Ionic liquid for treating androgen-derived alopecia as well as preparation method and application of ionic liquid

Abstract

The invention discloses an ionic liquid for treating androgen-derived alopecia as well as a preparation method and application thereof, and belongs to the technical field of medicines. Through the composite molecular structure design of the quaternary ammonium salt-taurine ionic liquid, the ionic liquid integrates ROS removal, stem cell activation, efficient transdermal and long-acting retention, so that the ionic liquid can directly act on a core pathological link of androgen-derived alopecia; and safe and efficient treatment without drug combination is effectively achieved.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical technology and relates to an ionic liquid for treating androgenetic alopecia, its preparation method and application, particularly an ionic liquid for treating androgenetic alopecia with its own hair growth-promoting activity, its preparation method and application. Background Technology

[0002] Androgenetic alopecia (AGA) is the most common type of hair loss in clinical practice. It is caused by non-cyclical upregulation of androgens, leading to damage to hair follicle function and significantly impacting patients' appearance and mental health. However, clinical treatment often focuses on oral finasteride and topical minoxidil, which have limitations such as low transdermal absorption efficiency, limited bioavailability, and the potential for skin irritation with long-term use.

[0003] Currently, drug delivery systems using ionic liquids as carriers can effectively improve the problems encountered in the above-mentioned drug treatments and have become a research hotspot in hair loss treatment. For example, using choline-ferulic acid ionic liquids as delivery carriers to load hydrophobic drugs can promote transdermal drug penetration and reduce skin irritation by disrupting the lipid bilayer of the scalp stratum corneum; using choline-geranic acid ionic liquids as delivery carriers can prolong the local residence time on the scalp and improve bioavailability by increasing drug solubility. In addition, current ionic liquids are all focused on the "carrier function," that is, improving drug delivery efficiency through the structural properties of ionic liquids. However, their own hair growth-promoting activity is insufficient, and they need to be used in combination with drugs to achieve the ideal therapeutic effect.

[0004] However, the therapeutic strategy of using ionic liquids as delivery carriers in combination with drugs still has shortcomings. For example, ionic liquids have poor compatibility with some drugs, limiting their applicability. Combination therapy may also pose safety risks and increase the complexity of the development process. Most importantly, it cannot fundamentally solve the problem of drug irritation to the skin, making it difficult to simultaneously meet the treatment needs of "high efficacy" and "safety". Summary of the Invention

[0005] This application discloses an ionic liquid for the treatment of androgenetic alopecia. Specifically, through molecular structure design, the ionic liquid is endowed with excellent hair growth-promoting activity, thus achieving a highly effective therapeutic effect without the need for combination with exogenous drugs. This is expected to simplify the development process and reduce the risk of side effects, providing a new technical path for the treatment of androgenetic alopecia.

[0006] To achieve the above objectives, the technical solution provided in this application is as follows:

[0007] The first aspect of this application provides an ionic liquid for treating androgenetic alopecia with its own hair growth-promoting activity, the ionic liquid for treating androgenetic alopecia comprising a quaternary ammonium salt-taurine ionic liquid.

[0008] According to the disclosure of the first aspect, the quaternary ammonium salt component in the quaternary ammonium salt-taurine ionic liquid is one of choline hydroxide, choline bicarbonate and betaine.

[0009] According to the disclosure of the first aspect, the molar ratio of the quaternary ammonium salt component to the taurine component in the quaternary ammonium salt-taurine ionic liquid is 1:1.

[0010] According to the disclosure of the first aspect, the quaternary ammonium salt-taurine ionic liquid is administered transdermally via topical application and can promote hair growth by activating hair follicle stem cells.

[0011] According to the disclosure of the first aspect, the dosage of the quaternary ammonium salt-taurine ionic liquid is 10 to 100 mg / kg.

[0012] According to the disclosure of the first aspect, the dosage of the quaternary ammonium salt-taurine ionic liquid is 50 mg / kg.

[0013] The second aspect of this application also discloses a method for preparing an ionic liquid for treating androgenetic alopecia with its own hair growth-promoting activity, comprising the following steps:

[0014] Taurine aqueous solution and quaternary ammonium salt aqueous solution are reacted at low temperature with stirring for 3-5 hours, and then the aqueous solvent is removed to obtain the ionic liquid for treating androgen-induced alopecia with its own hair growth-promoting activity.

[0015] According to the disclosure of the second aspect, the temperature of the low-temperature stirring is maintained at 0~20 ℃, and the stirring speed is 400~800 r / min.

[0016] According to the disclosure of the second aspect, the method for removing water solvent includes freeze drying, vacuum distillation, and vacuum drying.

[0017] The third aspect of this application also discloses the use of the ionic liquid for treating androgenetic alopecia, which has its own hair growth-promoting activity, in the preparation of drugs for the prevention and / or treatment of androgenetic alopecia.

[0018] Compared with the prior art, the advantages or beneficial effects of this application include at least the following:

[0019] This invention utilizes the design of a composite molecular structure of quaternary ammonium salt and taurine to create an ionic liquid with multiple targeting mechanisms, achieving a safe and highly effective treatment for androgenetic alopecia at its root. Specifically: First, the ionic liquid can specifically remove excess ROS in the hair follicle microenvironment and promote the activation of hair follicle stem cells, thus efficiently promoting hair growth without the need for combination with hair loss treatment drugs; second, the ionic liquid possesses high self-penetration efficiency and a long local retention time on the scalp, maintaining an effective concentration without relying on exogenous drugs, thereby effectively avoiding side effects such as skin irritation caused by combination with drugs; third, the ionic liquid also exhibits excellent biocompatibility and a mild metabolic pathway, ensuring the safety of treatment; fourth, the ionic liquid can be loaded with both hydrophobic and hydrophilic drugs, effectively expanding the compatibility between the ionic liquid and drugs. In summary, this invention, through the design of this composite molecular structure, integrates "ROS clearance, stem cell activation, highly efficient transdermal absorption, and long-lasting retention" into an ionic liquid. This allows the ionic liquid to directly target the core pathological aspects of androgenetic alopecia, effectively achieving safe and efficient treatment without the need for combined drug use. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The 1H NMR spectrum of CTIL1 provided in this application;

[0022] Figure 2 The infrared spectrum of CTIL1 provided in this application;

[0023] Figure 3 The cytotoxicity test results of CTIL1 provided for this application;

[0024] Figure 4 Fluorescent images and semi-quantitative analysis diagrams of CTIL1 clearing cellular ROS provided in this application;

[0025] Figure 5 Quantitative results of CTIL1 percutaneous penetration provided for this application;

[0026] Figure 6 Inverted fluorescence microscope images and semi-quantitative analysis diagrams of CTIL1 loaded with different fluorescent molecules percutaneously provided for this application;

[0027] Figure 7Comparison of hair regeneration in androgenetic alopecia model mice after different treatments provided in this application;

[0028] Figure 8 H&E staining and semi-quantitative analysis of hair follicle length in mice with androgenetic alopecia model provided in this application after different treatments;

[0029] Figure 9 SOX9 staining and semi-quantitative analysis of the back skin of mice with androgenetic alopecia model provided in this application after different treatments;

[0030] Figure 10 The images show DHE staining and semi-quantitative analysis of the back skin of mice with androgenetic alopecia model provided in this application after different treatments. Detailed Implementation

[0031] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments described in this application without creative effort are within the scope of protection of this application.

[0032] In the following description of this application, the term "and / or" is used to describe the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, B existing alone, and A and B existing simultaneously. Here, A and B can be singular or plural; the symbol " / " means "or".

[0033] In the following description of this application, the term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions mean any combination of such items, including any combination of single or plural items. For example, "at least one of A, B or C", or "at least one of A, B and C", can mean any one of A, B, and C, or A+B, or A+C, or B+C, or A+B+C, where A, B, and C can be single or multiple.

[0034] In the following description of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and does not constitute any limitation on the execution process of this application.

[0035] In the following description of this application, the numerical range should be understood to also specifically disclose each intermediate value between the upper and lower limits of the range. Any intermediate value within a stated range, as well as any other stated value or each smaller range between intermediate values ​​within a stated range, are also included in this embodiment, and the upper and lower limits of the smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, the technical / scientific terms used in this application have the meanings commonly understood by one of ordinary skill in the art. While this application describes only preferred materials and methods, similar or equivalent methods and materials may be used in specific embodiments or test cases. All references to this application are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this application shall prevail.

[0037] To address the shortcomings of using ionic liquids as delivery carriers in combination with hair loss treatment drugs when treating androgenetic alopecia, the first aspect of this application provides an ionic liquid for treating androgenetic alopecia with its own hair growth-promoting activity, wherein the ionic liquid for treating androgenetic alopecia is selected as a quaternary ammonium salt-taurine ionic liquid.

[0038] This application's embodiments utilize the design of a composite molecular structure of quaternary ammonium salt and taurine to enable the ionic liquid to possess multiple targeting mechanisms, achieving a safe and highly effective treatment for androgenetic alopecia from its root cause. Specifically:

[0039] (1) The composite molecular structure ionic liquid has specific antioxidant active sites, which can specifically remove excess ROS in the hair follicle microenvironment and promote the activation of hair follicle stem cells, thus promoting hair growth efficiently without the need to be used in combination with hair loss treatment drugs.

[0040] (2) The composite molecular structure ionic liquid has a high self-penetration efficiency and a long local skin retention time. It can maintain an effective concentration without relying on exogenous drugs, thus effectively avoiding side effects such as skin irritation caused by combined use with drugs.

[0041] (3) This composite molecular structure ionic liquid is formed by combining natural choline and taurine, thus possessing excellent biocompatibility and a mild metabolic pathway, which can ensure the safety of treatment.

[0042] (4) The composite molecular structure ionic liquid can also be used to load hydrophobic drugs and hydrophilic drugs respectively, effectively expanding the compatibility between ionic liquids and drugs.

[0043] In summary, this invention utilizes a composite molecular structure to integrate "ROS clearance, stem cell activation, highly efficient transdermal absorption, and long-lasting retention" into an ionic liquid. This allows the ionic liquid to directly target the core pathological aspects of androgenetic alopecia, effectively achieving safe and efficient treatment without the need for combined drug therapy.

[0044] In possible disclosed examples, the quaternary ammonium salt component in the quaternary ammonium salt-taurine ionic liquid of the present invention is preferably one of choline hydroxide, choline bicarbonate, and betaine.

[0045] In a possible disclosed example, the molar ratio of the quaternary ammonium salt component to the taurine component in the quaternary ammonium salt-taurine ionic liquid of the present invention is 1:1.

[0046] In possible public examples, the quaternary ammonium salt-taurine ionic liquid of the present invention is administered transdermally via topical application and can promote hair growth by activating hair follicle stem cells.

[0047] In possible disclosed examples, the dosage of the quaternary ammonium salt-taurine ionic liquid of the present invention is 10~100 mg / kg, which can be 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, preferably 50 mg / kg.

[0048] In a second aspect, embodiments of this application also provide a method for preparing the ionic liquid for treating androgenetic alopecia with its own hair growth-promoting activity, comprising the steps of:

[0049] Taurine aqueous solution and quaternary ammonium salt aqueous solution are reacted at low temperature with stirring for 3-5 hours, and then the aqueous solvent is removed to obtain the ionic liquid for treating androgen-induced alopecia with its own hair growth-promoting activity.

[0050] It should be noted that the present invention does not particularly limit the concentration of the taurine aqueous solution and the quaternary ammonium salt aqueous solution, as long as the concentration is sufficient to fully dissolve the taurine and the quaternary ammonium salt. These concentrations can be prepared by the user or obtained commercially. For example, the taurine aqueous solution can be 46.9 mg / mL; the choline hydroxide aqueous solution can be 44% w / w; and the choline bicarbonate aqueous solution can be 80% w / w. Meanwhile, the volume of the taurine aqueous solution and the quaternary ammonium salt aqueous solution is calculated based on a molar ratio of taurine to quaternary ammonium salt of 1:1.

[0051] In possible disclosed examples, the temperature for low-temperature stirring in this invention is preferably maintained at 0~20 ℃, which can be 0 ℃, 5 ℃, 10 ℃, 15 ℃, 20 ℃ and any one of the ranges; the stirring speed is preferably 400~800 r / min, which can be 400 r / min, 500 r / min, 600 r / min, 700 r / min, 800 r / min and any one of the ranges.

[0052] In possible public examples, the present invention can remove water solvents by various well-known dehydration measures suitable for removing ionic liquids, such as freeze drying, vacuum distillation, and vacuum drying. Furthermore, this application does not limit the specific operation of various dehydration measures, and can reasonably select them according to the actual situation.

[0053] Thirdly, embodiments of this application also provide the application of the ionic liquid for treating androgenetic alopecia, which has its own hair growth-promoting activity, as described above, in the preparation of drugs for the prevention and / or treatment of androgenetic alopecia. Since the ionic liquid for treating androgenetic alopecia of this invention has its own hair growth-promoting activity, it can safely and effectively treat androgenetic alopecia without needing to be used in combination with other hair loss treatment drugs, fundamentally solving the various shortcomings and disadvantages faced when using ionic liquids as delivery carriers in combination with hair loss treatment drugs.

[0054] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0055] Example 1

[0056] This example provides a method for preparing choline hydroxide-taurine ionic liquid (CTIL1), the specific steps of which are as follows:

[0057] S1: 1877.2 mg of taurine (Tau) was ultrasonically dissolved in deionized water to prepare an aqueous solution of taurine with a concentration of 46.9 mg / mL;

[0058] S2: Under the stirring conditions of ice-water bath at 0℃ and 500r / min, 44% w / w choline hydroxide aqueous solution was added dropwise to the taurine aqueous solution prepared in the previous step at a molar ratio of choline hydroxide to taurine of 1:1. After the addition was completed, stirring was continued for 4 h. After the stirring was completed, the mixture was concentrated at 60℃ for 2 h using a rotary evaporator, and then freeze-dried for 24 h to obtain a light yellow transparent viscous choline hydroxide-taurine ionic liquid (CTIL1).

[0059] Example 2

[0060] This example provides a method for preparing a choline bicarbonate-taurine ionic liquid (CTIL2), the specific steps of which are as follows:

[0061] S1: 1877.2 mg of taurine (Tau) was ultrasonically dissolved in deionized water to prepare an aqueous solution of taurine with a concentration of 46.9 mg / mL;

[0062] S2: Under the stirring conditions of ice-water bath at 0℃ and 500r / min, an 80% w / w aqueous solution of bicarbonate choline was added dropwise to the taurine aqueous solution prepared in the previous step at a molar ratio of bicarbonate choline to taurine of 1:1. After the addition was completed, stirring was continued for 4 h. After the stirring was completed, the mixture was concentrated at 60℃ for 2 h using a rotary evaporator, and then freeze-dried for 24 h to obtain the bicarbonate choline-taurine ionic liquid (CTIL2).

[0063] Example 3

[0064] This example provides a method for preparing a betaine-taurine ionic liquid (CTIL3), the specific steps of which are as follows:

[0065] S1: 1877.2 mg of taurine (Tau) was ultrasonically dissolved in deionized water to prepare an aqueous solution of taurine with a concentration of 46.9 mg / mL;

[0066] S2: Under the stirring conditions of ice-water bath at 0℃ and 500r / min, the betaine aqueous solution was added dropwise to the taurine aqueous solution prepared in the previous step at a molar ratio of betaine to taurine of 1:1. After the addition was completed, the mixture was stirred for 8 hours. After stirring was completed, the mixture was concentrated at 60℃ for 2 hours using a rotary evaporator. Then, the mixture was vacuum dried for 24 hours to obtain a colorless, transparent, and viscous betaine-taurine ionic liquid (CTIL3).

[0067] To illustrate the technical effects of the quaternary ammonium salt-taurine ionic liquid prepared by the present invention, this application also provides Comparative Example 1.

[0068] Comparative Example 1

[0069] This comparative example provides a method for preparing a saturated aqueous solution of taurine, the specific steps of which are as follows:

[0070] Add excess taurine (Tau) to deionized water, and shake at 100 rpm for 24 hours at a constant temperature of 37°C. Filter the solution using a 0.22 μm membrane filter, collect the supernatant, and dilute it to obtain a saturated aqueous solution of taurine.

[0071] Test case

[0072] 1.1 Characterization of the composite molecular structure

[0073] The choline hydroxide-taurine ionic liquid (CTIL1) prepared in Example 1 was characterized by 1H NMR and IR spectroscopy, respectively. The results were as follows: Figures 1 to 2 As shown. Among them, Figure 1 The 1H NMR spectrum of the choline hydroxide-taurine ionic liquid (CTIL1) is shown. Figure 2 The infrared spectrum of choline hydroxide-taurine ionic liquid (CTIL1).

[0074] according to Figure 1 It can be seen that the ionic liquid contains both methylene (a, b) of taurine and methyl (c) and methylene (d, e) of choline hydroxide, indicating the successful preparation of the choline hydroxide-taurine ionic liquid.

[0075] according to Figure 2 It can be seen that, compared with the monomer components (Tau, ChOH), the spectral bands of the ionic liquid (CTIL1) exhibit broadening and shifting, specifically the sulfonate (S=O, ≈1040-1180 cm⁻¹). -1 The occurrence of stretching displacement indicates an interaction between choline hydroxide and taurine; changes in the fingerprint region indicate the emergence of a unique new vibrational mode in CTIL1, suggesting the formation of a choline hydroxide-taurine ionic liquid.

[0076] 1.2 Taurine content test

[0077] The taurine content in the above-mentioned choline hydroxide-taurine ionic liquid (CTIL1) and saturated taurine aqueous solution was determined by high performance liquid chromatography (HPLC), and the results are recorded in Table 1. The chromatographic conditions were as follows: C18 column, 250 × 4.6 mm, 5 μm column, temperature 25 ℃; flow rate 1 mL / min; injection volume 20 μL; detection wavelength 254 nm; mobile phase: sodium acetate buffer solution (15 mM, pH 4.2): acetonitrile = 70:30.

[0078] Table 1: Taurine content in different systems

[0079]

[0080] As shown in Table 1, the taurine content in the choline hydroxide-taurine ionic liquid is much greater than that in the saturated aqueous solution, indicating that the choline hydroxide-taurine ionic liquid provided by the present invention can promote the dissolution of taurine.

[0081] 1.3 Cytotoxicity test

[0082] This application investigated the toxicity of CTIL1 to L929 (mouse fibroblasts) and HUVEC (human umbilical vein endothelial cells) using the MTT assay. Specifically, cells were seeded in 96-well plates, and different concentrations of CTIL (equivalent Tau concentrations of 1–2000 μg / mL) were prepared using serum-free medium. Cells were injected when the cell density reached approximately 60%, with four replicates per group. 24 h after injection, the culture plates were removed, the supernatant was discarded, and MTT solution (0.5 mg / mL, 100 μL / well, prepared using serum-free medium) was added. The plates were then incubated for another 4 h. Afterward, the 96-well plates were removed, inverted to discard the solution in each well, and 150 μL of dimethyl sulfoxide was added to each well. The plates were then incubated at 37 ℃ for 10 min on a shaker. Finally, the absorbance of each well at 490 nm was read using a microplate reader, and cell viability was calculated according to formula (1). The results were as follows: Figure 3 As shown. Among them, Figure 3 The results of the cytotoxicity test for the choline hydroxide-taurine ionic liquid CTIL1 are shown.

[0083] (1)

[0084] Among them, A x A0 is the absorbance of the experimental well, and A0 is the absorbance of the zeroing well. c The absorbance of the negative control well is shown.

[0085] according to Figure 3 It can be seen that the survival rate of L929 and HUVEC cells after CTIL1 treatment is above 80%, indicating that the choline hydroxide-taurine ionic liquid has good biocompatibility.

[0086] 1.4 In vitro antioxidant experiment of choline hydroxide-taurine ionic liquid CTIL1

[0087] This application uses the DCFH-DA probe to evaluate the ability of CTIL1 to scavenge ROS. Specifically, L929 cells were seeded in 12-well plates; CTIL1 was dissolved in serum-free medium containing 500 μM hydrogen peroxide, with the serum-free medium containing hydrogen peroxide serving as a control; when the cell density reached approximately 60%, the medium was replaced with a mixture of 2000 μg / mL CTIL and 500 μM hydrogen peroxide and incubated for 4 h. The supernatant was then discarded, the cells were washed three times with PBS, and then incubated with the DCFH-DA probe for another 30 min. The ROS status of the cells was qualitatively observed using a fluorescence microscope. The results were as follows: Figure 4 As shown. Among them, Figure 4 Fluorescent images and semi-quantitative analysis of CTIL1 clearing cellular ROS.

[0088] according to Figure 4 It is evident that intracellular ROS levels significantly increased after hydrogen peroxide stimulation, while the presence of CTIL1 effectively cleared ROS and prevented cellular oxidative stress damage.

[0089] 1.5 In vitro transdermal and retention experiments of choline hydroxide-taurine ionic liquid CTIL1

[0090] This application investigates the in vitro transdermal efficacy of CTIL1 using the Franz diffusion cell method. Specifically, 6-7 week old male C57BL / 6 mice were anesthetized, their back hair was removed with a razor, and they were euthanized by neck dislocation. Immediately afterward, the hairless skin was separated using a scalpel and forceps to remove subcutaneous tissue, blood vessels, and fat. The skin was repeatedly rinsed with physiological saline and then immersed in physiological saline for later use. Care was taken to ensure that the skin remained intact throughout the process.

[0091] The water temperature in the Franz diffusion cell was adjusted to maintain a constant temperature of 32 ± 1 °C throughout the experiment. Mouse skin samples were removed from the saline solution, dried with absorbent paper, and then fixed between the supply and receiving cells, with the stratum corneum facing the supply cell, resulting in an effective permeation area of ​​1.77 cm². 2 The receiving cell was filled with PBS (PBS) to prevent air bubbles. The PBS concentration was 14 mL, and the magnetic stirrer was set to 350 rpm. 400 μL of the ionic liquid was applied to the skin, and the top of the receiving cell was covered with plastic wrap to prevent evaporation. Taurine suspension (Tau sus) served as a control. Subsequently, at 1, 2, 4, 8, 12, and 24 h, 2 mL of the receiving solution was collected in EP tubes. An equal volume of receiving solution was replenished promptly after sampling to prevent air bubbles. The collected samples were derivatized and then analyzed by HPLC to calculate the taurine content and cumulative permeability. Finally, a time (h) versus cumulative permeability per unit area (µg / cm²) plot was generated. 2 The curve, the result is Figure 5 As shown in Table 2.

[0092] in, Figure 5 This is the quantitative result of CTIL1 percutaneous penetration.

[0093] Table 2: Quantitative results of CTIL1 and Taussus percutaneous penetration

[0094]

[0095] according to Figure 5As shown in Table 2, the amount of Tau permeation in both the CTIL1 group and the control group gradually increased over time, but the amount of permeation in the CTIL1 group was always greater than that in the control group. At 24 h, the cumulative permeation and retention in the CTIL1 group were significantly higher than those in the control group, indicating that the choline hydroxide-taurine ionic liquid can effectively promote the transdermal permeation and retention of Tau, which is beneficial to improving the efficacy of hair regeneration.

[0096] 1.6 Transdermal Experiments of Choline Hydroxide-Taurine Ionic Liquids Loaded with Different Fluorescent Molecules

[0097] This application uses Did as a representative hydrophobic molecule and RhB as a representative hydrophilic molecule to investigate the transdermal penetration of different molecules promoted by CTIL1 using an inverted fluorescence microscope. Specifically, 6-7 week old male C57BL / 6 mice were anesthetized and their backs were shaved with rosin paraffin. After 24 hours of skin recovery, a PBS solution of free fluorescein or an ionic liquid formulation of fluorescein was applied topically. The mice were divided into four groups, named Free Did, Did@IL, Free RhB, and RhB@IL. 24 hours after administration, the mice were euthanized by cervical dislocation, and the skin was quickly removed. The skin at the administration site was washed with PBS and then fixed in 4% paraformaldehyde for 24 hours. Frozen sections were prepared, and fluorescence images were taken to examine the transdermal penetration of different formulations. The results were as follows: Figure 6 As shown. Among them, Figure 6 Inverted fluorescence microscope images and semi-quantitative analysis diagrams of CTIL1 loaded with different fluorescent molecules percutaneously.

[0098] according to Figure 6 It is evident that the PBS solution and ionic liquid formulations of the two fluorescent molecules exhibit different distributions in the dermis after transdermal administration. The PBS solution group is mainly distributed in the outermost stratum corneum, while the ionic liquid group penetrates further into the dermis, indicating that the ionic liquid promotes the transdermal effect of the two fluorescent molecules.

[0099] 1.7 Evaluation of the therapeutic efficacy of choline hydroxide-taurine ionic liquid on androgenetic alopecia mice

[0100] 17.1 Animal model construction and dosing regimen:

[0101] Six-week-old male C57BL / 6 mice were acclimatized for one week and then randomly divided into four groups: a healthy control group (Control), a model group (Model), a minoxidil group (Minoxidil), and an ionic liquid group (CTIL1). The healthy control group received no treatment, while the other three groups received intraperitoneal injections of DHT (1 mg / mouse) daily for four consecutive days. On day 5, all mice underwent dorsal hair removal using rosin paraffin. After one day of skin recovery, daily dorsal application of the drug was initiated, with dosages of CTIL1 = 50 mg / kg and Minoxidil (5%) = 100 μL / kg. The first treatment was recorded as day 1, and the treatment continued for four consecutive days with a one-day break. Intraperitoneal injections of DHT continued throughout the treatment process until the end of the experiment.

[0102] Efficacy evaluation: During the experiment, photographs were taken of the backs of mice to observe skin changes and hair growth at different time points. Midway through the experiment, back skin was harvested; a portion was used for H&E sectioning to observe hair growth in mice after different treatments and to quantitatively analyze hair follicles. The other portion underwent immunofluorescence staining, including SOX9 and DHE staining, to examine hair follicle activation and oxidative stress. The results were as follows: Figures 7-10 As shown. Among them, Figure 7 Comparison of hair regeneration in androgen-induced alopecia model mice after different treatments; Figure 8 H&E staining and semi-quantitative analysis of hair follicle length in mice with androgenetic alopecia model after different treatments; Figure 9 SOX9 staining and semi-quantitative analysis of dorsal skin in mice with androgenetic alopecia after different treatments; Figure 10 This image shows DHE staining and semi-quantitative analysis of the back skin of mice with androgenetic alopecia after different treatments.

[0103] according to Figure 7 As can be seen, on the first day of treatment, the skin of mice in all groups was pink, indicating that the hair follicles in the hair-removed areas of mice in all groups were in the resting phase at this time; after treatment with different protocols, there was obvious hair regrowth compared with the model group.

[0104] according to Figure 8 It can be seen that on day 10, many hair bulbs began to swell and melanin was produced in all groups. However, the hair follicles in the healthy group and the CTIL1-treated group were more swollen and longer. Quantitative analysis showed that the hair follicles in the CTIL1 group were significantly longer than those in the model group and the minoxidil group, indicating that choline hydroxide-taurine ionic liquid has a better effect on promoting hair growth.

[0105] according to Figure 9 As can be seen, the SOX9 fluorescence intensity in the CTIL1 group was significantly enhanced compared to the model group, indicating that hair follicle stem cells were activated.

[0106] according to Figure 10 It is evident that the DHE probe can be oxidized in the presence of reactive oxygen species in the tissue. The oxidation products bind to intracellular nucleic acids to produce fluorescence, and the intensity of the fluorescence reflects the level of reactive oxygen species in the tissue. This indicates that the choline hydroxide-taurine ionic liquid effectively removes reactive oxygen species in the hair follicle microenvironment, improves the oxidative stress microenvironment, and promotes hair regeneration.

[0107] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0108] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. An ionic liquid for treatment of androgen-source alopecia having self-promoting hair growth activity, characterized by, The ionic liquid comprises a quaternary ammonium salt-taurine.

2. The ionic liquid for use in the treatment of androgenetic alopecia according to claim 1, characterized in that, The quaternary ammonium salt component in the quaternary ammonium salt-taurine ionic liquid is one of choline hydroxide, choline bicarbonate and betaine.

3. The ionic liquid for use in the treatment of androgenetic alopecia according to claim 1, characterized in that, The molar ratio of the quaternary ammonium salt component to the taurine component in the quaternary ammonium salt-taurine ionic liquid is 1:

1.

4. The ionic liquid for use in the treatment of androgenetic alopecia according to claim 3, characterized in that The quaternary ammonium salt-taurine ionic liquid is applied topically for transdermal administration and can promote hair growth by activating hair follicle stem cells.

5. The ionic liquid for use in the treatment of androgenetic alopecia according to claim 4, characterized in that The administration dose of the quaternary ammonium salt-taurine ionic liquid is 10-100 mg / kg.

6. The ionic liquid for use in the treatment of androgenetic alopecia according to claim 5, characterized in that The administration dose of the quaternary ammonium salt-taurine ionic liquid is 50 mg / kg.

7. A method for producing the ionic liquid for treating androgenic alopecia having self-promoting hair growth activity according to any one of claims 1 to 6, characterized by, The method comprises: The aqueous taurine solution is contacted with the aqueous quaternary ammonium salt solution under low-temperature stirring for 3-5 hours, and then the aqueous solvent is removed to obtain the ionic liquid for treating androgenetic alopecia with self-promoting hair growth activity.

8. The preparation method according to claim 7, characterized in that, The temperature of the low-temperature stirring is kept at 0-20 ℃, and the stirring speed is 400-800 r / min.

9. The method of claim 6 wherein the step of forming the first and second layers comprises the step of: The method for removing the aqueous solvent comprises freeze-drying, reduced-pressure distillation and vacuum drying. ​ 10. Use of the ionic liquid for treating androgenetic alopecia with self-promoting hair growth activity according to any one of claims 1-5 in the preparation of a medicine for preventing and / or treating androgenetic alopecia.

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