNA-RNA heteroduplex.

50. The method of claim 49, wherein the composition administered to the subject comprises 4.5 mg / ml, 5 mg / ml, or 5.5 mg / ml of the DNA-RNA heteroduplex.

51. The method according to any one of claims 31-50, wherein the method comprises administering 1-100 mg of the DNA-RNA heteroduplex once a week.

52. The method of claim 51, wherein the method comprises administering 1-30 mg of the DNA-RNA heteroduplex once weekly.

53. The method according to any one of claims 31-50, wherein the method comprises administering 5-10 mg of the DNA strand or the RNA strand once a week.

54. The method of claim 51, wherein the method comprises administering 1-10 mg of the DNA strand or the RNA strand once a week.

55. The method according to any one of claims 31-54, the method comprising per scalp area (cm²) 2 ) Administer 1-100 mg of the DNA-RNA heteroduplex once a week.

56. The method of claim 54, wherein the method comprises per scalp area (cm²) 2 ) Administer 1-50 mg of the DNA-RNA heteroduplex once a week.

57. The method of claim 55, wherein the method comprises per scalp area (cm²) 2 ) Administer 5-20 mg of the DNA-RNA heteroduplex once a week.

58. The method of claim 57, wherein the method comprises per scalp area (cm²) 2 ) Administer 8-10 mg of the DNA-RNA heteroduplex once a week.

59. The method according to any one of claims 31-54, the method comprising per scalp area (cm²) 2 Apply 0.5-5 ml of the DNA-RNA heteroduplex once a week.

60. The method of claim 59, wherein the method comprises per scalp area (cm²) 2 Apply 1-2 ml of the DNA-RNA heteroduplex once a week.

61. The method of claim 60, wherein the method comprises per scalp area (cm²) 2 Apply 1-1.5 ml of the DNA-RNA heteroduplex once a week.

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