A transfersome composition and its preparation method and application

Abstract

The present invention discloses a transfersome composition, its preparation method, and application. The transfersome composition comprises the following raw materials, calculated by mass: 5-20 parts of an active drug; 2-40 parts of lecithin; and 2-40 parts of sodium cholate. The active drug comprises a protein drug or a small molecule drug. The present invention coats the protein drug or small molecule drug with lecithin and sodium cholate, resulting in a drug-coated transfersome composition with higher transdermal efficiency, shortened onset time, improved therapeutic efficacy, and avoided drug side effects.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a transfersome composition, a preparation method and an application thereof. Background Art

[0002] Transdermal drug delivery refers to applying or applying drugs to the surface of the skin, allowing the drugs to pass through the skin (stratum corneum, epidermis and dermis), reach the subcutaneous tissue, circulate through the body and finally reach the lesion, achieving the effect of drug treatment.

[0003] Protein drugs are receiving great attention in today's pharmaceutical industry, but due to their large molecular weight and other issues, they are not easy to penetrate the skin and act on specific parts of the body. Traditional routes of administration, such as intravenous administration and subcutaneous injection, are inconvenient and can easily cause pain to patients, making them unsuitable for specific populations. Transdermal administration is convenient and can accurately target the site of action, but it faces the problem of poor transdermal effect due to the large molecular weight of protein drugs, making transdermal administration impossible. At the same time, some clinically observed drugs also have poor water solubility, resulting in poor efficacy. Among them, some drugs also have obvious adverse reactions, which may cause adverse reactions such as skin redness, itching, and allergies.

[0004] The skin's barrier function is a key factor in determining drug penetration rate. Therefore, overcoming this barrier and promoting transdermal drug penetration to achieve a therapeutic dose within a specified timeframe is a key issue in the research of many transdermal drug delivery systems. Different drug molecules with different properties require the screening of different types of transdermal absorption enhancers. There is an urgent need for a broad-spectrum transdermal drug delivery system that can effectively deliver small molecule drugs or protein drugs through the skin. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a transfersome composition that can effectively improve the transdermal efficiency of drugs, shorten the onset time, and enhance the therapeutic effect.

[0006] A second aspect of the present invention also provides a method for preparing the transfersome composition.

[0007] The third aspect of the present invention also provides a medicine.

[0008] The fourth aspect of the present invention also provides the use of the transfersome composition.

[0009] According to a first aspect of the present invention, there is provided a transfersome composition comprising the following raw materials calculated by mass: 5 to 20 parts of active drug; 2 to 40 parts of lecithin; 2 to 40 parts of sodium cholate;

[0010] The active drugs include protein drugs or small molecule drugs.

[0011] The transfersome composition according to the embodiments of the present invention has at least the following beneficial effects:

[0012] The present invention uses lecithin and sodium cholate to coat protein or small molecule drugs, so that the drug-coated transfersome composition has higher transdermal efficiency, shortens the onset time, improves the therapeutic effect and avoids the side effects of the drug.

[0013] According to some embodiments of the present invention, the mass ratio of the lecithin to the sodium cholate is 1:20 to 19:1.

[0014] According to some embodiments of the present invention, the mass ratio of the lecithin to the sodium cholate is 1 to 19:1.

[0015] According to some embodiments of the present invention, the mass ratio of the lecithin to the sodium cholate is 4 to 10:1.

[0016] According to some embodiments of the present invention, the protein drug includes at least one of albumin, insulin, and growth hormone.

[0017] According to some embodiments of the present invention, the small molecule drug includes at least one of minoxidil (MXD), opioid receptor drugs, and tetracycline drugs.

[0018] According to some embodiments of the present invention, the opioid receptor drug includes at least one of morphine, pethidine, fentanyl, dezocine, butorphanol, pentazocine or naloxone.

[0019] According to some embodiments of the present invention, the tetracycline drug includes at least one of chlortetracycline, oxytetracycline, tetracycline, minocycline, methacycline, demeclocycline, doxycycline or metamycin.

[0020] According to some embodiments of the present invention, the small molecule drug comprises minoxidil.

[0021] The transfersome composition according to the present invention takes effect much faster than the commercially available 5% MXD. Hair begins to grow on the 10th day and reaches a normal hair growth rate after 17 days, giving patients with alopecia the ability to grow hair normally. Furthermore, because lecithin and sodium cholate in the transfersome composition according to the present invention are both biocompatible materials, the side effects of 5% MXD, which may cause irritation and contact dermatitis, are avoided.

[0022] The method for preparing the transfersome composition according to the second embodiment of the present invention comprises the following steps:

[0023] S1. Dissolving lecithin and sodium cholate in an organic solvent in a certain proportion, and freeze-drying the mixture to obtain a transfersome;

[0024] S2. Add the transfersome to the active drug solution, and obtain it by ultrasonication and freeze-thawing.

[0025] According to some embodiments of the present invention, in step S1, the freeze-drying conditions include: temperature of -60°C to -100°C; time of 5 hours to 36 hours.

[0026] According to some embodiments of the present invention, in step S2, the mass volume ratio of the transfersomes to the active drug solution is 1 mg-100 mg: 3 mL-10 mL.

[0027] According to some embodiments of the present invention, in step S2, the mass-to-volume ratio of the transfersomes to the active drug solution is 100 mg:1 mL.

[0028] According to some embodiments of the present invention, the mass concentration of the active drug solution is 1% to 50%.

[0029] According to some embodiments of the present invention, the mass concentration of the active drug solution is 2% to 5%.

[0030] According to some embodiments of the present invention, the organic solvent includes at least one of ethanol, acetone, and dimethyl sulfoxide.

[0031] According to some embodiments of the present invention, in step S2, the ultrasonic conditions include: an ultrasonic frequency of 80 to 100 kHz, and an ultrasonic time of 2 to 180 minutes.

[0032] According to some embodiments of the present invention, the transfersome composition is a topical preparation.

[0033] According to some embodiments of the present invention, the transfersome composition is a transdermal administration formulation.

[0034] According to some embodiments of the present invention, the transdermal administration preparation is a liquid or semisolid preparation.

[0035] According to some embodiments of the present invention, the semisolid preparation is a suspension, an emulsion, a microemulsion, a cream, a gel, a foam or an ointment.

[0036] The fourth aspect of the present invention provides the use of the transfersome composition in the preparation of a drug;

[0037] According to some embodiments of the present invention, the medicament is used for preventing and / or treating alopecia, cardiovascular diseases, and skin diseases.

[0038] According to some embodiments of the present invention, the hair loss disorder is seborrheic alopecia, telogen effluvium, or anagen hair loss syndrome.

[0039] Additional features and advantages of the present invention will be set forth in the detailed description which follows, and in part will be obvious from the detailed description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0041] Figure 1 is a graph showing the transdermal efficiency of the transfersome compositions of Examples 1 to 4;

[0042] Figure 2 is an in vivo fluorescence imaging image of the transfersome composition of Example 1 after application to mice;

[0043] Figure 3 is a graph showing the particle size of the transfersome composition of Example 10 of the present invention;

[0044] Figure 4 This figure shows the effects of the 5% MXD solution of Comparative Example 1 and the transfersome composition of Example 10 on hair growth in C57BL / 6 mice;

[0045] Figure 5 3. The morphology of the cell tissues during hair growth in C57BL / 6 mice is shown in Figure 1. The 5% MXD solution of Comparative Example 1 and the transfersome composition of Example 10 are shown in Figure 2. a) shows hair follicle staining and b) shows mast cells stained with toluidine blue.

[0046] Figure 6 This is a graph showing body weight monitoring of C57BL / 6 mice after administration of the 5% MXD solution of Comparative Example 1 and the transfersome composition of Example 10;

[0047] Figure 7 Graphs showing the effects of the 5% MXD solution of Comparative Example 1 and the transfersome composition of Example 10 on hair growth-related components in AGA mice; wherein, a is a graph showing plasma DHT concentration; b is a graph showing mouse IGF-1 mRNA levels; c is a graph showing VEGF mRNA levels; and d is a graph showing the expression of mouse hair growth-related factors (IGF-1, VEGF, and LEF-1); DETAILED DESCRIPTION

[0048] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described in conjunction with the embodiments, but the present invention is not limited to these embodiments.

[0049] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0050] Albino laboratory mice (BALB / c mice) were purchased from Guangdong Animal Center;

[0051] Phosphatidylcholine and sodium cholate were purchased from MacLean Biotechnology;

[0052] BSA and FITC were purchased from Sigma-Aldrich;

[0053] Fluorescently modified bovine serum albumin (BSA-FITC): BSA (1-10 mg / mL) and FITC were reacted in Heppes buffer (pH 8.0) in the dark for 24 hours.

[0054] MXD, hematoxylin, erythrocytes, and toluidine blue were purchased from Sigma-Aldrich;

[0055] Dihydrotestosterone was purchased from Energy Chemical Company;

[0056] The dihydrotestosterone enzyme-linked immunosorbent assay kit was purchased from Lunchangshuo Biotechnology Co., Ltd., Xiamen, China;

[0057] One-step qRT-PCR kit and SYBR Green I were purchased from Shanghai Xinfeng Technology Co., Ltd.;

[0058] PVDF membrane was purchased from Millipore;

[0059] Anti-LEF-1, anti-VEGF, anti-GAPDH antibodies, and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Arigo Biolabs;

[0060] Organic solvents such as xylene, anhydrous ethanol, and dimethyl sulfoxide, as well as inorganic salt reagents such as sodium chloride, were purchased from Guangzhou Chemical Reagent Factory.

[0061] C57BL / 6 mice were purchased from Guangdong Animal Center:

[0062] The detection methods used in the embodiments and comparative examples are as follows:

[0063] Hematoxylin-hemagglutinin (H&E) staining:

[0064] On days 0 and 17 of treatment, mice were depilated and sacrificed, and skin was removed from the treated area on the back. Skin specimens were fixed in 10% formalin for 24 hours, embedded in paraffin, and sectioned according to standard techniques. Histological changes were observed using H&E staining. Dewaxed and hydrated sections were immersed in hematoxylin solution for 10 minutes to stain cell nuclei, then rinsed in tap water for approximately 10 minutes to remove residual hematoxylin. Sections were immersed in 70% ethanol containing 1% hydrochloric acid for several seconds and then rinsed in pH 7.4 PBS to neutralize the blue color. Sections were then immersed in 1% eosin solution for 2 minutes to stain the cytoplasm. After rinsing in tap water, sections were dehydrated in a gradient of 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, 100% ethanol I, and 100% ethanol II for 1 minute each. Sections were then cleared in xylene I and xylene II for 1-2 minutes. Finally, after the xylene had evaporated slightly, a drop of neutral resin was added, and the film was sealed with a coverslip. The quantity, extension and depth of high-frequency signals were observed under a light microscope.

[0065] Toluidine blue staining:

[0066] Observe MCs using toluidine blue staining. Dewaxed and hydrated sections were soaked in toluidine blue staining solution for 10 minutes and washed twice with distilled water for decolorization. Sections were soaked in glacial acetic acid differentiation solution for approximately 30 seconds, and the clarity of the particles was monitored under a microscope. After rinsing with tap water, sections were dehydrated in a gradient of 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, and 100% ethanol for 1 minute each. Sections were transparentized in xylene I and xylene II for 1 to 2 minutes, respectively. Finally, after the xylene evaporated slightly, a drop of neutral resin was added, and the film was sealed with a coverslip. The number of dermal and subcutaneous MCs was counted under a light microscope.

[0067] Enzyme-linked immunosorbent assay (ELISA)

[0068] On day 17 of treatment, blood was collected from the orbits of AGA mice using an EP tube containing 1% sodium heparin, and the supernatant was collected by centrifugation (8000 rpm, 5 minutes). Plasma DHT levels in each group of mice were measured using a dihydrotestosterone enzyme-linked immunosorbent assay (DHT-ELISA). The ELISA was performed according to the manufacturer's instructions for the DHT ELISA kit. Briefly, the supernatant was added to a microtiter plate pre-coated with an anti-mouse DHT monoclonal antibody. The microtiter plate was incubated at 37°C in a humidified environment for 1 hour. After 1 hour, the supernatant was discarded and washed six times with washing solution. Substrate A and reagent B were added to the wells and incubated at 37°C in a humidified environment for 15 minutes. Stop solution was added, and the wells were incubated at 37°C in a humidified environment for 15 minutes. The absorbance was measured at a wavelength of 450 nm using a microplate reader. The OD450nm value was plotted on the vertical axis, and the DHT standard concentration was plotted on the horizontal axis. A scatter plot was then fitted. The DHT concentration in each group of samples was calculated based on the fitted curve.

[0069] Real-time quantitative PCR (qRT-PCR)

[0070] On day 17 of treatment, AGA mice were sacrificed by cervical dislocation, and skin samples from the treated area were collected for testing. IGF-1 and VEGF mRNA levels in each group were measured using qRT-PCR. Total RNA was extracted from the skin using chloroform-isopropanol-ethanol extraction and Trizol reagent. qRT-PCR reactions were performed according to the manufacturer's instructions for the one-step qRT-PCR kit. Briefly, 0.1 μg of total RNA was mixed with LightCycler FastStart DNA Master SYBR Green I and RT enzyme mix. Then, the following components were added to a final volume of 20 μL: 0.5 pmol of specific primers for IGF-1 (for 5'-ctggtcctgtgtgtgtgtgc-3 (seq_1), REV 5'-GGGGACTTCTGAGTCTT-3 (seq_2)), VEGF (for 5'-caacttctgggctcttctcg-3 (seq_3), REV 5'-cctctcctcttccttcttcttcc-3 (seq_4)), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (for 5'-tgcaccaccaactgcttagc-3 (seq_5), REV 5'-ggcatggactgtggtcatag-3 (seq_6)). The mixture was incubated at 42°C for 30 min and 95°C for 10 min before reverse transcription. Amplification was then quantified using 50 cycles of denaturation (95°C for 10 seconds), annealing (66°C for IGF-1, 64°C for VEGF, or 66 or 64°C for GAPDH) for 10 seconds, and extension (72°C for 5 seconds). The differences in IGF-1 / VEGF threshold cycles and GAPDH threshold cycles were used to calculate IGF-1 and VEGF mRNA levels in AGA mice.

[0071] Western blot (WB)

[0072] On day 17 of treatment, AGA mice were sacrificed, and skin samples from the treated areas were collected for testing. Western blotting was used to assess the expression of hair growth-related factors, LEF-1 and VEGF, in each group. Skin tissue was homogenized in cold RIPA buffer containing the protease inhibitor PMSF, centrifuged at 14,000 rpm for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA assay. To detect LEF-1 and VEGF protein expression, total protein (10 μg / well) was separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked in blocking buffer for 1 hour and then rinsed seven times with TBST containing Tris-buffered saline and Tween 20. The membrane was then incubated with anti-LEF-1, anti-VEGF, and anti-GAPDH antibodies overnight at 4°C. After washing again seven times with TBST, the membrane was incubated with an HRP-conjugated secondary antibody for 1 hour at room temperature. Finally, the membrane was rinsed seven more times with TBST and blotted dry with an enhanced chemiluminescence reagent. The membrane was visualized using a chemiluminescence imaging system (Tanon-4600SF) to observe the protein bands.

[0073] Example 1

[0074] Example 1 provides a transfersome composition comprising the following raw materials, calculated by weight: 8 parts of fluorescently modified bovine serum albumin; 35 parts of lecithin; and 5 parts of sodium cholate. The preparation method of the transfersome composition is as follows:

[0075] S1. Dissolve lecithin and sodium cholate in anhydrous ethanol at a mass ratio of 7:1, and add the fluorescently modified bovine serum albumin solution to obtain a mixture; the volume ratio of the mixture to the fluorescently modified bovine serum albumin solution is 1:10;

[0076] S2. The mixture was ultrasonicated for 60 min at a frequency of 100 kHz and then frozen and thawed to obtain a transfersome composition.

[0077] Example 2

[0078] Example 2 provides a transfersome composition comprising the following raw materials calculated by mass: 8 parts of fluorescently modified bovine serum albumin; 38 parts of lecithin; and 2 parts of sodium cholate. The preparation method of the transfersome composition is as follows:

[0079] S1. Dissolving lecithin and sodium cholate in anhydrous ethanol at a mass ratio of 19:1 to obtain a mixed solution, and mixing the fluorescently modified bovine serum albumin solution and the mixed solution to obtain a mixture; the volume ratio of the mixed solution to the fluorescently modified bovine serum albumin solution is 1:10;

[0080] S2. The mixture was ultrasonicated for 60 min at a frequency of 100 kHz and then frozen and thawed to obtain a transfersome composition.

[0081] Example 3

[0082] Example 3 provides a transfersome composition comprising the following raw materials calculated by mass: 8 parts of fluorescently modified bovine serum albumin; 20 parts of lecithin; and 5 parts of sodium cholate. The preparation method of the transfersome composition is as follows:

[0083] S1. Dissolve lecithin and sodium cholate in anhydrous ethanol at a mass ratio of 4:1, and add the fluorescently modified bovine serum albumin solution to obtain a mixture; the volume ratio of the mixture to the fluorescently modified bovine serum albumin solution is 1:10;

[0084] S2. The mixture was ultrasonicated for 60 min at a frequency of 100 kHz and then frozen and thawed to obtain a transfersome composition.

[0085] Example 4

[0086] Example 4 provides a transfersome composition comprising the following raw materials, calculated by weight: 8 parts of fluorescently modified bovine serum albumin; 5 parts of lecithin; and 20 parts of sodium cholate. The preparation method of the transfersome composition is as follows:

[0087] S1. Dissolve lecithin and sodium cholate in anhydrous ethanol at a mass ratio of 0.25:1, and add the fluorescently modified bovine serum albumin solution to obtain a mixture; the volume ratio of the mixture to the fluorescently modified bovine serum albumin solution is 1:10;

[0088] S2. The mixture was ultrasonicated for 60 min at a frequency of 100 kHz and then frozen and thawed to obtain a transfersome composition.

[0089] Example 5

[0090] Example 5 provides a transfersome composition comprising the following raw materials calculated by mass: 8 parts of fluorescently modified bovine serum albumin; 40 parts of lecithin; and 4 parts of sodium cholate. The preparation method of the transfersome composition is as follows:

[0091] S1. Dissolve lecithin and sodium cholate in anhydrous ethanol at a mass ratio of 10:1, and add the fluorescently modified bovine serum albumin solution to obtain a mixture; the volume ratio of the mixture to the fluorescently modified bovine serum albumin solution is 1:10;

[0092] S2. The mixture was ultrasonicated for 60 min at a frequency of 100 kHz and then frozen and thawed to obtain a transfersome composition.

[0093] Example 6

[0094] Example 6 provides a transfersome composition, the component contents and preparation method are the same as those in Example 1, except that the volume ratio of the mixed solution in Example 6 to the fluorescent-modified bovine serum albumin solution is 1:20.

[0095] Example 7

[0096] Example 7 provides a transfersome composition, the component contents and preparation method are the same as those in Example 1, except that the volume ratio of the mixed solution in Example 7 to the fluorescent-modified bovine serum albumin solution is 1:5.

[0097] Example 8

[0098] Example 8 provides a transfersome composition, the component contents and preparation method are the same as those in Example 1, except that the volume ratio of the mixed solution in Example 8 to the fluorescent-modified bovine serum albumin solution is 1:1.

[0099] Example 9

[0100] Example 9 provides a transfersome composition, the component contents and preparation method are the same as those in Example 1, except that the volume ratio of the mixed solution in Example 9 to the fluorescent-modified bovine serum albumin solution is 1:0.5.

[0101] Performance Testing

[0102] BSA-FITC was used as a protein model drug. Therefore, the transfersome compositions of Examples 1 to 4 of the present invention were tested for their transdermal efficiency using a small animal imaging method.

[0103] Specifically, BALB / c mice were acclimated to a 12-hour light / 12-hour dark environment with free access to food and water for one week. The mice were then shaved on their backs and randomly divided into groups. The transfersome compositions of Examples 1 to 4 were applied to the shaved areas. At pre-set time points (2, 5, and 8 hours), the mice were anesthetized and observed for transdermal penetration using a live animal imaging system and photographed. Before each injection, the dorsal area of ​​the mice was rinsed with water to ensure that the fluorescence obtained was emitted by BSA-FITC that had been absorbed by the skin.

[0104] The results are as follows Figure 1 As shown, from Figure 1 It can be found that when the transfersome composition is prepared with lecithin and sodium cholate in a mass ratio of 7:1, its transdermal efficiency is the highest.

[0105] Furthermore, the present invention used BSA-FITC alone as a control, the transfersome composition prepared in Example 1, and the non-freeze-dried transfersome composition of Example 1 as experimental groups. BALB / c mice were acclimated to a 12-hour light / 12-hour dark environment with free access to food and water for one week. The mice were then shaved on their backs and randomly divided into three groups: Group 1: BSA-FITC was applied to the shaved area; Group 2: The transfersome composition prepared in Example 1 was applied to the shaved area; and Group 3: The non-freeze-dried transfersome composition of Example 1 was applied to the shaved area. At pre-set time points (0, 2, 5, 8, and 24 hours), the mice were anesthetized and their transdermal delivery was observed and photographed using a live animal imaging system. Before each injection, the dorsal area of ​​the mice was rinsed with water to ensure that the fluorescence was emitted by BSA-FITC absorbed by the skin.

[0106] The results are as follows Figure 2 As shown, using BSA-FITC alone as a control, based on the fluorescence properties of BSA-FITC, different agents were applied to the shaved area, and the absorption behavior of BSA-FITC was imaged at predetermined time points. The administration site was rinsed before imaging. Strong fluorescence was observed 5 hours after administration and maintained thereafter. Compared to the BSA-FITC group, the non-freeze-dried Example 1 group and the Example 1 group showed stronger fluorescence accumulation 5 hours after administration and persisted for up to 24 hours. The fluorescence of the latter two groups was more pronounced than that of the control group. The fluorescence of the Example 1 group was stronger than that of the non-freeze-dried Example 1 group, demonstrating that the transfersome composition of Example 1 of the present invention has a high transdermal effect.

[0107] Example 10

[0108] Example 10 provides a transfersome composition, comprising the following raw materials calculated by weight: 8 parts minoxidil; 35 parts lecithin; 5 parts sodium cholate; and the preparation method is as follows:

[0109] S1. Dissolve lecithin and sodium cholate in anhydrous ethanol at a mass ratio of 7:1 and freeze-dry using a freeze dryer to obtain transfersomes.

[0110] S2. According to the mass volume ratio of transfersomes to minoxidil solution of 100 mg:1 mL, 5% minoxidil solution was added to the transfersomes, and the transfersome preparation was obtained by ultrasonication for 60 minutes at a frequency of 100 kHz and freeze-thawing.

[0111] like Figure 3 As shown, transmission electron microscopy and DLS characterization show that its particle size is around 100nm-200nm.

[0112] Comparative Example 1

[0113] Comparative Example 1 provides a 5% minoxidil solution, which is prepared as follows:

[0114] Minoxidil was weighed and dissolved in a mixed solvent of propylene glycol, water and ethanol in a volume ratio of 20:30:50, and vortex mixed to obtain a 5% minoxidil solution.

[0115] Performance testing

[0116] The therapeutic effect of the transfersome composition of Example 10 on hair loss:

[0117] Six-week-old male C57BL / 6 mice were selected and acclimated to a 12-hour light / 12-hour dark environment with free access to food for one week. To establish the AGA mouse model, the back hair of the C57BL / 6 mice was shaved and 0.2 mL of 15% dihydrotestosterone dissolved in DMSO was injected subcutaneously. Treatment began 24 hours after the AGA mouse model was established.

[0118] AGA mice were randomly divided into 3 groups (n=6) and administered according to the following conditions: 1) 200 μL 0.9% NaCl; 2) 200 μL 5% minoxidil solution of Comparative Example 1; 3) 200 μL transfersome composition of Example 1. Healthy shaved mice (n=6) served as positive controls. The healthy mouse group was not treated and remained normal. Hair growth was monitored using a digital camera (Cannon, Japan) 1 hour after daily administration, and time points with significant hair growth were selected for display. The grayscale changes in the shaved area (2 cm × 2 cm, 4 cm) were evaluated using image analysis software Image J to evaluate changes in the hair growth area of ​​each group of mice up to the last measurement point (day 17).

[0119] Effects of the transfersome composition of Example 10 on DHT-induced hair growth in AGA mice:

[0120] To evaluate the ability of the transfersome composition to regenerate hair in DHT-induced AGA mice, the mice were randomly divided into three groups (n=6). 200 μL of each of 0.9% NaCl, the 5% minoxidil solution from Comparative Example 1, and the transfersome composition from Example 1 were administered to the dorsal skin of the model mice once daily for 17 consecutive days. A group of healthy, shaved C57BL / 6 mice (n=6) served as controls for normal hair growth rates. Skin pigmentation is considered evidence of hair growth; during the resting phase, the skin appears bright pink, while during the anagen phase, it turns gray / black. Because melanocytes are exclusively present in hair follicles, melanin synthesis closely matches the hair growth cycle.

[0121] like Figure 4As shown in the figure, during the experiment, the dorsal surface of the mice in the model group continued to be pink, indicating that DHT treatment significantly delayed hair regeneration in the mice. In contrast, the skin of the control group mice turned gray on the 10th day after hair removal, followed by the appearance of short hair shafts. The mice in the transfersome composition group of Example 10 also developed gray skin on the 10th day after administration, but the grayness was weaker than that of the healthy mice and significantly stronger than that of the mice in the 5% MXD group of Comparative Example 1 ( Figure 4 ), which indicates that the transfersome composition of Example 1 significantly shortens the effective time of MXD. In addition, as the treatment time is extended to 17 days, the hair growth area of ​​the transfersome composition group of Example 10 is close to that of the control group ( Figure 4 ), indicating that the transfersome composition of Example 10 can restore the hair growth rate of AGA mice to normal levels.

[0122] Furthermore, for histological evaluation of hair follicles, skin tissue was excised from the back on day 17 of the experiment and stained with hematoxylin-eosin. The number of hair follicles, high-frequency depth, and dermal thickness of the 5% MXD group of Comparative Example 1 and the transfersome composition group of Example 10 were observed under an optical microscope. Compared with the model group and the 5% MXD group of Comparative Example 1, the number of hair follicles, high-frequency depth, and dermal thickness of the control group and the transfersome composition group of Example 10 were significantly increased, which supports the morphological observation that the transfersome composition of Example 10 promotes hair growth ( Figure 5 ). As hair growth progresses, hair follicles gradually migrate to the subcutaneous tissue, the epidermis becomes thinner, and the dermis and subcutaneous layer become thicker. Mast cells (MCs) are distributed around the hair follicles, and the mouse hair cycle is closely related to MCs degranulation. Degranulated MCs significantly decrease after hair growth and significantly increase before the onset of hair degeneration. The number of MCs can be measured by toluidine blue staining. In this study, after the 17th day of treatment, the number of MCs in the control group and the transfersome composition group of Example 10 was significantly lower than that in the model group and the 5% MXD group of Comparative Example 1 ( Figure 5 ). Therefore, the transfersome composition of Example 10 significantly prolonged the growth period and promoted hair growth. In order to verify the safety of the transfersome composition of Example 10, the body weight of mice was monitored and no significant difference was found in body weight ( Figure 6 ), and the experiment of treating mice with the transfersome composition of Example 10 ( Figure 4 ), no adverse reactions were observed. In summary, morphological and histological observations revealed that the transfersome composition of Example 10 significantly promoted hair growth in patients with alopecia, and even restored the hair growth rate to normal levels. Furthermore, no inflammation or MXD powder residue was observed on the skin after treatment with the transfersome composition of Example 10.

[0123] Changes in the levels of hair growth-related factors in AGA mice after treatment with the transfersome composition of Example 10:

[0124] The changes in DHT levels in mice treated with the transfersome composition of Example 10 were studied. DHT levels in the blood of AGA mice were measured using an ELISA kit after repeated administration. DHT induces hair follicles to enter a regression phase associated with apoptosis by increasing the level of transforming growth factor (TGF)-β2. Figure 7 As shown, after 17 days of treatment, the transfersome composition of Example 10 significantly inhibited DHT in AGA-molded mice compared with the model group and 5% MXD of Control Example 1, demonstrating that the transfersome composition of Example 10 can increase the amount of MXD introduced into the body, thereby significantly reducing the level of dihydrotestosterone.

[0125] Real-time fluorescence quantitative PCR (RT-PCR) and immunoblotting (WB) were also used to detect changes in the expression levels of hair growth-related proteins in AGA mice. It is well known that insulin-like growth factor-1 (IGF-1) activates hair root cells and inhibits the degeneration of the hair growth cycle. Vascular endothelial growth factor (VEGF) can trigger hair growth, angiogenesis and vasculogenesis, while lymphocyte enhancer binding factor-1 (LEF-1) can promote the normal development of hair by regulating the interaction between epithelial cells and mesenchymal cells. Therefore, we selected IGF-1, VEGF and LEF-1 as target proteins to demonstrate the therapeutic effect of the transfersome composition of Example 10. After treatment with the transfersome composition of Example 10, the IGF-1 and VEGF mRNA levels in AGA mice were higher than those in the model group and the 5% MXD group of Comparative Example 1, and the VEGF and LEF-1 protein levels were higher than those in the 5% MXD group of Comparative Example 1 ( Figure 7 ). This shows that the transfersome composition of Example 10 can improve the therapeutic effect.

[0126] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the scope of the present invention.

Claims

1. A transfersome composition, characterized in that The preparation comprises the following raw materials calculated by weight: 5 to 20 parts of active drug; 2 to 40 parts of lecithin; 2 to 40 parts of sodium cholate; The active drug includes a protein drug or a small molecule drug; the protein drug is selected from albumin; the small molecule drug is selected from minoxidil; The mass ratio of the lecithin to the sodium cholate is 4 to 10:1; The transfersome composition is prepared by the following method: S1, dissolving lecithin and sodium cholate in an organic solvent, and freeze-drying to obtain transfersomes; S2. Adding the transfersome to the active drug solution, and performing sonication and freeze-thawing to obtain a transfersome composition.

2. The transfersome composition according to claim 1, wherein The mass volume ratio of the transfersome to the active drug solution is 1 mg-100 mg: 3 mL-10 mL.

3. The transfersome composition according to claim 2, wherein The mass concentration of the drug solution is 1% to 50%.

4. The transfersome composition according to claim 1, wherein The organic solvent includes at least one of ethanol, acetone, and dimethyl sulfoxide.

5. A drug, characterized in that A transfersome composition comprising the transfersome composition according to any one of claims 1 to 4.

6. The drug according to claim 5, characterized in that The medicine is an external preparation.

7. The drug according to claim 5, characterized in that The medicine is a transdermal preparation.

8. Use of the transfersome composition according to any one of claims 1 to 4 in the preparation of a medicament.

9. The use according to claim 8, characterized in that The medicine is used for preventing and / or treating alopecia, cardiovascular diseases and skin diseases.

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