Use of flibanserin in the preparation of a medicament for the treatment of androgenetic alopecia

Abstract

The present invention discloses the use of flibanserin in the preparation of a drug for treating androgenetic alopecia.The results of cell model show that low concentrations of flibanserin can inhibit the proliferation of Lncap cells, indicating pharmacodynamic activity at the cell level.The results of animal experiments show that flibanserin can improve androgen-mediated androgenetic alopecia disorder, and can treat androgenetic alopecia to restore hair growth.Therefore, flibanserin can be used in the preparation of a drug for treating androgenetic alopecia.

Description

[Technical field]

[0001] The present invention belongs to the field of small molecule drugs, and relates to the use of flibanserin in the preparation of a drug for the treatment of androgenetic alopecia. [Background technology]

[0002] Androgenetic alopecia (AGA) is a disease characterized by a progressive decrease in hair density, typically beginning with a receding frontal hairline on both sides of the head, followed by diffuse thinning from the vertex, and finally complete baldness from the center of the head. It is an autosomal dominant genetic disease. Compared with hair follicles located in the occipital region, the androgen receptor and 5α-reductase type I and type II activity levels are relatively high in the frontal region of the scalp in men and women. The activity of α-reductase type I and type II in the forehead hair follicles is three times higher in men than in women. Thus, male androgenetic alopecia is considered to be an androgen-dependent disease, and in China, the overall prevalence among men is 21.3%, but increases with age. AGA severely impacts the social and psychological aspects of patients, which encourages them to seek treatment. The pathological characteristics of AGA include miniaturization of hair follicles, shortening of the anagen phase and prolongation of the telogen phase. Clinically, it is manifested primarily by replacement of terminal hairs with villus (defined as villi <30 μm in diameter, <30 μm in length, transitional hair width between 30-40 μm, terminal hair >40 μm) and a decrease in total hair density (hairs / cm2). Histologically, it is characterized by replacement of terminal hair follicles with villous follicles, perifollicular infiltration of macrophages, increased apocrine size, and thinning of the dermis. The cyclical growth of hair follicles is divided into four stages: anagen, in which the hair matrix keratin surrounding dermal papilla cells (DPCs) undergoes cellular proliferation and the hair shaft grows; catagen, in which tightly controlled apoptosis and extracellular matrix regeneration occur in the hair follicle; telogen, in which the hair shaft is attached to the bulbous base of the hair follicle epithelium and may fall out due to combing or washing the face; and exogen, in which the hair in the telogen stage falls out. Testosterone (T) is one of the main sex hormones in the human body, and 5α-reductase (5GR, 5a-reductase) expressed in DPCs promotes the conversion of T to the more active 5α-dihydrotestosterone (DHT, 5α-dihvdrotestosterone).DHT binds to androgen receptors (AR) on hair matrix cells, forms DHT-AR complexes, enters the cell nucleus, and functions as a transcription factor that induces hair follicle dermal papilla cells to secrete multiple cytokines, such as transforming growth factor B (TGF-B), interleukin-1a (1L-1a), and tumor necrosis factor a (TNF-a). It may induce premature termination of the hair follicle anagen phase, leading to AGA. Androgenetic alopecia is the result of scalp hair follicles being highly sensitive to circulating androgens, and androgen induction increases the expression of androgen receptors, which in turn alters the mesenchymal-epithelial cell interactions within the hair follicle, affecting hair growth, dermis size, dermal cells, and keratinocyte and melanocyte activity, and Wnt signaling channels may regulate cells in the dermis and play an important role in the action of androgens on hair growth. However, the underlying molecular mechanisms of androgen-related actions remain largely unknown.

[0003] At present, only two drugs have been approved by the FDA for the treatment of androgenetic alopecia: finasteride, an oral drug, and minoxidil, an external application. Finasteride is a 5α-reductase inhibitor that treats hair loss by reducing the amount of DHT in the body. Minoxidil is a vasodilator that improves the microcirculation of hair follicles, thereby treating hair loss. Other treatment methods include non-pharmacological treatment methods such as laser treatment, hair transplantation, and stem cell therapy. However, all of these treatment methods have significant side effects, such as sexual dysfunction, hirsutism, and high costs. Therefore, it is important to find new androgenetic alopecia treatment drugs with fewer side effects. Summary of the Invention [Problem to be solved by the invention]

[0004] The object of the present invention is to provide the use of flibanserin in the preparation of a medicament for the treatment of androgenetic alopecia in response to the above-mentioned shortcomings of the prior art. [Means for solving the problem]

[0005] The object of the present invention can be achieved by the following technical solutions. Use of flibanserin in the preparation of a medicament for the treatment of androgenetic alopecia.

[0006] As a preferred option of the present invention, flibanserin can increase hair length, anagen to telogen ratio, epidermal to dermal thickness ratio, number of hair follicles per unit area and hair follicle diameter.

[0007] As a preferred option of the present invention, flibanserin is used in the preparation of a medicament for improving androgen-mediated androgenetic alopecia disorders, treating androgenetic alopecia, and restoring hair growth.

[0008] Flibanserin is used in the preparation of drugs that inhibit the androgen receptor dimer and prevent it from entering the cell nucleus.

[0009] The use of androgen receptor dimer as a therapeutic target in screening for androgenetic alopecia treatment drugs. Flibanserin is a compound for treating androgenetic alopecia that is obtained by screening using computer simulation technology in combination with the structure of androgen receptor dimer. Effect of the Invention

[0010] The beneficial effects are as follows: The present invention combines computer simulation screening technology with in vitro and in vivo drug screening experiments to obtain the inhibitor of androgen receptor dimer, while this compound structurally has binding activity to the structure of androgen receptor dimer, has a clear target of action for minoxidil, and has high specificity.This compound is an inhibitor of androgen receptor dimer, and can inhibit the entry of androgen receptor dimer into the nucleus by binding with androgen receptor dimer, inhibit the mobilization of downstream activating factors of this complex and the binding with androgen receptor response components, thereby blocking downstream gene response and treating androgenetic alopecia.Compared with finasteride, this drug has milder action conditions and does not cause downstream response mediated by androgen receptor at other sites, so that it can greatly reduce the side effects of androgenetic alopecia treatment drugs.

[0011] The results of the cell model show that low concentrations of flibanserin can inhibit the proliferation of Lncap cells, indicating pharmacodynamic activity at the cell level. The results of the animal experiment show that, compared with the model group, the hair of the mice in the flibanserin and finasteride groups was restored to different degrees, and was close to the hair growth situation in the control group and the complete blank group. The hair recovery effectiveness rate of the animals in the flibanserin group reached 85.7%, and that of the finasteride group reached 71.4%. Compared with the model group, the staining of HE tissue sections shows that the hair length of the flibanserin group increased, the ratio of anagen to telogen increased, the ratio of the thickness of the epidermis to the dermis of the mice increased, the number of hair follicles in a unit area increased, and the diameter of the hair follicles increased. Therefore, the results show that flibanserin can improve androgen-mediated androgenetic alopecia disorders and can treat androgenetic alopecia to restore hair growth. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the inhibition of proliferation of prostate cancer Lncap cells by dihydrotestosterone stimulation. [Diagram 2] FIG. 2 is a statistical graph showing hair length in flibanserin-treated AGA model mice. [Diagram 3] Figure 3 is a histogram of hair regrowth rate in flibanserin-treated AGA model mice. [Figure 4] FIG. 4 is a histogram showing the ratio of anagen to telogen in hair follicles in AGA model mice treated with flibanserin. [Diagram 5] FIG. 5 is a histogram showing the ratio of the thickness of the dermis layer to the epidermis layer in AGA model mice treated with flibanserin. [Figure 6] FIG. 6 is a statistical histogram showing the number of hair follicles per unit area in flibanserin-treated AGA model mice. [Figure 7] FIG. 7 is a statistical histogram showing the diameter of hair follicles in flibanserin-induced AGA model mice. [Figure 8] FIG. 8 shows hair recovery in AGA model mice treated with flibanserin. [Figure 9] FIG. 9 is a nucleoplasmic segregation protein map showing that flibanserin inhibits androgen receptor dimer entry into the nucleus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Example 1 Reagents and cells involved in cell-related experiments: Dihydrotestosterone, Lot No.: CHB16A802100G; DMSO, Lot No.: #RNBK5094; Flibanserin, Lot No.: W16O8Z45834; 5mg of Flibanserin was dissolved in 1.28mL of DMSO to prepare a mother solution with a concentration of 10000uM; Lncap cell line is a human prostate cancer cell line purchased from Xiamen Yimou Biotechnology Co., Ltd., Sample No.: 20210106-06, Biosafety Level: BSL-1; epithelioid adherent growth, single cells and loosely adherent cell clusters. Culture conditions were 88 ml RPMI Medium 1640 (Invitrogen, 11875093) + 10 ml FBS (Gibco) + 1 ml Glutamax (Invitrogen, 35050) + 1 ml Sodium Pyruvate 100 mM Solution (Invitrogen, 11360070) and the cells respond to dihydrotestosterone (which regulates growth and produces acid phosphatase ACP).

[0014] Dihydrotestosterone was dissolved in DMSO to prepare a 0.01 mol / L mother solution, which was then diluted in medium for 10 min. -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 The concentration of dihydrotestosterone was 1000 M, diluted to a concentration gradient, added to the attached Lncap cells, and cultured for 48, 72, and 96 hours. The proliferation of Lncap cells was measured by MTT, and the response of downstream genes KLK3 and KLK2 at different concentrations was examined in combination with RT-PCR. The proliferation-promoting effect was most obvious at the concentration of dihydrotestosterone 1000 M, which corresponds most significantly to the response of downstream genes. -9 M was the optimal androgen-induced modeling concentration of Lncap cells. After androgen-induced modeling, Lncap cells were treated with 100 mg of drug with different concentration gradients. -3 uM, 10 -1The inhibitory rate of flibanserin on Lncap cell proliferation was measured by MTT assay after co-culture for 24h, 48h, and 96h, respectively. The results showed that low concentrations of flibanserin could inhibit Lncap cell proliferation, indicating pharmacodynamic activity at the cellular level.

[0015] The results of this experiment were processed using Excel software and are shown in Figure 1 and the table below.

[0016] Table 1 Different concentrations of DHT promote Lncap cell proliferation for 48 h. JPEG0007634783000001.jpg60170

[0017] The experimental results showed that different concentrations of DHT promoted the proliferation of Lncap cells differently. -9 The results show that the promotion of Lncap cell proliferation by DHT at uM is most obvious, and therefore this concentration was selected as the optimal concentration for drug modeling.

[0018] Table 2. Different concentrations of Fibanserin inhibit the proliferation of Lncap cells for 48 h. JPEG0007634783000002.jpg67170

[0019] The experimental results are as follows: -9 After addition of different concentrations of flibanserin, modeling with DHT of M showed that the inhibition on Lncap cell proliferation increased with increasing flibanserin concentration. Thus, at the cellular level, flibanserin has pharmacodynamic activity.

[0020] Example 2 Reagents related to animal testing: Testosterone Propionate, Lot No.: T818615; Soybean Oil, Lot No.: A23GS146219; 80mg of Testosterone Propionate was dissolved in 40mL of soybean oil to prepare a 2mg / mL solution; Finasteride, Lot No.: A16GS145548; 0.28mg of Finasteride was dissolved in 20mL of saline to prepare a 1.4X10 -2 A solution of 1.6X10 mg / mL of Flibanserin was prepared by dissolving 0.32 mg of Flibanserin in 20 mL of saline. -2 A mg / mL solution was prepared.

[0021] The test animals were male C58BL / 6 mice, outbred closed group, weighing 18-22g, 5 weeks old, certificate number: No. 202207119, provided by Yangzhou University Comparative Medicine Center. The room temperature was 20-22℃, the relative humidity was 40%-60%, ventilated with a ventilator, natural light source was 12h / day, and the mice were kept in cages, 7 mice per cage, and the cages were cleaned once every 2 days.

[0022] Before modeling by injecting testosterone propionate, mice were first fixed, the hair on the back of the mouse was cut with an electric clipper, and the villi were removed with a depilatory cream. The mice were divided into groups of 7 mice per cage into the following groups: complete blank group (no treatment other than hair removal), control group (0.2 mL of soybean oil was injected + 0.1 mL of saline was injected into the stomach), model group (0.2 mL of testosterone propionate solution was injected + 0.1 mL of saline was injected into the stomach), model group + finasteride (0.2 mL of testosterone propionate solution was injected + 0.1 mL of finasteride solution was injected into the stomach), and model group + flibanserin (0.2 mL of testosterone propionate solution was injected + 0.1 mL of flibanserin solution was injected into the stomach). Before intragastric injection, the mice were first injected with testosterone propionate solution and modeled for 21 days, and the growth of the hair on the back of the mice was observed to determine the success rate of modeling. After modeling is successful, continue to inject testosterone propionate solution and carry out intragastric administration, administering for 4 weeks.After administration is completed, calculate the effective rate of the drug (the percentage of the ratio of the mice that have recovered hair to the total number of mice), calculate the ratio of the area of ​​the hair growth area of ​​the mice to the area of ​​the hair loss area, take the hair of the mice from different points on the back of the mice that have recovered hair, measure and calculate the length of the hair, dislocate the mice cervically, kill them, take the skin of the back, freeze a part of it at -80℃, extract protein, and carry out nucleoplasm separation experiment to investigate the inhibition of the drug on the entry of androgen receptor dimer into the nucleus. A portion of the mice was fixed in 4% paraformaldehyde (Product No. AF030) for 48 hours, then histological sections were cut and stained with HE. The number of hair follicles in a unit area of ​​the skin on the back of the mice, the diameter of the hair papilla, and the ratio of the growth phase and the resting phase of the hair follicles were calculated under a microscope, and the efficacy of flibanserin was calculated by comparing with the model group, the complete blank group, the control group, and the positive drug finasteride. As for the criteria, the area of ​​hair loss on the back of the mice after hair removal was measured and recorded as A, and the area of ​​hair growth on the back of the mice after the administration was completed was recorded as B, and the average value of A / B in each group was taken as the hair recovery efficacy rate of the animals.

[0023] The results show that, compared with the model group, the mice in the flibanserin and finasteride groups recovered hair to varying degrees, and were close to the hair growth status in the control and complete blank groups (Figure 2). The hair recovery effectiveness rate of the animals in the flibanserin group reached 85.7%, and that of the finasteride group reached 71.4% (Figure 3). Compared with the model group, the staining of HE tissue sections shows that the hair length of the flibanserin group increased, the ratio of anagen to telogen increased, the ratio of epidermis to dermis thickness increased, the number of hair follicles in a unit area increased, and the diameter of hair follicles increased (Figures 4 to 7). Therefore, the results show that flibanserin can improve androgen-mediated androgenetic alopecia disorders and can treat androgenetic alopecia to restore hair growth (Figure 8).

[0024] Table 3. Efficacy rates of animals in the control group, finasteride and flibanserin JPEG0007634783000003.jpg13170

[0025] Example 3 Primary mouse dermal papilla cells were extracted using an enzyme digestion method. The culture conditions were 88 ml of L-DMEM (Keiji Bio) + 10 ml of FBS (Morikawa Bio) + 1 ml of Glutamax (Invitrogen, 35050). The cells were cultured for 10 days, and then expanded and subcultured until the primary cells filled a T25 culture flask. A portion of the cells was used in a flow cytometry experiment to identify cell surface markers. The identification results showed that CD56, CD133, and CD140a were positive, and CD73 and CD90 were negative, indicating that the cells were dermal papilla cells. The cells were seeded into a 12-well plate and divided into a blank group, a DHT group (10 -8 mol / L), DHT(10 -8 mol / L) + finasteride group (5umol / L), DHT (10 -8 mol / L) + high dose flibanserin (5umol / L), DHT (10 -8The mice were treated with either the low-dose flibanserin group (1 umol / L) or the low-dose flibanserin group (1 umol / L). After 48 hours, a nucleoplasmic separation experiment was performed using a Biyuntian nuclear protein and cytoplasmic protein separation kit according to the manufacturer's instructions, and the separated nuclear proteins and cytoplasmic proteins were subjected to SDS-PAGE gel electrophoresis.

[0026] The experimental results show that H3 is the internal standard of nuclear protein, and β-Tublin is the internal standard of cytoplasmic protein. From the protein map, it can be seen that there is only H3 protein in the nuclear protein group, and only β-Tublin protein in the cytoplasmic protein group, and the nucleoplasmic separation experiment was successful. Compared with the blank group, the DHT group had increased expression of androgen receptor. Compared with the DHT group, the finasteride group and the two dosages of flibanserin groups had no significant difference in the content of androgen receptor, indicating that finasteride and flibanserin do not change the total amount of androgen receptor. However, the content of androgen receptor in the cell nucleus of the two dosages of flibanserin group was both decreased compared with the finasteride group and the DHT group. The content in the cytoplasm was both relatively increased, indicating that flibanserin can inhibit the entry of androgen receptor into the nucleus (Figure 9).

Claims

1. 13. Use of flibanserin in the preparation of a drug for the treatment of androgenetic alopecia.

2. 2. The use according to claim 1, characterized in that flibanserin is capable of increasing hair length, anagen to telogen ratio, epidermal to dermal thickness ratio, number of hair follicles per unit area and diameter of hair follicles.

3. 2. The use according to claim 1, characterized in that flibanserin is used in the preparation of a medicament for improving androgen-mediated androgenetic alopecia disorders, treating androgenetic alopecia and restoring hair growth.

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