WO2025119007A1 - Group of antisense oligonucleotides that specifically inhibit androgen receptor, and use thereof - Google Patents

Abstract

A group of antisense oligonucleotides that specifically inhibit an androgen receptor, and the use thereof. The antisense oligonucleotides have a length of 14-22 bases. The antisense oligonucleotides (ASO) have the following uses: (1) preparing a preparation that inhibits the expression level of an AR protein; and (2) preparing a drug or a pharmaceutical composition for treating androgen-induced alopecia; or (3) preparing a drug or a pharmaceutical composition for treating androgen-induced acne; or (4) inhibiting the expression or activation of the AR protein; and (5) treating diseases caused by the overexpression or excessive activation of the AR protein, wherein the diseases include, but are not limited to, tumors, alopecia or acne caused by the overexpression or excessive activation of the AR protein.

Description

A group of antisense oligonucleotides specifically inhibiting androgen receptor and their application Technical Field

The present invention belongs to the field of biotechnology, and specifically relates to a group of antisense oligonucleotides for specifically inhibiting androgen receptors and applications thereof.

Background Art

The epidemiological population of hair loss indications is increasing day by day, among which androgen-induced hair loss is the most common type of hair loss, with an incidence of 50% for men aged 50, a lifetime incidence of 90%, and a lifetime incidence of 50% for women. However, there are limitations such as few treatment methods, poor efficacy, and large side effects. New nucleic acid drugs are potential treatments for such skin diseases. Since existing nucleic acid delivery systems have not been successfully used to treat skin diseases such as hair loss, the development of new transdermal nucleic acid drug (even protein drugs and small molecule drugs) delivery systems may expand the methods for treating skin diseases, which is of great significance for the development of drugs for the treatment of hair loss indications.

AR (Androgen Receptor) is a nuclear receptor that exists in the cytoplasm when not activated. It forms a complex with heat shock proteins and other molecular chaperone proteins in the form of dimers. After AR binds to androgen, the complex dissociates, the AR dimer is phosphorylated, binds to other regulatory proteins, and then translocates into the cell nucleus. Subsequently, the DNA binding domain in AR binds to the DNA sequence of the androgen response element (ARE; the most typical is TGTTCT), transcribes downstream genes, and then plays its role in regulating the growth, immune and endocrine systems. What is more special is that only in the hair papilla cells of the hair follicles on the top of the head, AR can combine with testosterone and the highly active metabolite dihydrotestosterone (DHT) converted by type II 5α reductase to produce an effect, shortening the hair growth period, miniaturizing and even shrinking the hair follicles, and ultimately causing hair loss.

In recent years, the development direction of AR-targeted drugs has been small molecule inhibitors or agonists for tumor indications, and the overall competition is relatively fierce. The treatment of androgen-induced alopecia and acne with nucleic acid drugs targeting this target is still in the early stages of development, but the research enthusiasm is increasing and the development potential is relatively large.

Based on this, the present invention is proposed.

Summary of the invention

The present invention firstly relates to a group of antisense oligonucleotides (ASO) that specifically inhibit androgen receptor (AR), wherein the antisense oligonucleotides are 14-22 bases in length, the target gene is AR mRNA, and the ASO specifically pairs with a specific region of the target gene, wherein the starting site of the specific region is located at:

(1) 5'UTR region: 132-144, 164-167, 191-229, 266-298;

(2) Exon 1: 1199-1201, 1591-1610, 1892, 2209-2210, 2306, 2572-2574;

(3) Exon 2: 2777-2782;

(4) Exon 2-exon 3: 2878-2882;

(5) Exon 3: 2981-2983;

(6) Exon 4: 3113-3116;

(7) Exon 5: 3397-3401;

(8) Exon 5-Exon 6: 3432-3443;

(9) Exon 6: 3464, 3505-3506;

(10) Exon 6-Exon 7: 3563-3573;

(11) Exon 7: 3612, 3614-3616, 3618/3619, 3627, 3629, 3631, 3633;

(12) Exon 8: 3749-3759, 3818-3863;

(13) 3′UTR region: 7346-7354, 7394, 8139, 8418-8423, 10358-10363, 10386-10425;

The AR target gene is numbered: ENST00000374690.9, and the sequence is shown in SEQ ID NO.63.

Further, the ASO comprises a chemical modification;

Preferably, the modifications are: thiolation of phosphate bonds, methoxyethyl modification (2'-MOE modification) of the 2' position of the base, and 5-methyl modification of cytosine;

More preferably, the modifications are: monothiolation of the phosphate bonds of all nucleotides in the ASO sequence, 2'-MOE modification of 3 to 5 bases symmetrically at the 3' and 5' ends, and 5-methyl modification of all cytosines.

Further, the ASO is selected from the oligonucleotides shown in any sequence in the following table;

Preferably, the phosphate bonds of all nucleotides in the ASO described in the table are monothiolated, and there are symmetrical 4-5 bases at the 3' end and 5' end for MOE modification and 5-methyl modification of all cytosines.

More preferably, the motif of the ASO is 4-8-4, 4-9-4, 5-8-5, 5-10-5, or 5-12-5.

Furthermore, the present invention also relates to the following applications of the antisense oligonucleotide (ASO) or its modified form:

(1) preparing a preparation for inhibiting the expression of AR protein;

(2) preparing a medicament or pharmaceutical composition for treating androgen-induced alopecia; or

(3) preparing a drug or pharmaceutical composition for treating androgen-induced acne; or

(4) inhibiting the expression or activation of AR protein;

(5) Treating diseases caused by overexpression or overactivation of AR protein, including but not limited to: tumors, hair loss or acne caused by overexpression or overactivation of AR protein; preferably, the tumor is prostate cancer.

The medicine or pharmaceutical composition comprises a therapeutically effective amount of the antisense oligonucleotide (ASO) or a modified form thereof, and necessary pharmaceutical excipients or delivery carriers.

The present invention also relates to a pharmaceutical composition comprising the antisense oligonucleotide (ASO) or a modified form thereof, wherein the pharmaceutical composition comprises: a therapeutically effective amount of the antisense oligonucleotide (ASO) or a modified form thereof, and necessary pharmaceutical excipients or delivery carriers; preferably, the pharmaceutical composition is a transdermal administration preparation.

Furthermore, the present invention also relates to an externally applied smear-type preparation containing ASO/ASO modification, wherein the smear-type preparation comprises:

(1) the antisense oligonucleotide (ASO) or the chemically modified antisense oligonucleotide (ASO): 1-50 mg/mL; preferably 2-20 mg/mL; more preferably 4-10 mg/mL;

(2) Sodium 8-(2-hydroxybenzamido)octanoate (SNAC): 5-50 mg/mL, preferably 30 mg/mL;

(3) dimethyl isosorbide (DMI): 5-60 mg/mL, preferably 40 mg/mL;

(4) Vitamin E polyethylene glycol succinate (TPGS): 2-20 mg/mL, preferably 10 mg/mL;

(5) Vitamin C: 2-20 mg/mL, preferably 10 mg/mL.

Preferably, the chemically modified antisense oligonucleotide (ASO) in the smearable preparation is

(1) LT-0390 (ASO No. 289): T*G*C*C*A*g*t*g*a*a*c*a*t*A*C*A*T*A*HSA, which is modified as follows:

*：phosphorothioate internucleoside linkage; phosphorothioate;

A, G, C, T: 2’-O-methoxyethyl; 2’-MOE modification;

a, g, c, t: 2′-deoxy;

C base or c base are both 5-methyl modified cytosine;

HSA is a modification module: or

(2) LT-0449 (ASO No. 289): T*G*CCA*g*t*g*a*a*c*a*t*ACA*T*A*HSA, wherein the modification method is:

*: phosphorothioate internucleoside linkage;

A, G, C, T: 2’-O-methoxyethyl;

a, g, c, t: 2′-deoxy;

C base or c base are both 5-methyl modified cytosine;

HSA is a modification module: or

(3) LT-0410 (ASO No. 204): G*G*A*A*A*g*t*t*g*t*a*g*t*a*g*T*C*G*C*G*RA, wherein the modification is:

*: phosphorothioate internucleoside linkage;

A, G, C, T: 2’-O-methoxyethyl;

a, g, c, t: 2′-deoxy;

C base or c base are both 5-methyl modified cytosine;

RA is a modification module:

The present invention also relates to a method for preparing the smearable preparation, which specifically comprises the following steps:

(1) Dissolved oxygen balance: Take purified water and fill it with nitrogen at a nitrogen pressure of 0.1-0.2 MPa to balance the dissolved oxygen in the water;

(2) Dissolving the auxiliary materials and adjusting the pH value: Add SNAC, DMI, vitamin C, and TPGS to an appropriate amount of water that has been balanced with dissolved oxygen, and stir until dissolved; adjust the pH value to 7.5-9.0, and refill with nitrogen to balance the dissolved oxygen;

(3) Dissolution of the active pharmaceutical ingredient (API): Add the API (the ASO or ASO modification), dissolve it and make up to volume with oxygen-balanced water. After making up to volume, nitrogen is added again to balance the oxygen, thereby obtaining the ASO/ASO modification-containing spreadable preparation.

And an optional step (4) canning and labeling: transferring the preparation into a borosilicate vial, filling it with nitrogen, covering it with a rubber stopper and an aluminum cap, sealing it, pressing the cap and labeling it.

The present invention also relates to the following applications of the smearable preparation:

(1) preparing a drug or pharmaceutical composition for treating male pattern baldness; or

(2) preparing a drug or pharmaceutical composition for treating acne; or

(3) Treatment of hair loss or acne caused by overexpression or excessive activation of AR protein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. After cells were treated with antisense oligonucleotides (ASO), WB experiments were used to detect the inhibitory effect on AR protein expression.

Figure 2. After cells were treated with antisense oligonucleotides (ASO), WB experiments were used to detect the inhibitory effect on AR protein expression.

Figure 3. Electrophoresis detection of serum stability of ASO.

FIG4 . After cells were treated with antisense oligonucleotides (ASO), the mRNA expression of cytokines related to inflammatory response was detected.

DETAILED DESCRIPTION

Example 1: Design, synthesis and modification of ASO targeting AR target gene

AR (ENST00000374690.9) in the Ensembl database was selected as the target gene. The sequence of the target gene is shown in SEQ ID NO.63.

7650 antisense oligonucleotide sequences (ASOs) were designed for the mRNA of the AR target gene, and 349 ASO sequences were selected for synthesis and testing of in vitro efficacy. The specific locations and information of these ASOs are shown in Table 1;

Furthermore, 349 of the above ASO sequences were chemically modified in the following ways: monothiolation of the phospholipid bonds of all nucleotides in the sequence, and MOE modification of 3 to 5 bases at the 3' and 5' ends respectively (i.e., methoxyethyl modification of the 2' position of the base).

Table 1 Detailed information of ASO molecules

Example 2: Testing the activity of ASO in in vitro cell models (HaCaT cells, human immortalized epidermal cells or LNCaP human prostate cancer cells)

In this example, the inhibitory effects of some of the ASO molecules listed in Table 1 on androgen receptor (AR) expression in HaCaT cells or LNCaP cells were verified. The specific experimental process is as follows:

(1) Preparation of suspension transfection reagent: Dissolve ASO dry powder in sterile water to a concentration of 10 μM. Dilute 10 μM ASO stock solution to the required concentration with reduced serum medium for transfection (Basalmedia, L530KJ), dilute Lipofectamine 2000 transfection reagent (Invitrogen, 11668-019) with reduced serum medium, mix the transfection reagent diluent and ASO diluent to prepare ASO transfection complexes of preset concentration or concentration gradient, pipette 10 times to mix, and let stand at room temperature for 20 min.

(2) Cell treatment: Cells were plated one day before transfection at 6×10 ³ cells/well in a 96-well plate, and 100 μL of DMEM medium containing 10% FBS (HaCaT) or RPMI 1640 medium containing 10% FBS, 1% sodium pyruvate, and 1% glutamine (LNCaP) was added to each well. Before transfection, the confluence of HaCaT cells or LNCaP cells should be >70% under microscopy. The old medium was discarded and replaced with 50 μL of DMEM medium containing 10% FBS (HaCaT) or RPMI 1640 medium containing 10% FBS (LNCaP). The ASO transfection complex prepared in step (1) was added to the 96-well plate and cultured in a 37°C, 5% CO ₂ incubator. After 5 hours, the medium was replaced with new medium.

(3) 24 hours after transfection, total RNA was extracted from the cells, and the expression of AR mRNA in the cells was detected by quantitative real-time PCR. The PCR primers and probes used to amplify the internal reference gene Actin and the target gene AR are shown in Table 2:

Table 2 PCR primer and probe sequences

The relative gene expression was calculated using the 2^-ΔΔCT method (Livak method), and the inhibition rate of antisense oligonucleotide mRNA expression level was calculated according to the following equation:

Inhibition rate = (1-2^-ΔΔCT) × 100%.

The experimental groups are:

Cells treated with the modified ASOs indicated by the respective numbers;

The blank control group was cells that were not treated with any ASO.

The efficiency of the ASO sequences described in some of Table 1 in inhibiting AR mRNA in HaCaT or LNCaP cells is shown in the following Tables 3-1 to 3-4.

The results show that the ASO sequences shown in Table 1 have a significant inhibitory effect on the transcription of AR mRNA

Table 3-1 Inhibition rate of AR mRNA after antisense oligonucleotide treatment of HaCaT cells (%)



Note: Motif indicates the MOE modification of the 3' and/or 5' bases, for example,
4-8-4, means: 4 bases at the 3' end are MOE-modified, and 4 bases at the 5' end are MOE-modified;
5-10-5 means: 5 bases at the 3' end are MOE modified, and 5 bases at the 5' end are MOE modified.

Table 3-2 Inhibition rate of AR mRNA after antisense oligonucleotide treatment of HaCaT cells (%)

Table 3-3 Inhibition rate of AR mRNA after antisense oligonucleotide treatment of LNCaP cells (%)

Table 3-4 Inhibition rate of AR mRNA after antisense oligonucleotide treatment of LNCaP cells (%)

Example 3: Testing the activity of ASO in an in vitro cell model (LNCaP human prostate cancer cells)

In this example, the inhibitory effect of the ASO molecules shown in Table 1 on the AR protein level of LNCaP cells was verified. The transfection reagent was prepared as in Example 2, and the experimental steps were as follows:

(1) Use 6-well plates and use 1.5×10 ⁵ cells/well;

(2) The concentration of ASO molecules used to treat cells was 10 nM;

(3) 72 h after ASO treatment, total protein was extracted from treated LNCaP cells using RIPA lysis buffer (Beyotime, P0013B) supplemented with PMSF (phenylmethylsulfonyl fluoride, Beyotime, ST505) and protease inhibitors;

(4) Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. After blocking with 5% skim milk powder, the membrane was incubated with AR antibody (Abcam, AB133273) and Actin antibody (Abcam, ab49900) at 4°C overnight. The next day, the membrane was incubated with secondary antibody (CST, 7074S) at room temperature for 1 h. The expression of proteins was observed using a multifunctional imaging system (Tanon 5200Multi).

The effects of the ASOs described in Table 1 on inhibiting AR protein expression in LNCaP human prostate cancer cells are shown in FIG. 1 and Table 4 (only some of the results are shown).

The results show that the ASO sequences shown in Table 1 have a significant inhibitory effect on the expression level of AR protein in LNCaP cells.

Table 4. Inhibition rate of ASO on AR protein in LNCaP cells (%) (WB experiment)

Example 4. Testing the activity of antisense oligonucleotide-fatty acid construct (ASO-S1) in in vitro cell model (HaCaT cells, human immortalized epidermal cells/NCaP cells, human prostate cancer cells)

In this example, the inhibitory effect of the fatty acid chain-modified construct (ASO-S1) of the ASO molecule shown in Table 1 on the expression of the androgen receptor (AR) in HaCaT/LNCaP cells was verified. The specific experimental process is as follows:

(1) Preparation of incubation reagents: Prepare DMEM (HaCaT) or RPMI 1640 (LNCaP) culture medium containing 1% FBS and 1% insulin-transferrin-selenium-aminoethanol (GIBCO, 51500056). Dissolve ASO-S1 dry powder in sterile water to a concentration of 1 mM. Dilute 1 mM ASO-S1 stock solution with the prepared culture medium to prepare ASO-S1 incubation complexes of a preset concentration or concentration gradient, and pipette 10 times to mix.

(2) Cell treatment: Cells were plated one day before incubation, and 1.5×10 ⁵ cells/well were inoculated into 6-well plates. 2000 μL of DMEM medium containing 10% FBS (HaCaT) or RPMI 1640 medium containing 10% FBS, 1% sodium pyruvate, and 1% glutamine (LNCaP) was added to each well. Before transfection, the confluence of HaCaT cells and LNCaP cells should be >70% under microscopy. The old medium was discarded, and 2000 μL of the incubation reagent prepared in step (1) was added to each well. The cells were placed in a 37°C, 5% CO ₂ incubator for 24 h, and then replaced with complete medium.

(3) After incubation for 72 h, the total protein of the cells was extracted, and the subsequent steps were the same as those in Example 3.

The efficiency of the ASO-S1 constructs of the ASO sequences described in Table 1 in inhibiting AR protein in HaCaT/LNCaP cells is shown in Table 5 and Figure 2 (only part of the results are shown).

The results show that ASO-S1 shown in Table 1 has a significant inhibitory effect on AR protein when the incubation concentration is 10 μM.

Table 5. Inhibition rate of AR protein of HaCaT/LNCaP cells by ASO-S1 construct (%)

Example 6. Serum stability of antisense oligonucleotides (ASOs) (fetal bovine serum and rat serum)

In this example, it was verified that the ASO molecules shown in Table 1 can be stably present in serum for at least 48 hours.

(1) Serum with a concentration of 10% or 50% was prepared with sterile water. 0.3 μg of ASO solution was added to 50 μL of serum of each concentration and mixed evenly. The mixture was then incubated at 37°C for 0 h, 4 h, or 24 h.

(2) After incubation at all time points, DNA was extracted with DNA extraction solution (Solarbio, P1021), precipitated with a mixture of 100% ethanol, 3M sodium acetate, and glycogen, and dissolved with ddH ₂ O.

(3) Separate DNA by 15% TBE-UREA PAGE gel electrophoresis, stain the gel with a safe nucleic acid dye (Monad, ME20301), and observe the amount of DNA using a multifunctional imaging system (Tanon 5200Multi).

The stability of some of the ASOs described in Table 1 and the corresponding ASO-S1 constructs in serum is shown in FIG3 .

The results show that the ASOs shown in Table 1 and the corresponding ASO-S1 constructs can be stably present in serum for at least 24 hours.

Example 7: In vitro cell model (PBMC peripheral blood mononuclear cells) to detect the levels of cytokines associated with inflammatory response after treatment with ASO and the corresponding ASO-S1 construct

In this example, it was verified that after PBMC cells were treated with some ASO/ASO-S1 shown in Table 1, the levels of cytokines related to inflammatory response were significantly lower than those of Yangshen. The specific experimental process is as follows:

(1) Cell treatment: Cells were revived and plated one day before incubation. 1×10 ⁶ cells/well were plated in a 12-well plate. 100 μL of RPMI 1640 medium containing 1% FBS and 1% insulin-transferrin-selenium-aminoethanol (GIBCO, 51500056) was added to each well.

(2) Incubation: Add the corresponding volume of ASO/ASO-S1 directly to each well in (1) to a final concentration of 10 μM, or add the corresponding volume of Yangshen Poly IC (Sigma, P9582) to a final concentration of 2 μg/mL, mix well, and culture in a 37°C, 5% _CO2 incubator.

(3) After incubation for 24 h, total RNA was extracted from the cells, and the expression of each cytokine mRNA in the cells was detected by quantitative real-time PCR. The PCR primers used to amplify the internal reference gene RPL13A and the target genes IL-6 and IL-1β are shown in Table 6.

Table 6. PCR primers and probe sequences for cytokines

The results are shown in Figure 4: After treatment with the tested ASO/ASO-S1, the effect on inflammatory factors was lower.

Example 8: Testing the tumor cell killing effect of ASO-S1 using an in vitro cell model (LNCaP human prostate cancer cells)

In this example, it was verified that the construct (ASO-S1) of the ASO molecule shown in Table 1 after being modified with a fatty acid chain had a certain killing effect on prostate cancer cells LNCaP. The specific experimental process is as follows:

(1) Preparation of incubation reagents: Prepare RPMI 1640 medium containing 1% FBS and 1% insulin-transferrin-selenium-aminoethanol (GIBCO, 51500056). Dissolve ASO-S1 dry powder in sterile water to a concentration of 1 mM. Dilute 1 mM ASO-S1 stock solution with the prepared medium to prepare a concentration gradient of ASO-S1 incubation complexes. Pipet 10 times to mix.

(2) Cell treatment: Cells were plated one day before incubation, and 1×10 ³ cells/well were inoculated into a 96-well plate. 100 μL of RPMI 1640 medium containing 10% FBS, 1% sodium pyruvate, and 1% glutamine was added to each well. The state of LNCaP cells was observed under a microscope before treatment, the old medium was discarded, and 100 μL of the incubation reagent prepared in step (1) was added to each well, and the cells were cultured in a 37°C, 5% CO ₂ incubator.

(3) After incubation for 72 h, CCK-8 assay was performed.

Table 7. Growth inhibition rate of LNCaP cells by ASO-S1 (%)

The results are shown in Table 7. After the tested ASO-S1 was treated with prostate cancer cells, it also played an inhibitory effect on the growth of cancer cells.

Example 9: Preparation of external smear preparation

1. Prepare the ASO/ASO modified body smearable preparation for external use according to the prescription in Table 8

Table 8. Formulation of ASO/ASO-modified smear-type preparations

Sodium 8-(2-hydroxybenzamido)octanoate (SNAC)

Isosorbide dimethyl ether (DMI)

Vitamin E polyethylene glycol succinate (TPGS)

The ASO or chemically modified ASO is selected from the ASO or chemically modified products thereof in the aforementioned examples.

2. The preparation method of the smearable preparation containing ASO/ASO modification comprises the following steps:

2.1 Dissolved oxygen balance

Take purified water (prepared by a pure water machine), insert the nitrogen tube below the liquid surface, open the nitrogen main valve, slowly rotate the nitrogen slave valve, adjust the nitrogen pressure to about 0.1-0.2MPa (repeat the following nitrogen filling operation for 6 hours) to balance the dissolved oxygen in the water.

2.3 Dissolution

Add the weighed SNAC, DMI, vitamin C and TPGS into an appropriate amount of oxygen-balanced water and stir until dissolved.

2.4. Adjust pH

Adjust the pH value to 7.5-9.0 and fill with nitrogen for 2 hours to balance the dissolved oxygen.

2.5 API Dissolution

To the solution obtained in step 2.4 above, add API (ASO or modified form of ASO) and dissolve it.

2.6. Fixed volume

The volume is made up with oxygen-balanced water, and nitrogen is filled for 2 hours after the volume is made up to balance, thereby preparing the ASO/ASO modified body-containing smearable preparation.

2.7. Canning and labeling

The preparation was transferred into a borosilicate vial, filled with nitrogen, and sealed with a rubber stopper and an aluminum cap, capped, and labeled.

Example 10: Testing of the hair growth function of external smear preparations

In this example, three external application preparations containing different ASO modifications prepared according to the above-mentioned Example 9 were selected to investigate their hair growth function.

This IIT (Investigator Initiated Clinical Trial) study was conducted at West China Hospital, Sichuan University.

1. Ethics approval number: Approval from the Biomedical Ethics Review Committee of West China Hospital, Sichuan University, 2024 (80).

2. Product Usage: Males clinically diagnosed with androgenic alopecia (aged 18-60 years old, Hamilton-Norwood grade IIIa, III, IIIv, IV, IVa, V) were selected for this IIT study. This product is for topical application, with a single dose of 1 mL (1 tube), once a day for the first 3 days, and then once a week. It is recommended to use it at the same time each week, and the continuous use time is not less than 3 months; for some patients with more severe hair loss, the single dose is increased to 2 mL (2 tubes) according to the advice of professionals, and the frequency of use is the same as above. The placebo group is a blank excipient without API.

This product is a local external product applied with a massage applicator. Before each use, you need to wash your hair and blow dry it with hot air before applying it immediately. When using, pour the product into the applicator, apply the product evenly on the hair loss area on the head with the applicator, and massage with the applicator for 3 to 5 minutes. After application, wait for the scalp to dry naturally. It is also recommended not to wash your hair within 24 hours.

The non-vellus hair density of the application area of the patients before and after use was counted. The hair growth effects of each medication group are shown in the following Tables 9-1 to 9-7

Table 9-1. Statistical table of information of placebo patients before and after medication

The average increase in non-vellus hair density in the placebo group was -2.44%.

The calculation formula is: (183.6-188.2)/188.2*100% (the same below).

Thirty percent of patients did not experience a reduction in non-vellus hair density after taking placebo.

Table 9-2. Statistical table of information before and after medication of patients in the preparation (4 mg/mL LT010-0390) group

The average increase in non-vellus hair density after use of this preparation group was -0.11%.

Mean increase in non-vellus hair density compared to placebo: 2.33%

50% of the patients had increased non-vellus hair density after use, and the average increase in non-vellus hair density among these effective patients was 6.53%.

Table 9-3. Statistical table of information before and after medication of patients in the preparation (10 mg/mL LT010-0390) group

The average increase in non-vellus hair density after use of this preparation was 1.77%.

Mean increase in non-vellus hair density compared to placebo: 4.21%

46% of the patients had an increase in non-vellus hair density after use, and the average increase in non-vellus hair density in these effective patients was 7.06%.

Table 9-4. Statistical table of information before and after medication of patients in the preparation (4 mg/mL LT010-0410) group

The average increase in non-vellus hair density after use of this preparation was 2.60%.

Mean increase in non-vellus hair density compared to placebo: 5.04%

65% of patients had increased non-vellus hair density after use, and the average increase in non-vellus hair density among these effective patients was 5.34%.

Table 9-5. Statistical table of information before and after medication of patients in the preparation (10 mg/mL LT010-0410) group

The average increase in non-vellus hair density after use of this preparation was 3.64%.

Mean increase in non-vellus hair density compared to placebo: 6.09%

62.5% of patients had increased non-vellus hair density after use, and the average increase in non-vellus hair density among these effective patients was 7.49%.

Table 9-6. Statistical table of information before and after medication of patients in the preparation (4 mg/mL LT010-0449) group

The average increase in non-vellus hair density after use of this preparation was 3.44%.

Mean increase in non-vellus hair density compared to placebo: 5.88%

65% of the patients had an increase in non-vellus hair density after use, and the average increase in non-vellus hair density in these effective patients was 7.80%.

Table 9-7. Statistical table of information before and after medication of patients in the preparation (10 mg/mL LT010-0449) group

The average increase in non-vellus hair density after use of this preparation was 1.91%.

Mean increase in non-vellus hair density compared to placebo: 4.35%

65% of patients had increased non-vellus hair density after use, and the average increase in non-vellus hair density among these effective patients was 8.38%.

The results show that after using the product of the present invention, the effective rate (the number of patients with increased vellus hair density) and the average increase rate of vellus hair density are significantly improved compared with the blank preparation group.

The API structures in the three formulations used in this study are:

(1)LT-0390 (ASO No. 289): T*G*C*C*A*g*t*g*a*a*c*a*t*A*C*A*T*A*HSA

The modification methods are:

*：phosphorothioate internucleoside linkage; phosphorothioate;

A, G, C, T: 2’-O-methoxyethyl; 2’-MOE modification;

a, g, c, t: 2′-deoxy;

C base or c base are both 5-methyl modified cytosine;

HSA is a modification module:

The specific API can be hydrogen form, sodium salt form or S ^- form, and the structure is shown below

LT-0390 Hydrogen Type:

LT-0390 sodium salt type:

LT-0390S ^- Model:

(2)LT-0449 (ASO No. 289): T*G*CCA*g*t*g*a*a*c*a*t*ACA*T*A*HSA

The modification methods are:

*：phosphorothioate internucleoside linkage

A, G, C, T: 2’-O-methoxyethyl

C base or c base are both methyl type C bases

a, g, c, t: 2′-deoxy;

C base or c base are both 5-methyl modified cytosine;

HSA is a modification module:

The specific API can be hydrogen form, sodium salt form or S ^- form, and the structure is shown below

LT-0449 Hydrogen Type:

LT-0449 sodium salt type:

LT-0449S ^- Model:

(3)LT-0410 (ASO No. 204): G*G*A*A*A*g*t*t*g*t*a*g*t*a*g*T*C*G*C*G*RA

The modification methods are:

*：phosphorothioate internucleoside linkage

A, G, C, T: 2’-O-methoxyethyl

a, g, c, t: 2′-deoxy;

C base or c base are both 5-methyl modified cytosine;

RA is a modification module:

The specific API can be hydrogen form, sodium salt form or S ^- form, and the structure is shown below

LT-0410 Hydrogen Type:

LT-0410 sodium salt type:

LT-0410S ^- Model:

Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the present invention and are not used to limit the protection scope of the present invention.

Claims (11)

A group of antisense oligonucleotides (ASOs) that specifically inhibit androgen receptor (AR), wherein the antisense oligonucleotides are 14-22 bases in length, the target gene is AR mRNA, and the ASOs specifically pair with a specific region of the target gene, wherein the starting site of the specific region is located at: (1) 5'UTR region: 132-144, 164-167, 191-229, 266-298; (2) Exon 1: 1199-1201, 1591-1610, 1892, 2209-2210, 2306, 2572-2574; (3) Exon 2: 2777-2782; (4) Exon 2-exon 3: 2878-2882; (5) Exon 3: 2981-2983; (6) Exon 4: 3113-3116; (7) Exon 5: 3397-3401; (8) Exon 5-Exon 6: 3432-3443; (9) Exon 6: 3464, 3505-3506; (10) Exon 6-Exon 7: 3563-3573; (11) Exon 7: 3612, 3614-3616, 3618/3619, 3627, 3629, 3631, 3633; (12) Exon 8: 3749-3759, 3818-3863; (13) 3′UTR region: 7346-7354, 7394, 8139, 8418-8423, 10358-10363, 10386-10425; The AR target gene is: ENST00000374690.9, and the sequence is shown in SEQ ID NO.63. The antisense oligonucleotide according to claim 1, characterized in that the antisense oligonucleotide comprises a chemical modification; Preferably, the modifications are: thiolation of phosphate bonds, methoxyethyl modification (MOE modification) of the 2' position of the base and 5-methyl modification of cytosine; More preferably, the modification is: the phosphate bonds of all nucleotides in the antisense oligonucleotide sequence are monothiolated, 3 to 5 bases at the 3' end and 5' end are symmetrically MOE-modified and all cytosines are 5-methyl-modified. The antisense oligonucleotide according to claim 1 or 2, characterized in that the antisense oligonucleotide is selected from the oligonucleotide shown in any sequence in the following table;


Preferably, the modifications include: monothiolation of phosphate bonds, methoxyethyl modification (2'MOE modification) of the 2' position of the base, and 5-methyl modification of cytosine; More preferably, the modification comprises: monothiolation of the phosphate bonds of all nucleotides in the ASO sequence, 2'-MOE modification of 3 to 5 bases symmetrically at the 3' and 5' ends, and 5-methyl modification of all cytosines; after modification, the motif of the ASO is 4-8-4, 4-9-4, 5-8-5, 5-10-5, 5-12-5. The present invention also relates to the following use of the antisense oligonucleotide or its chemically modified form according to any one of claims 1 to 3: (1) preparing a preparation for inhibiting the expression of AR protein; (2) preparing a medicament or pharmaceutical composition for treating androgen-induced alopecia; or (3) preparing a drug or pharmaceutical composition for treating androgen-induced acne; or (4) inhibiting the expression or activation of AR protein; or (5) Treating diseases caused by overexpression or overactivation of AR protein, including but not limited to: tumors, alopecia or acne caused by overexpression or overactivation of AR protein; preferably, the tumor is prostate cancer; The medicine or pharmaceutical composition comprises a therapeutically effective amount of the antisense oligonucleotide (ASO) or a modified form thereof, and necessary pharmaceutical excipients or delivery carriers. A pharmaceutical composition comprising the antisense oligonucleotide (ASO) or a chemically modified form thereof according to any one of claims 1 to 3, wherein the pharmaceutical composition comprises: a therapeutically effective amount of the antisense oligonucleotide (ASO) or a modified form thereof, and necessary pharmaceutical excipients or delivery carriers; preferably, the pharmaceutical composition is a transdermal preparation. An external smearable preparation comprising the antisense oligonucleotide (ASO) or a chemically modified form thereof according to any one of claims 1 to 3, wherein the smearable preparation comprises: (1) The antisense oligonucleotide (ASO) or chemically modified antisense oligonucleotide (ASO) according to any one of claims 1 to 3: 1-50 mg/mL; preferably 2-20 mg/mL; more preferably 4-10 mg/mL; (2) Sodium 8-(2-hydroxybenzamido)octanoate (SNAC): 5-50 mg/mL, preferably 30 mg/mL; (3) 1,3-dimethyl-2-imidazolidinone (DMI): 5-60 mg/mL, preferably 40 mg/mL; (4) Vitamin E polyethylene glycol succinate (TPGS): 2-20 mg/mL, preferably 10 mg/mL; (5) Vitamin C: 2-20 mg/mL, preferably 10 mg/mL. The smear-type preparation according to claim 6, characterized in that the chemically modified antisense oligonucleotide (ASO) in the smear-type preparation is (1) LT-0390 (ASO No. 289): T*G*C*C*A*g*t*g*a*a*c*a*t*A*C*A*T*A*HSA, which is modified as follows: *: phosphorothioate internucleoside linkage; A, G, C, T: 2’-O-methoxyethyl; a, g, c, t: 2′-deoxy; C base or c base are both 5-methyl modified cytosine; HSA is a modification module: or (2) LT-0449 (ASO No. 289): T*G*CCA*g*t*g*a*a*c*a*t*ACA*T*A*HSA, wherein the modification method is: *: phosphorothioate internucleoside linkage; A, G, C, T: 2’-O-methoxyethyl; a, g, c, t: 2′-deoxy; C base or c base are both 5-methyl modified cytosine; HSA is a modification module: or (3) LT-0410 (ASO No. 204): G*G*A*A*A*g*t*t*g*t*a*g*t*a*g*T*C*G*C*G*RA, wherein the modification is: *: phosphorothioate internucleoside linkage; a, g, c, t: 2′-deoxy; C base or c base are both 5-methyl modified cytosine; RA is a modification module: The smearable preparation according to claim 7, characterized in that the chemically modified antisense oligonucleotide (ASO) is a hydrogen type, a sodium salt type or an S ^- type, and its specific structure is: LT-0390 Hydrogen Type: LT-0390 sodium salt type: LT-0390 S ^- Type: LT-0449 Hydrogen Type: LT-0449 sodium salt type: LT-0449 S ^- Type: LT-0410 Hydrogen Type: LT-0410 sodium salt type: LT-0410 S ^- Type: The method for preparing the spreadable preparation according to any one of claims 6 to 8, comprising the following steps: (1) Dissolved oxygen balance: Take purified water and fill it with nitrogen at a nitrogen pressure of 0.1-0.2 MPa to balance the dissolved oxygen in the water; (2) Dissolving the auxiliary materials and adjusting the pH value: Add SNAC, DMI, vitamin C, and TPGS to an appropriate amount of water that has been balanced with dissolved oxygen, and stir until dissolved; adjust the pH value to 7.5-9.0, and refill with nitrogen to balance the dissolved oxygen; (3) API dissolution: Add API (ASO or ASO modification), dissolve it and make up to volume with oxygen-balanced water. After making up to volume, nitrogen is added again to balance the oxygen, thus obtaining the ASO/ASO modification-containing spreadable preparation. The method according to claim 9 is characterized in that it also comprises the step (4) of canning and labeling: transferring the preparation into a borosilicate vial, filling it with nitrogen, covering it with a rubber stopper and an aluminum cap, sealing it, pressing the cap and labeling it. The following application of the smearable preparation according to any one of claims 6 to 8: (1) preparing a drug or pharmaceutical composition for treating male pattern baldness; or (2) preparing a drug or pharmaceutical composition for treating acne; or (3) Treatment of hair loss or acne caused by overexpression or excessive activation of AR protein.