Group of antisense oligonucleotides that specifically inhibit androgen receptor, and use thereof

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 applications Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a group of antisense oligonucleotides that specifically inhibit androgen receptors and applications thereof. Background Art

[0002] The epidemiological population of hair loss indications is increasing, among which androgen-induced hair loss is the most common type of hair loss, with an incidence rate of 50% and a lifetime incidence rate of 90% in men at the age of 50, and a lifetime incidence rate of 50% in women. However, there are limitations such as limited treatment options, poor efficacy, and large side effects. New nucleic acid drugs are potential treatments for this type of skin disease. Since existing nucleic acid delivery systems have not been successfully used to treat skin diseases such as hair loss, the development of new transdermal delivery systems for nucleic acid drugs (even protein drugs and small molecule drugs) may expand the methods for treating skin diseases and is of great significance for the development of drugs to treat hair loss indications.

[0003] AR (Androgen Receptor) is a nuclear receptor that exists in the cytoplasm when inactive, forming a complex with heat shock proteins and other molecular chaperone proteins as a dimer. After AR binds to androgen, the complex dissociates, and the AR dimer becomes phosphorylated, binds to other regulatory proteins, and then translocates into the cell nucleus. Subsequently, the DNA binding domain of AR binds to the DNA sequence of the androgen response element (ARE; the most typical is TGTTCT), transcribes downstream genes, and then exerts its role in regulating the hormone, immune, and endocrine systems. More specifically, AR can bind to testosterone and its highly active metabolite, dihydrotestosterone (DHT), converted by type II 5α-reductase, only within the dermal papilla cells of the hair follicles on the top of the head, resulting in a shortened anagen phase, miniaturization, and even atrophy of hair follicles, ultimately causing hair loss.

[0004] 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. However, nucleic acid drugs targeting this target for the treatment of androgen-induced alopecia and acne are still in the early stages of development, but the research enthusiasm is increasing and the development potential is relatively large.

[0005] Based on this, the present invention is proposed. Summary of the Invention

[0006] The present invention first relates to a group of antisense oligonucleotides (ASOs) that specifically inhibit androgen receptor (AR). The antisense oligonucleotides are 14-22 bases in length, and the target gene is AR mRNA. The ASOs specifically pair with a specific region of the target gene, and the starting site of the specific region is located at:

[0007] (1) 5'UTR region: 132-144, 164-167, 191-229, 266-298;

[0008] (2) Exon 1: 1199-1201, 1591-1610, 1892, 2209-2210, 2306, 2572-2574;

[0009] (3) Exon 2: 2777-2782;

[0010] (4) Exon 2-exon 3: 2878-2882;

[0011] (5) Exon 3: 2981-2983;

[0012] (6) Exon 4: 3113-3116;

[0013] (7) Exon 5: 3397-3401;

[0014] (8) Exon 5-Exon 6: 3432-3443;

[0015] (9) Exon 6: 3464, 3505-3506;

[0016] (10) Exon 6-Exon 7: 3563-3573;

[0017] (11) Exon 7: 3612, 3614-3616, 3618 / 3619, 3627, 3629, 3631, 3633;

[0018] (12) Exon 8: 3749-3759, 3818-3863;

[0019] (13) 3'UTR region: 7346-7354, 7394, 8139, 8418-8423, 10358-10363, 10386-10425;

[0020] The AR target gene is numbered ENST00000374690.9, and its sequence is shown in SEQ ID NO.63.

[0021] Furthermore, the ASO comprises a chemical modification;

[0022] 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;

[0023] More preferably, the modifications are: monothiolation of the phosphate bonds of all nucleotides in the ASO sequence, 2'-MOE modification of 3 to 5 symmetrical bases at the 3' and 5' ends, and 5-methyl modification of all cytosines.

[0024] Furthermore, the ASO is selected from the oligonucleotides shown in any sequence in the following table;

[0025] Preferably, the phosphate bonds of all nucleotides in the ASO described in the table are monothioated, and 4-5 bases at the 3' end and 5' end are symmetrically MOE-modified and all cytosines are 5-methyl-modified.

[0026] More preferably, the ASO has a sequence motif of 4-8-4, 4-9-4, 5-8-5, 5-10-5, or 5-12-5.

[0027] Furthermore, the present invention also relates to the following applications of the antisense oligonucleotide (ASO) or its modified form:

[0028] (1) preparing a preparation for inhibiting the expression of AR protein;

[0029] (2) preparing a medicament or pharmaceutical composition for treating androgen-induced alopecia; or

[0030] (3) preparing a drug or pharmaceutical composition for treating androgen-induced acne; or

[0031] (4) inhibiting the expression or activation of AR protein;

[0032] (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.

[0033] The medicine or pharmaceutical composition comprises a therapeutically effective amount of the antisense oligonucleotide (ASO) or its modified form, and necessary pharmaceutical excipients or delivery carriers.

[0034] 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 vehicles; preferably, the pharmaceutical composition is a transdermal preparation.

[0035] Furthermore, the present invention also relates to an externally applied smear-type preparation containing ASO / ASO modifications, wherein the smear-type preparation comprises:

[0036] (1) The antisense oligonucleotide (ASO) or chemically modified antisense oligonucleotide (ASO): 1-50 mg / mL; preferably 2-20 mg / mL; more preferably 4-10 mg / mL;

[0037] (2) Sodium 8-(2-hydroxybenzamido)octanoate (SNAC): 5-50 mg / mL, preferably 30 mg / mL;

[0038] (3) dimethyl isosorbide (DMI): 5-60 mg / mL, preferably 40 mg / mL;

[0039] (4) Vitamin E polyethylene glycol succinate (TPGS): 2-20 mg / mL, preferably 10 mg / mL;

[0040] (5) Vitamin C: 2-20 mg / mL, preferably 10 mg / mL.

[0041] Preferably, the chemically modified antisense oligonucleotide (ASO) in the smearable preparation is

[0042] (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:

[0043] *:phosphorothioate internucleoside linkage; phosphorothioate;

[0044] A, G, C, T: 2'-O-methoxyethyl; 2'-MOE modification;

[0045] a, g, c, t: 2′-deoxy;

[0046] C base or c base are both 5-methyl modified cytosine;

[0047] HSA is the modification module: or

[0048] (2) LT-0449 (ASO No. 289): T*G*CCA*g*t*g*a*a*c*a*t*ACA*T*A*HSA, wherein the modification is:

[0049] *: phosphorothioate internucleoside linkage;

[0050] A, G, C, T: 2'-O-methoxyethyl;

[0051] a, g, c, t: 2′-deoxy;

[0052] C base or c base are both 5-methyl modified cytosine;

[0053] HSA is the modification module: or

[0054] (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:

[0055] *: phosphorothioate internucleoside linkage;

[0056] A, G, C, T: 2'-O-methoxyethyl;

[0057] a, g, c, t: 2′-deoxy;

[0058] C base or c base are both 5-methyl modified cytosine;

[0059] RA is the modification module:

[0060] The present invention also relates to a method for preparing the smear-type preparation, which specifically comprises the following steps:

[0061] (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;

[0062] (2) Dissolving excipients and adjusting the pH: Add SNAC, DMI, vitamin C, and TPGS to an appropriate amount of oxygen-balanced water and stir until dissolved; adjust the pH to 7.5-9.0 and refill with nitrogen to balance the dissolved oxygen;

[0063] (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 dissolved oxygen. This is the spreadable preparation containing the ASO / ASO modification.

[0064] And the optional step (4) canning and labeling: transfer the preparation into a borosilicate vial, fill it with nitrogen, cover it with a rubber stopper and an aluminum cap, press the cap and label it.

[0065] The present invention also relates to the following applications of the smear-type preparation:

[0066] (1) preparing a medicament or pharmaceutical composition for treating male pattern baldness; or

[0067] (2) preparing a drug or pharmaceutical composition for treating acne; or

[0068] (3) Treating hair loss or acne caused by overexpression or excessive activation of AR protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1. After cells were treated with antisense oligonucleotides (ASOs), WB assay was used to detect the inhibitory effect on AR protein expression.

[0070] Figure 2. After cells were treated with antisense oligonucleotides (ASOs), WB assay was used to detect the inhibitory effect on AR protein expression.

[0071] Figure 3. Electrophoresis detection of serum stability of ASO.

[0072] Figure 4. After cells were treated with antisense oligonucleotides (ASOs), the mRNA expression of cytokines related to inflammatory responses was detected. DETAILED DESCRIPTION

[0073] Example 1: Design, synthesis and modification of ASO targeting AR target gene

[0074] 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.

[0075] 7650 antisense oligonucleotide sequences (ASOs) were designed targeting 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;

[0076] Furthermore, 349 of the above ASO sequences were chemically modified. Specifically, the modifications were: 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 (i.e., methoxyethyl modification of the 2' position of the base).

[0077] Table 1 Detailed information of ASO molecules

[0078] Example 2: Testing the activity of ASO in an in vitro cell model (HaCaT cells, human immortalized epidermal cells, or LNCaP human prostate cancer cells)

[0079] 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:

[0080] (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 desired 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 concentrations or concentration gradients. Pipet 10 times to mix thoroughly and let stand at room temperature for 20 minutes.

[0081] (2) Cell treatment: Cells were plated one day before transfection at a concentration of 6 × 10 3 Cells / well were plated 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 a microscope. 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 incubated at 37°C, 5% CO2 incubator. After 5 hours, the medium was replaced with new medium.

[0082] (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:

[0083] Table 2 PCR primer and probe sequences

[0084] 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:

[0085] Inhibition rate = (1-2^-ΔΔCT) × 100%.

[0086] The experimental groups are:

[0087] Cells treated with the modified ASOs indicated by the respective numbers;

[0088] The blank control group consisted of cells not treated with any ASO.

[0089] 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.

[0090] The results show that the ASO sequences shown in Table 1 have a significant inhibitory effect on the transcription of AR mRNA

[0091] Table 3-1 Inhibition rate of AR mRNA after antisense oligonucleotide treatment of HaCaT cells (%) Note: The motif indicates the MOE modification of bases at the 3' and / or 5' ends. For example, 4-8-4 indicates: 4 bases at the 3' end and 4 bases at the 5' end are MOE modified; 5-10-5 indicates: 5 bases at the 3' end and 5 bases at the 5' end are MOE modified.

[0092] Table 3-2 Inhibition rate of AR mRNA after antisense oligonucleotide treatment of HaCaT cells (%)

[0093] Table 3-3 Inhibition rate of AR mRNA expression after antisense oligonucleotide treatment of LNCaP cells (%)

[0094] Table 3-4 Inhibition rate of AR mRNA expression after antisense oligonucleotide treatment of LNCaP cells (%)

[0095] Example 3: Testing the activity of ASO using an in vitro cell model (LNCaP human prostate cancer cells)

[0096] In this example, the inhibitory effect of the ASO molecules shown in Table 1 on the AR protein level in LNCaP cells was verified. The transfection reagent was prepared as in Example 2, and the experimental steps were as follows:

[0097] (1) Use 6-well plates and use 1.5×10 cells. 5 / hole;

[0098] (2) The concentration of ASO molecules used to treat cells was 10 nM;

[0099] (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;

[0100] (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 hour. Protein expression was observed using a multifunctional imaging system (Tanon 5200Multi).

[0101] The effects of the ASOs described in Table 1 on inhibiting AR protein expression in LNCaP human prostate cancer cells are shown in FIG1 and Table 4 (only part of the results are shown).

[0102] The results showed that the ASO sequences shown in Table 1 had a significant inhibitory effect on the expression level of AR protein in LNCaP cells.

[0103] Table 4. Inhibition rate of ASO on AR protein in LNCaP cells (%) (WB experiment)

[0104] Example 4. In vitro cell model (HaCaT cells, human immortalized epidermal cells / NCaP cells, human prostate cancer cells) testing the activity of the antisense oligonucleotide-fatty acid construct (ASO-S1)

[0105] In this example, the inhibitory effect of the fatty acid chain-modified ASO construct (ASO-S1) 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:

[0106] (1) Incubation reagent preparation: 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 the 1 mM ASO-S1 stock solution with the prepared culture medium to prepare ASO-S1 incubation complexes of a predetermined concentration or concentration gradient. Mix by pipetting 10 times.

[0107] (2) Cell treatment: Cells were plated one day before incubation at a concentration of 1.5 × 105 Cells were seeded / well in a 6-well plate, and 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 a microscope. The old medium was discarded, and 2000 μL of the incubation reagent prepared in step (1) was added to each well. The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours, and then replaced with complete medium.

[0108] (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.

[0109] The efficiency of the ASO-S1 constructs with 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).

[0110] 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.

[0111] Table 5. Inhibition rate of AR protein of HaCaT / LNCaP cells by ASO-S1 construct (%)

[0112] Example 6. Serum Stability of Antisense Oligonucleotides (ASOs) (Fetal Bovine Serum and Rat Serum)

[0113] 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.

[0114] (1) Serum was prepared with sterile water at concentrations of 10% and 50%. 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, and 24 h, respectively.

[0115] (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 ddH2O.

[0116] (3) DNA was separated by 15% TBE-UREA PAGE gel electrophoresis, the gel was stained with a safe nucleic acid dye (Monad, ME20301), and the amount of DNA was observed using a multifunctional imaging system (Tanon 5200 Multi).

[0117] The stability of some of the ASOs described in Table 1 and the corresponding ASO-S1 constructs in serum is shown in FIG3 .

[0118] 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.

[0119] Example 7: In vitro cell model (PBMC) detection of cytokine levels associated with inflammatory response after treatment with ASO and corresponding ASO-S1 constructs

[0120] 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:

[0121] (1) Cell treatment: Cells were revived and plated one day before incubation, and 1×10 6 Cells were plated per well in a 12-well plate, and 100 μL of RPMI 1640 medium containing 1% FBS and 1% insulin-transferrin-selenium-aminoethanol (GIBCO, 51500056) was added to each well.

[0122] (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.

[0123] (3) After 24 h of incubation, 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.

[0124] Table 6. PCR primers and probe sequences for cytokines

[0125] The results are shown in FIG4 : After treatment with the tested ASO / ASO-S1, the effects on inflammatory factors were low.

[0126] Example 8: Testing the Tumor Cell Killing Effect of ASO-S1 in an In Vitro Cell Model (LNCaP Human Prostate Cancer Cells)

[0127] In this example, it was verified that the fatty acid chain-modified ASO molecule (ASO-S1) shown in Table 1 had a certain killing effect on prostate cancer cells LNCaP. The specific experimental process is as follows:

[0128] (1) Incubation reagent preparation: 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 the 1 mM ASO-S1 stock solution with the prepared medium to prepare a concentration gradient of ASO-S1 incubation complexes. Mix thoroughly by pipetting 10 times.

[0129] (2) Cell treatment: Cells were plated one day before incubation at a concentration of 1×10 3 Cells were seeded into a 96-well plate at 100 μL per well, and 100 μL of RPMI 1640 medium containing 10% FBS, 1% sodium pyruvate, and 1% glutamine was added to each well. Observe the LNCaP cells under a microscope before treatment, discard the old medium, and add 100 μL of the incubation reagent prepared in step (1) to each well. The cells were then cultured in a 37°C, 5% CO2 incubator.

[0130] (3) After incubation for 72 h, CCK-8 assay was performed.

[0131] Table 7. Growth inhibition rate of LNCaP cells by ASO-S1 (%)

[0132] The results are shown in Table 7. After the tested ASO-S1 was treated with prostate cancer cells, the growth of cancer cells was also inhibited.

[0133] Example 9: Preparation of a topical smear formulation

[0134] 1. Prepare the topical ASO / ASO modified form smear formulation according to the following Table 8:

[0135] Table 8. Formulation of ASO / ASO-modified smear-type preparations

[0136] Sodium 8-(2-hydroxybenzamido)octanoate (SNAC)

[0137] Isosorbide dimethyl ether (DMI)

[0138] Vitamin E polyethylene glycol succinate (TPGS)

[0139] The ASO or chemically modified ASO is selected from the ASO or chemically modified forms thereof in the aforementioned examples.

[0140] 2. The preparation method of the smearable preparation containing ASO / ASO modification comprises the following steps:

[0141] 2.1 Dissolved oxygen balance

[0142] 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.

[0143] 2.3 Dissolution

[0144] Add the weighed SNAC, DMI, vitamin C, and TPGS into an appropriate amount of oxygen-balanced water and stir until dissolved.

[0145] 2.4. Adjust pH

[0146] Adjust the pH value to 7.5-9.0 and aerate with nitrogen for 2 hours to balance the dissolved oxygen.

[0147] 2.5 API Dissolution

[0148] To the solution obtained in step 2.4 above, add API (ASO or modified form of ASO) and dissolve.

[0149] 2.6. Fixed volume

[0150] The volume was adjusted to the desired volume using oxygen-balanced water, and nitrogen was added for 2 hours to prepare the ASO / ASO modified body-containing smearable preparation.

[0151] 2.7. Canning and labeling

[0152] The preparation was transferred into a borosilicate vial, filled with nitrogen, and sealed with a rubber stopper and an aluminum cap, capped, and labeled.

[0153] Example 10: Testing of the Hair Growth Function of Externally Used Spreadable Preparations

[0154] In this example, three topical smear-type preparations containing different ASO modifications prepared according to the aforementioned Example 9 were selected to investigate their hair growth function.

[0155] This IIT (Investigator Initiated Clinical Research) study was conducted at West China Hospital, Sichuan University.

[0156] 1. Ethical approval number: Approval from the Biomedical Ethics Review Committee of West China Hospital, Sichuan University, 2024 (80).

[0157] 2. Product Usage: Men clinically diagnosed with androgenic alopecia (aged 18-60 years, Hamilton-Norwood grade IIIa, III, IIIv, IV, IVa, V) were enrolled in this IIT study. This product is for topical application, with a single dose of 1 mL (one vial) for once daily use for the first three days, followed by weekly use. It is recommended to use the product at the same time each week for at least three months. For patients with more severe alopecia, the single dose may be increased to 2 mL (two vials) based on professional recommendations, with the same frequency of use. The placebo group received a blank excipient without the API.

[0158] This product is a topical product that is applied with a massage applicator. Before each use, you need to wash your hair and blow dry it with hot air immediately. When using, pour the product into the applicator, use the applicator to evenly apply the product to the hair loss area on the head 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.

[0159] The density of non-vellus hair in the application area of ​​the patients before and after use was calculated. The hair growth effects of each medication group are shown in Tables 9-1 to 9-7 below.

[0160] Table 9-1. Statistics of information of placebo patients before and after medication

[0161] The average increase in non-vellus hair density in the placebo group was -2.44%.

[0162] The calculation formula is: (183.6-188.2) / 188.2*100% (the same below).

[0163] Thirty percent of patients in the placebo group experienced no reduction in non-vellus hair density.

[0164] Table 9-2. Statistics of patients in the preparation (4 mg / mL LT010-0390) group before and after medication

[0165] The average increase in non-vellus hair density in patients in this preparation group after use was -0.11%.

[0166] Average increase in non-vellus hair density compared to placebo: 2.33%

[0167] 50% of the patients had increased non-vellus hair density after use, and the average increase in non-vellus hair density in these effective patients was 6.53%.

[0168] Table 9-3. Statistics of patients in the preparation (10 mg / mL LT010-0390) group before and after medication

[0169] The average increase in non-vellus hair density in patients in the preparation group after use was 1.77%.

[0170] Average increase in non-vellus hair density compared to placebo: 4.21%

[0171] 46% of patients had increased non-vellus hair density after use, and the average increase in non-vellus hair density in these effective patients was 7.06%.

[0172] Table 9-4. Statistical table of information before and after medication for patients in the preparation (4 mg / mL LT010-0410) group

[0173] The average increase in non-vellus hair density in patients in the preparation group after use was 2.60%.

[0174] Average increase in non-vellus hair density compared to placebo: 5.04%

[0175] 65% of patients had increased non-vellus hair density after use, and the average increase in non-vellus hair density in these effective patients was 5.34%.

[0176] Table 9-5. Statistics of patients in the preparation (10 mg / mL LT010-0410) group before and after medication

[0177] The average increase in non-vellus hair density in patients in the preparation group after use was 3.64%.

[0178] Average increase in non-vellus hair density compared to placebo: 6.09%

[0179] 62.5% of patients had increased non-vellus hair density after use, and the average increase in non-vellus hair density in these effective patients was 7.49%.

[0180] Table 9-6. Statistics of patients in the preparation (4 mg / mL LT010-0449) group before and after medication

[0181] The average increase in non-vellus hair density in patients in the preparation group after use was 3.44%.

[0182] Average increase in non-vellus hair density compared to placebo: 5.88%

[0183] 65% of patients had increased non-vellus hair density after use, and the average increase in non-vellus hair density in these effective patients was 7.80%.

[0184] Table 9-7. Statistics of patients in the preparation (10 mg / mL LT010-0449) group before and after medication

[0185] The average increase in non-vellus hair density in patients in the preparation group after use was 1.91%.

[0186] Mean increase in non-vellus hair density compared to placebo: 4.35%

[0187] 65% of patients had increased non-vellus hair density after use, and the average increase in non-vellus hair density in these effective patients was 8.38%.

[0188] 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.

[0189] The API structures in the three formulations used in this study are:

[0190] (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

[0191] The modification methods are:

[0192] *:phosphorothioate internucleoside linkage; phosphorothioate;

[0193] A, G, C, T: 2'-O-methoxyethyl; 2'-MOE modification;

[0194] a, g, c, t: 2′-deoxy;

[0195] C base or c base are both 5-methyl modified cytosine;

[0196] HSA is the modification module:

[0197] The specific API can be hydrogen, sodium salt or S - Type, the structure is as follows

[0198] LT-0390 Hydrogen Type:

[0199] LT-0390 sodium salt type:

[0200] LT-0390S - type:

[0201] (2)LT-0449 (ASO No. 289): T*G*CCA*g*t*g*a*a*c*a*t*ACA*T*A*HSA

[0202] The modification methods are:

[0203] *: phosphorothioate internucleoside linkage

[0204] A, G, C, T: 2'-O-methoxyethyl

[0205] C base or c base are both methyl type C bases

[0206] a, g, c, t: 2′-deoxy;

[0207] C base or c base are both 5-methyl modified cytosine;

[0208] HSA is the modification module:

[0209] The specific API can be hydrogen, sodium salt or S - Type, the structure is as follows

[0210] LT-0449 Hydrogen Type:

[0211] LT-0449 sodium salt type:

[0212] LT-0449S - type:

[0213] (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

[0214] The modification methods are:

[0215] *: phosphorothioate internucleoside linkage

[0216] A, G, C, T: 2'-O-methoxyethyl

[0217] a, g, c, t: 2′-deoxy;

[0218] C base or c base are both 5-methyl modified cytosine;

[0219] RA is the modification module:

[0220] The specific API can be hydrogen, sodium salt or S - Type, the structure is as follows

[0221] LT-0410 Hydrogen Type:

[0222] LT-0410 sodium salt type:

[0223] LT-0410S - type:

[0224] 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 scope of protection of the present invention.

Claims

1. 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.

2. 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.

3. The antisense oligonucleotide according to claim 1 or 2, characterized in that The antisense oligonucleotide is selected from the oligonucleotides 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.

4. 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.

5. 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.

6. 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.

7. The spreadable preparation according to claim 6, characterized in that 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; 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 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:

8. The spreadable preparation according to claim 7, characterized in that The chemically modified antisense oligonucleotide (ASO) is a hydrogen form, a sodium salt form or a S - Type, its specific structure is: LT-0390 Hydrogen Type: LT-0390 sodium salt type: LT-0390S - type: LT-0449 Hydrogen Type: LT-0449 sodium salt type: LT-0449S - type: LT-0410 Hydrogen Type: LT-0410 sodium salt type: LT-0410S - type:

9. A 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, thereby obtaining the ASO / ASO modification-containing spreadable preparation.

10. The method according to claim 9, characterized in that The method further 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.

11. The following use of the smear-type 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.

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