Composition and microneedle for treating alopecia as well as preparation method and application of composition and microneedle

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a composition and a microneedle for treating alopecia as well as a preparation method and application of the composition and the microneedle. The composition for treating alopecia comprises arginine and a pentacyclic triterpenoid compound, the composition is a co-amorphous substance. The arginine and the pentacyclic triterpenoid have a synergistic effect, and compared with a suspension formed by directly mixing the arginine and the pentacyclic triterpenoid which are independently used, the treatment effect on alopecia is remarkably improved. A co-amorphous system formed by the arginine and the pentacyclic triterpenoid can up-regulate a Wnt / beta-catenin pathway, improve the microenvironment of oxidative stress around hair follicles and insufficient angiogenesis, provide favorable conditions for growth of the hair follicles, and can effectively prevent and treat alopecia. In addition, the co-amorphous system is further processed into the microneedle, so that the treatment effect can be improved. Meanwhile, due to the separable design of the microneedle, the medicine can be effectively retained in the skin and continuously released, and the administration frequency can be reduced.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to a composition for treating hair loss, a microneedle, and a preparation method and application thereof. Background Art

[0002] Hair loss is a common disease in contemporary society, and its causes are complex and diverse, covering multiple aspects such as physiological aging, disease effects, and social pressure. In recent years, the incidence of hair loss has been rising year by year, and it is showing a trend of younger age, which has had a serious impact on the physical and mental health of patients.

[0003] At present, the treatments for hair loss are limited, mainly including drug therapy, physical therapy (such as low-energy laser and scalp microneedle) and surgical treatment (such as hair transplantation). However, these treatments have varying degrees of deficiencies in terms of cost, effectiveness and safety. Taking drug therapy as an example, at present, the only drugs approved by the US Food and Drug Administration (FDA) for the treatment of hair loss are topical minoxidil and oral finasteride. Although these drugs can alleviate the symptoms of hair loss to a certain extent, their side effects cannot be ignored. For example, ingredients such as ethanol and propylene glycol in minoxidil preparations may cause dryness and irritation of the scalp, while the oral form of finasteride may cause adverse reactions such as sexual dysfunction. These side effects not only affect the patient's treatment experience, but may also pose a threat to the patient's overall health. Therefore, there is an urgent need for safer and more effective treatments to alleviate the troubles that hair loss brings to patients' lives.

[0004] Therefore, it is of great significance to provide a composition for treating hair loss which has good effect of promoting hair growth and is green and safe. Summary of the invention

[0005] The present invention aims to solve one or more technical problems existing in the above-mentioned prior art, and at least provide a beneficial choice or create conditions. Specifically, the present invention provides a composition for treating hair loss, which has a good effect of promoting hair growth, can significantly improve the treatment effect of hair loss, and is green and safe.

[0006] The inventive concept of the present invention: The composition for treating hair loss of the present invention comprises arginine and pentacyclic triterpenoid compounds; the composition is a co-amorphous substance. The arginine and pentacyclic triterpenoid compounds of the present invention have a synergistic effect, and compared with the use of the active ingredients alone, compared with the suspension formed by the mixture of arginine and pentacyclic triterpenoid compounds, the therapeutic effect of hair loss is significantly improved. In addition, the co-amorphous system formed by arginine and pentacyclic triterpenoid compounds can upregulate the Wnt / β-catenin pathway, improve the microenvironment of oxidative stress and insufficient angiogenesis around the hair follicles, provide favorable conditions for the growth of hair follicles, and effectively prevent and treat hair loss.

[0007] Accordingly, a first aspect of the present invention provides a composition for treating hair loss.

[0008] Specifically, the composition for treating hair loss includes arginine and pentacyclic triterpenoid compounds; the composition is a co-amorphous substance.

[0009] Specifically, compared with the suspension of arginine and pentacyclic triterpenoid compounds, the co-amorphous composition can improve the transdermal delivery efficiency of the active ingredients arginine and pentacyclic triterpenoid compounds, promote the proliferation of human hair papilla cells, reverse the effects of dihydrotestosterone on the expression of various factors in human dermal hair follicle papilla cells, resist oxidative stress, and promote the migration of human umbilical vein endothelial cells, thereby having a better effect in treating hair loss.

[0010] A co-amorphous substance is a unidirectional amorphous system composed of two or more small molecule components. These components can be connected by non-covalent bonds such as hydrogen bonds, ionic bonds, π-π stacking, or there may be no interaction forces. The co-amorphous form can significantly improve the solubility and dissolution rate of poorly soluble drugs, and can also improve the physical stability of amorphous drugs. After the two small molecules with active ingredients are made into a co-amorphous form and administered, the advantages of the combined use of arginine and pentacyclic triterpenoids for the treatment of hair loss can be fully utilized, which can significantly improve the therapeutic effect of hair loss compared to the use of active ingredients alone. In addition, the co-amorphous system has the special property of forming a gel in water, which can improve the hydrophobicity of pentacyclic triterpenoids and facilitate processing into other preparations.

[0011] Preferably, the pentacyclic triterpenoid compound includes at least one of oleanolic acid, glycyrrhetinic acid, ursolic acid, asiatic acid, betulinic acid and maslinic acid.

[0012] Preferably, the molar ratio of arginine to pentacyclic triterpenoids is (0.9-4.5):1; further preferably, the molar ratio of arginine to pentacyclic triterpenoids is (1-4):1.

[0013] The second aspect of the present invention provides a method for preparing the composition for treating hair loss according to the first aspect of the present invention.

[0014] Specifically, the method for preparing the composition for treating hair loss comprises the following steps:

[0015] The raw material components are mixed, ultrasonically treated and dried to prepare the composition.

[0016] Preferably, the method for preparing the composition for treating hair loss comprises the following steps:

[0017] (1) mixing arginine and water to obtain an arginine solution; mixing a pentacyclic triterpenoid compound, an alcohol and / or an ether to obtain a dispersion of the pentacyclic triterpenoid compound;

[0018] (2) The arginine solution obtained in step (1) and the dispersion of pentacyclic triterpenoid compounds are mixed, subjected to ultrasonic treatment, and dried to obtain the composition.

[0019] Preferably, in step (1), the alcohol includes at least one of anhydrous ethanol and methanol; further preferably, the alcohol includes anhydrous ethanol.

[0020] Preferably, in step (1), the ether comprises diethyl ether.

[0021] Preferably, in step (2), the ultrasonic treatment time is 20-40 min; further preferably, the ultrasonic treatment time is 28-33 min; further preferably, the ultrasonic treatment time is 30 min.

[0022] Preferably, in step (2), the drying comprises rotary evaporation and vacuum drying in sequence.

[0023] Preferably, the temperature of the rotary evaporation is 35-50°C, and the time of the rotary evaporation is 9-22 min; further preferably, the temperature of the rotary evaporation is 40-50°C, and the time of the rotary evaporation is 10-20 min.

[0024] Preferably, the vacuum drying temperature is 30-50°C, and the vacuum drying time is 40-55h; further preferably, the vacuum drying temperature is 35-45°C, and the vacuum drying time is 45-52h; further preferably, the vacuum drying temperature is 40°C, and the vacuum drying time is 48h.

[0025] Specifically, the composition obtained by the above preparation method is an amorphous substance. Compared with directly mixing arginine and pentacyclic triterpenoid compounds to prepare a suspension, the transdermal delivery efficiency of the active ingredients arginine and pentacyclic triterpenoid compounds can be significantly improved, thereby improving the effect of treating hair loss.

[0026] A third aspect of the present invention provides a hydrogel.

[0027] Specifically, the hydrogel includes the composition for treating hair loss described in the first aspect of the present invention.

[0028] Preferably, the method for preparing the hydrogel comprises the following steps:

[0029] The composition for treating hair loss and a solvent are mixed and ultrasonically treated to prepare the hydrogel.

[0030] Specifically, the composition having an amorphous system can be self-assembled to form a hydrogel through ultrasonic treatment.

[0031] A fourth aspect of the present invention provides a microneedle.

[0032] Specifically, the microneedle comprises a backing layer, a separation layer and a needle tip in sequence; the needle tip is composed of the hydrogel described in the third aspect of the present invention.

[0033] Preferably, the mass ratio of the backing layer, the separation layer and the needle tip is (1.5-4.5):(0.8-1.2):1; further preferably, the mass ratio of the backing layer, the separation layer and the needle tip is (1.8-2.5):(0.9-1.1):1; further preferably, the mass ratio of the backing layer, the separation layer and the needle tip is 2:1:1.

[0034] Preferably, the backing layer is composed of a solvent and a biodegradable polymer material.

[0035] Preferably, in the backing layer, the biodegradable polymer material includes at least one of hyaluronic acid and dextran; further preferably, in the backing layer, the biodegradable polymer material includes hyaluronic acid and dextran.

[0036] Preferably, in the backing layer, the hyaluronic acid includes 200-400 kDa hyaluronic acid and 37-56 kDa hyaluronic acid.

[0037] Preferably, the composition of the separation layer includes a solvent, a biodegradable polymer material and sucrose.

[0038] Preferably, in the separation layer, the biodegradable polymer material includes at least one of hyaluronic acid, sodium carboxymethyl cellulose, dextran, and polyvinyl alcohol; further preferably, in the separation layer, the biodegradable polymer material includes 3-10 kDa hyaluronic acid.

[0039] Preferably, the needle tip is further composed of a biodegradable polymer material.

[0040] Preferably, in the needle tip, the biodegradable polymer material comprises dextran.

[0041] The fifth aspect of the present invention provides a method for preparing the microneedle according to the fourth aspect of the present invention.

[0042] Specifically, the method for preparing the microneedle comprises the following steps:

[0043] The needle tip, the separation layer and the backing layer are sequentially molded in a mold to obtain the microneedle.

[0044] The sixth aspect of the present invention provides a use of the composition for treating hair loss described in the first aspect of the present invention, the hydrogel described in the third aspect of the present invention, or the microneedle described in the fourth aspect of the present invention in the preparation of a medicament for treating hair loss.

[0045] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0046] (1) The composition for treating hair loss of the present invention comprises arginine and pentacyclic triterpenoid compounds; the composition is a co-amorphous substance. The arginine and pentacyclic triterpenoid compounds of the present invention have a synergistic effect, and compared with the use of the active ingredients alone, compared with the suspension formed by the mixture of arginine and pentacyclic triterpenoid compounds, the therapeutic effect of hair loss is significantly improved. In addition, the co-amorphous system formed by arginine and pentacyclic triterpenoid compounds can upregulate the Wnt / β-catenin pathway, improve the microenvironment of oxidative stress and insufficient angiogenesis around hair follicles, provide favorable conditions for the growth of hair follicles, and effectively prevent and treat hair loss.

[0047] (2) The composition of the present invention is a co-amorphous substance, which has the property of self-assembling in water to form a supramolecular hydrogel, which provides convenience for further formulation design (for example, the gel form is convenient for making microneedles, and can be further designed into thermosensitive gels, pH-responsive gels, injectable gels, etc. by adding other materials). At the same time, the present invention further loads the hydrogel into a detachable microneedle, and the microneedle is designed to be detachable, which can achieve rapid separation of the needle tip and the backing layer, so that the drug in the needle tip is retained in the skin and slowly released, which not only improves the drug delivery efficiency, but also forms a drug reservoir under the skin, achieving the purpose of long-term treatment of hair loss. The present invention combines co-amorphous drugs and microneedles for the first time, expands the formulation form and administration method of co-amorphous drugs, and provides new ideas and directions for its future development and application.

[0048] (3) The composition of the present invention does not contain irritating ingredients such as ethanol and propylene glycol, and is more environmentally friendly and safe. At the same time, it can effectively reduce the frequency of administration and reduce the pain caused by frequent administration. In addition, the preparation process of the present invention is simple and convenient for large-scale production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is an appearance diagram of the hydrogel prepared in Example 4 of the present invention;

[0050] Figure 2 This is an appearance diagram of the microneedle prepared in Application Example 1 of the present invention;

[0051] Figure 3 This is an X-ray diffraction pattern of the composition for treating hair loss prepared in Example 1 of the present invention;

[0052] Figure 4This is a transmission electron microscopy image of the hydrogel prepared in Example 4 of the present invention;

[0053] Figure 5 This is a rheological curve of the hydrogel prepared in Example 4 of the present invention;

[0054] Figure 6 This is a diagram showing the mechanical properties of the microneedle prepared in Application Example 1 of the present invention;

[0055] Figure 7 This is a graph showing the porosity of the sealing membrane of the microneedle prepared in Application Example 1 of the present invention;

[0056] Figure 8 This is a diagram showing the microneedle separation results of the microneedles prepared in Application Example 1 of the present invention;

[0057] Fig. 9 This is a graph showing the in vitro drug release performance of oleanolic acid in the microneedles prepared in Application Example 1 of the present invention;

[0058] Fig.10 This is a graph showing the effect of the composition of Example 1 of the present invention, arginine, and oleanolic acid on the proliferation of human dermal hair follicle papilla cells;

[0059] Fig.11 This is the result diagram of the relative expression of CTNNBIP1 mRNA in human dermal hair follicle papilla cells;

[0060] Fig.12 The relative expression results of DKK1 mRNA in human dermal hair follicle papilla cells;

[0061] Fig.13 This is the result diagram of the relative expression of TGFB1 mRNA in human dermal hair follicle papilla cells;

[0062] Fig.14 This is the result of the relative expression of IGF1 mRNA in human dermal hair follicle papilla cells;

[0063] Fig.15 This is a graph showing the survival rate of human dermal hair follicle papilla cells in each treatment group under hydrogen peroxide conditions;

[0064] Fig.16 The figure is the result of fluorescence detection of reactive oxygen species (ROS) produced by human dermal hair follicle papilla cells in each treatment group under hydrogen peroxide conditions;

[0065] Fig.17 The diagram shows the promoting effect of each treatment group on the migration of human umbilical vein endothelial cells;

[0066] Fig.18 This is the statistical result of migration area of ​​human umbilical vein endothelial cells in each treatment group;

[0067] Fig.19 The figure shows the growth of back hair in each group of mice during the drug administration period;

[0068] Fig. 20 This is a statistical chart of the regenerated hair coverage rate of each group of mice after drug administration;

[0069] Fig.21 This is a statistical chart of the length of regenerated hair in each group of mice after drug administration. DETAILED DESCRIPTION

[0070] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0071] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0072] Example 1

[0073] A composition for treating hair loss comprises 0.27 g of arginine and 0.36 g of oleanolic acid.

[0074] A method for preparing a composition for treating hair loss comprises the following steps:

[0075] (1) dissolving 0.27 g of arginine in 2 mL of water to obtain an arginine solution; dispersing 0.36 g of oleanolic acid in 40 mL of anhydrous ethanol by ultrasonication to obtain a dispersion of oleanolic acid;

[0076] (2) Slowly adding the arginine solution to the oleanolic acid dispersion, ultrasonicating at room temperature for 30 minutes to obtain a colorless and transparent solution; then, the solution is rotary evaporated at 45° C. to remove most of the water and ethanol, and then the resultant is placed in a vacuum dryer at 40° C. for 48 hours to obtain a composition for treating hair loss.

[0077] Example 2

[0078] A composition for treating hair loss comprises 0.27 g of arginine and 0.74 g of glycyrrhetinic acid.

[0079] A method for preparing a composition for treating hair loss comprises the following steps:

[0080] (1) dissolving 0.27 g of arginine in 2 mL of water to obtain an arginine solution; dispersing 0.74 g of glycyrrhetinic acid in 40 mL of anhydrous ethanol by ultrasonication to obtain a dispersion of glycyrrhetinic acid;

[0081] (2) Slowly adding the arginine solution to the dispersion of glycyrrhetinic acid, ultrasonicating at room temperature for 30 minutes to obtain a colorless and transparent solution; then, the solution is rotary evaporated at 45° C. to remove most of the water and ethanol, and then the resultant is placed in a vacuum dryer at 40° C. for 48 hours to obtain a composition for treating hair loss.

[0082] Example 3

[0083] A composition for treating hair loss comprises 0.54 g of arginine and 0.36 g of ursolic acid.

[0084] A method for preparing a composition for treating hair loss comprises the following steps:

[0085] (1) dissolving 0.54 g of arginine in 2 mL of water to obtain an arginine solution; dispersing 0.36 g of ursolic acid in 40 mL of anhydrous ethanol by ultrasonication to obtain an ursolic acid dispersion;

[0086] (2) Slowly adding the arginine solution to the ursolic acid dispersion, ultrasonicating at room temperature for 30 minutes to obtain a colorless and transparent solution; then, the solution is rotary evaporated at 45° C. to remove most of the water and ethanol, and then the resultant is placed in a vacuum dryer at 40° C. for 48 hours to obtain a composition for treating hair loss.

[0087] Example 4

[0088] A hydrogel comprises 0.06 g of the composition for treating hair loss prepared in Example 1 and 1 g of water.

[0089] The method for preparing the hydrogel comprises the following steps:

[0090] The composition for treating hair loss prepared in Example 1 was added into water, stirred evenly, and then ultrasonically treated for 1 hour to allow self-assembly to obtain a hydrogel.

[0091] The appearance of the hydrogel obtained in Example 4 is as follows Figure 1 As shown, Figure 1 Figures (a) and (b) are respectively the appearance diagrams of the hydrogel of Example 4 under different placement states.

[0092] Example 5

[0093] A hydrogel comprises 0.1 g of the composition for treating hair loss prepared in Example 2 and 1 g of water.

[0094] The method for preparing the hydrogel comprises the following steps:

[0095] The composition for treating hair loss prepared in Example 2 was added into water, stirred evenly, and then ultrasonically treated for 1 hour to allow self-assembly to obtain a hydrogel.

[0096] Example 6

[0097] A hydrogel comprises 0.1 g of the composition for treating hair loss prepared in Example 3 and 1 g of water.

[0098] The method for preparing the hydrogel comprises the following steps:

[0099] The composition for treating hair loss prepared in Example 3 was added into water, stirred evenly, and then ultrasonically treated for 0.5 h to allow self-assembly to obtain a hydrogel.

[0100] Application Example 1

[0101] A detachable microneedle comprises a backing layer, a separation layer and a needle tip in sequence.

[0102] The composition of the backing layer (composition of the matrix of the backing layer) includes 0.1 g of hyaluronic acid having a molecular weight of 200-400 kDa, 0.1 g of hyaluronic acid having a molecular weight of 37-56 kDa, 0.1 g of dextran and 1 mL of water.

[0103] The method for preparing the matrix of the backing layer comprises the following steps:

[0104] Hyaluronic acid and dextran of two molecular weights were added into water, stirred thoroughly, and placed in a refrigerator overnight to allow them to fully swell to obtain a matrix of the backing layer. Bubbles were removed by centrifugation before use.

[0105] The composition of the separation layer (composition of the matrix of the separation layer) includes 0.4 g of hyaluronic acid having a molecular weight of 3-10 kDa, 0.1 g of sucrose and 1 mL of water.

[0106] The method for preparing the matrix of the separation layer comprises the following steps:

[0107] Add hyaluronic acid and sucrose into water, stir thoroughly, and place in a refrigerator overnight to allow them to fully swell to obtain a matrix of the separation layer. Remove bubbles by centrifugation before use.

[0108] The composition of the needle tip (composition of the matrix of the needle tip) includes 1.06 g of the hydrogel prepared in Example 4 and 0.12 g of dextran.

[0109] The method for preparing the needle tip matrix comprises the following steps:

[0110] Dextran was added to the hydrogel prepared in Example 4, stirred evenly, and then placed in a refrigerator overnight to allow it to fully swell to obtain a needle tip matrix. Bubbles were removed by centrifugation before use.

[0111] A method for preparing a detachable microneedle comprises the following steps:

[0112] (1) Preparation of needle tip: Add the needle tip matrix to the polydimethylsiloxane (PDMS) mold, centrifuge at 3750 rpm at 4°C for 2-4 times, each time for 3 min, and change the direction of the mold so that the needle tip matrix fills the needle tip cavity of the mold; scrape off the excess needle tip matrix with a flat spatula, centrifuge at 3750 rpm at 25°C for 30 min to fully deposit and dry the needle tip matrix to obtain the needle tip;

[0113] (2) Preparation of separation layer: The matrix of the separation layer was added to the PDMS mold, and centrifuged at 3750 rpm at 25° C. for 30 min to allow the matrix of the separation layer to be fully deposited and dried to obtain a separation layer;

[0114] (3) Preparation of backing layer: Add the matrix of the backing layer to the PDMS mold and centrifuge it twice at 3750 rpm at 4°C for 3 min each time. At the same time, change the direction of the mold so that the matrix of the backing layer is flattened in the mold to obtain the backing layer. Then put the whole into a dryer and dry it at 4°C for 48 h. Demold to obtain detachable microneedles.

[0115] The appearance of microneedles Figure 2 As shown. Among them, Figure 2 Figure (a) is a macroscopic image of the microneedle taken by a camera; Figure 2 Figure (b) is a picture of the microneedle taken under a microscope; Figure 2 Figure (c) is a scanning electron microscope image of the microneedle; Figure 2 Figure (d) shows a microneedle image taken under natural light after the tip and separation layer of the microneedle were labeled with different fluorescent dyes; Figure 2 Figure (e) is a picture of the tip of the microneedle under a fluorescence microscope; Figure 2 Figure (f) is a picture of the separation layer of the microneedle under a fluorescence microscope; Figure 2 Figure (g) is a picture of the microneedle tip and separation layer merged under a fluorescence microscope.

[0116] Depend on Figure 2 It can be seen that the tip of the microneedle is a regular tetrahedron + regular tetrahedron, the total length of the tip is 1200μm, the width of the bottom is 300μm, and the interval between microneedles is 600μm. Sodium fluorescein (which can emit green fluorescence) and rhodamine B (which can emit red fluorescence) are loaded into the separation layer and tip of the microneedle, respectively. The obvious double-layer structure of the microneedle can be seen under natural light and fluorescence microscope.

[0117] Comparative Example 1

[0118] An arginine and oleanolic acid suspension comprises 0.026 g of arginine, 0.034 g of oleanolic acid and 1 mL of water.

[0119] A method for preparing an arginine and oleanolic acid suspension comprises the following steps:

[0120] Add arginine and oleanolic acid into water and stir evenly to obtain arginine and oleanolic acid suspension.

[0121] Since the drug in Comparative Example 1 is in a state of particle suspension, it is difficult to disperse the drug particles evenly in water or in the matrix of the needle tip, so it is difficult to prepare an ideal microneedle. However, the co-amorphous composition formed by arginine and oleanolic acid in the present invention can self-assemble in water to form a gel and can be prepared as a microneedle.

[0122] Performance Testing

[0123] 1. X-ray diffraction analysis

[0124] The composition for treating hair loss prepared in Example 1 was subjected to X-ray diffraction analysis, and the results were as follows: Figure 3 As shown, the abscissa 2θ (degree) represents the diffraction angle 2θ (°).

[0125] Depend on Figure 3 It can be seen that no sharp diffraction peak appears on the X-ray diffraction pattern of the composition for treating hair loss prepared in Example 1, indicating that the co-amorphous material is successfully prepared.

[0126] 2. Transmission electron microscopy observation

[0127] The hydrogel prepared in Example 4 was observed by transmission electron microscopy, and the results were as follows: Figure 4 As shown. Figure 4 It can be seen that the hydrogel prepared in Example 4 has a lamellar cross structure.

[0128] 3. Rheological characteristics of hydrogels

[0129] The hydrogel formed by the self-assembly of the co-amorphous material in Example 1, i.e., the hydrogel in Example 4, was subjected to amplitude scanning and frequency scanning using an MCR 302e rheometer to investigate the rheological properties of the hydrogel. The specific method is as follows:

[0130] (1) Shear rate scan: set the temperature to 32°C and the initial shear rate to 1s -1 The end shear rate is 100 s -1 , record the corresponding viscosity at different shear rates to obtain the viscosity curve of the hydrogel;

[0131] (2) Amplitude scanning: Using a 20 mm diameter parallel plate fixture, the temperature was set to 32 °C, the angular frequency was 10 rad / s, and the amplitude range was 0.1-100%. The storage modulus (G') and loss modulus (G") of the sample were measured to determine the linear viscoelastic region of the sample;

[0132] (3) Frequency scanning: According to the range of the linear viscoelastic region, the strain was selected as 0.5%, a parallel plate fixture with a diameter of 25 mm was used, the temperature was set to 32 °C, and the frequency was scanned in the angular frequency range of 1-100 rad / s to measure the physical stability of the sample.

[0133] The rheological characteristics of the hydrogel of Example 4 are as follows Figure 5 As shown, Figure 5 Figure (a) is a viscosity curve of the hydrogel; Figure 5 The middle (b) figure is the amplitude scanning result of the hydrogel; Figure 5 The middle (c) figure is the angular frequency scanning result of the hydrogel; Figure 5 The ordinate G'G" (Pa) in Figures (b) and (c) represents the storage modulus and loss modulus. In Figures (b) and (c), G' represents the storage modulus, and G" represents the loss modulus.

[0134] Depend on Figure 5 From the viscosity curve, we can see that the hydrogel has the property of shear thinning. From the amplitude scanning and angular frequency scanning results, we can see that the hydrogel has good elastic properties and a relatively stable structure.

[0135] 4. Characterization of mechanical properties and separation of microneedles

[0136] The mechanical properties, sealing film porosity, and microneedle separability of the microneedles prepared in Example 1 were tested. The specific methods are as follows:

[0137] (1) Mechanical properties test: The mechanical strength of the microneedle was measured using a texture analyzer. The microneedle was placed on a stage with the tip facing upward. The probe was moved to the tip of the microneedle and the initial position was recorded. The probe was set to move downward at a speed of 1 mm / s from the initial position to completely break the microneedle body. The force and displacement curve of the probe from the initial position to the complete breaking of the microneedle was recorded. The mechanical properties of the microneedle were measured. Figure 6 As shown;

[0138] (2) Sealing film porosity test: The penetration depth of the microneedle was measured using a Parafilm sealing film. Each layer of sealing film was equivalent to 100 μm of skin thickness. Ten layers of sealing film were stacked to simulate human skin. The microneedle tip was placed on the sealing film with the tip facing downward. A digital push-pull force gauge was used to apply a force of 30 N vertically downward on the backing layer of the microneedle. The force was maintained for 1 minute and then the microneedle was removed. The porosity of each layer of sealing film was calculated. The porosity results of the microneedle sealing film are shown in the figure. Figure 7 As shown;

[0139] (3) Microneedle detachment test: Rhodamine B was loaded onto the tip of the microneedle to examine the skin puncture performance and drug delivery ability of the microneedle. The microneedle was placed on the pig skin with the tip facing downward. The microneedle was vertically inserted into the skin with a force of 30 N using a digital push-pull force gauge. The microneedle was pressed for 1 minute and then removed. The pig skin was then frozen and sectioned and photographed using a fluorescence microscope. The results of the microneedle detachment test are shown in Figure 2. Figure 8 shown.

[0140] Figure 8 The middle (a) picture shows the appearance of pig skin before microneedle puncture. Figure 8 The middle (b) picture shows the appearance of pig skin after microneedle puncture. Figure 8 The middle figure (c) shows the retention of the microneedle tip in the frozen section.

[0141] Depend on Figure 6 It can be seen that the microneedle did not break during the stress process and has good mechanical properties.

[0142] Depend on Figure 7 It can be seen that the microneedle can penetrate into the subcutaneous tissue to a depth of about 500 μm.

[0143] Depend on Figure 8 It can be seen that the microneedle can effectively puncture the skin and achieve rapid separation of the needle tip and the backing layer within 1 minute, so that the needle tip remains in the skin (fluorescence is added to the needle tip, Figure 8 The orange-red part in (c) is the retained needle tip).

[0144] 5. In vitro drug release experiment of microneedles

[0145] The microneedles prepared in Application Example 1 were used to examine the release characteristics of oleanolic acid from the microneedles.

[0146] In this experiment, the Franz diffusion cell method was used to investigate the release performance of the drug. A 0.45 μm organic nylon filter membrane was used as an artificial membrane to simulate the skin. The filter membrane was soaked in the release medium for 30 minutes before use. After the device was fixed, the microneedle was placed in the supply pool and 100 μL of PBS was added. PBS containing 2% oleyl alcohol polyether (Oleth-20) was selected as the receiving solution. The receiving solution was added to the receiving pool, and the diffusion pool was placed in the diffusion instrument. The temperature was set to 32°C and the speed was 250 rpm. 1 mL of release medium was taken at 4h, 8h, 12h, 24h, 36h, 48h, 72h, and 120h, and 1 mL of blank release medium was added. The samples were filtered through a 0.22 μm microporous filter membrane and the content of oleanolic acid was determined by high performance liquid chromatography. The cumulative release amount at each time point was calculated according to formula (1), and the cumulative release degree (%) was calculated according to the ratio of the cumulative release amount to the total content of oleanolic acid in the microneedle.

[0147]

[0148] Wherein, Cn is the drug concentration measured at the nth sampling, Ci is the drug concentration measured at each sampling, V represents the volume of the release medium, Vi represents the volume of each sampling, and Qn (μg) is the cumulative drug release.

[0149] Chromatographic conditions: the chromatographic column is an XB-C18 column (250×4.6 mm, 5 μm); the mobile phase is 0.1% phosphoric acid water: acetonitrile = 10:90 (v / v); flow rate: 1 mL / min; detection wavelength: 210 nm; injection volume: 20 μL.

[0150] In vitro drug release results of oleanolic acid from microneedles Fig. 9 As shown. Fig. 9 It can be seen that the in vitro release of oleanolic acid in the microneedles showed a sustained release trend, and the drug could be released continuously for 5 days. At 3 days, the release of oleanolic acid was 82.66±5.89%, and the release at 5 days could reach 94.48±4.38%, which was basically released completely.

[0151] 6. In vitro microneedle permeation experiment

[0152] The microneedles prepared in Application Example 1 were used to investigate the in vitro transdermal performance of oleanolic acid. The specific method is as follows:

[0153] The frozen rat skin was soaked in physiological saline to thaw, the moisture on the skin surface was absorbed with filter paper, and the integrity of the skin was checked; the microneedle group was pressed on the stratum corneum of the skin with a force of 30N using a digital push-pull dynamometer, and maintained for 3 minutes before being fixed with medical 3M tape; rat skin (stratum corneum facing the supply pool) and a gasket were placed on top of the supply pool in sequence, and the receiving pool was fixed; phosphate buffered saline (PBS) containing 2% oleth-20 (Oleth-20) was selected as the receiving solution, the receiving solution was added to the receiving pool, and the diffusion pool was placed in the diffusion instrument, the temperature was set to 32°C, and the rotation speed was 250rpm. Example 4 and Comparative Example 1 were selected as controls, and the supply pool was added to keep the dosage per unit area consistent with that of the microneedle group; 1 mL of receiving solution was taken out at 1, 2, 4, 8, 12, and 24 hours, and 1 mL of blank receiving solution was added to ensure that the volume of the receiving solution in the receiving pool was consistent; the obtained sample was filtered with a 0.22 μm microporous filter membrane, and the content of oleanolic acid therein was determined by high performance liquid chromatography. The cumulative permeation amount Qn of oleanolic acid per unit area was calculated according to formula (2).

[0154]

[0155] Where A is the transdermal area, Cn is the measured drug concentration at the nth sampling, V is the volume of the diffusion medium, Vi is the volume of each sampling, Ci is the concentration measured at each sampling, and Qn is the cumulative permeation of the drug per unit area.

[0156] After the in vitro transdermal experiment, the rat skin was removed from the diffusion cell, and the skin at the administration site was wiped three times with cotton balls dipped in water, methanol, and water to remove residual drugs; the skin at the administration site was cut off, placed in an EP tube after drying, cut into pieces, and the skin mass was weighed; then 1 mL of methanol was added to the EP tube, and the drug in the skin was extracted by ultrasound for 30 minutes, and then allowed to stand at room temperature for 2 hours; the supernatant was filtered with a 0.22 μm microporous filter membrane, and the content of oleanolic acid therein was determined by high performance liquid chromatography, which was the skin retention amount.

[0157] The in vitro transdermal results of oleanolic acid in the hydrogel of Example 4, the microneedles of Application Example 1, and the suspension of Comparative Example 1 are shown in Table 1.

[0158] Table 1: In vitro transdermal results of oleanolic acid in the hydrogel of Example 4, microneedles of Application Example 1, and suspension of Comparative Example 1

[0159]

[0160] Note: *** There is a statistical difference between the results of comparison 1 (P<0.001). ## There is a statistical difference between the above results and Example 4 (P<0.01). ### There was a statistical difference between the results of Example 4 (P<0.001).

[0161] As can be seen from Table 1, compared with the hydrogel of Example 4, the microneedles prepared by the present invention can well enhance the skin penetration and skin retention of oleanolic acid, which is beneficial to the effect of oleanolic acid in treating hair loss. For Comparative Example 1, since it is a suspension obtained by simply and directly mixing arginine and oleanolic acid, and no co-amorphous system is formed, the skin penetration of oleanolic acid cannot be detected, and the skin retention is also significantly lower than that of Example 4, and even lower than that of the microneedles of Application Example 1, that is, the skin penetration and skin retention of oleanolic acid in Comparative Example 1 are both poor, and the effect of oleanolic acid in treating hair loss cannot be exerted.

[0162] 7. Cellular pharmacodynamic evaluation of arginine-oleanolic acid co-amorphous

[0163] (1) Cell proliferation assay

[0164] Human dermal follicle papilla cells (HDPC) have an important influence on the regulation of hair follicle cycle. They promote hair follicle growth by division and proliferation, stop proliferation and cause hair follicle regression, and are in a dormant state during the resting period. In addition, they are also involved in the regeneration and repair process of hair follicles. In summary, the proliferation and differentiation of HDPC are of great significance for hair regeneration, so this study will investigate the effect of the arginine-oleanolic acid co-amorphous compound of Example 1 on the proliferation activity of HDPC, and the specific method is as follows:

[0165] HDPC in the logarithmic growth phase were digested and inoculated into a 96-well plate at a density of 8000 cells / well; the well plate was then placed in an incubator for overnight culture; arginine, oleanolic acid, and the co-amorphous substance prepared in Example 1 (i.e., the composition for treating hair loss) were diluted with complete DMEM medium to a concentration of 0.17-8.00 μg / mL and incubated with HDPC cells for 24 hours; the 96-well plate was then taken out of the incubator, the original medium was removed, and 120 μL of MTT diluent (containing 3-(4, 5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) 0.83 mg / mL), then the 96-well plate was wrapped with tin foil and placed in an incubator to culture in the dark; after 4 hours, the culture medium in the wells was removed, 150 μL of dimethyl sulfoxide (DMSO) was added to each well, and the wells were shaken on a shaker for 15-20 minutes to allow the crystals to fully develop color; the whole process was kept in the dark; finally, the absorbance value (OD) of each well in the 96-well plate was measured at 490 nm using an enzyme reader, and the cell survival rate was calculated according to the following formula:

[0166] Cell survival rate / %=(OD value of drug administration group-OD value of apoptosis group) / (OD value of control group-OD value of apoptosis group)×100%.

[0167] Among them, the drug administration group is a group that is both implanted with cells and administered with drugs, the control group is a group that is implanted with cells but not administered with drugs, and the zero adjustment group is a group that is neither implanted with cells nor administered with drugs.

[0168] The results of the effects of the composition of Example 1, arginine, and oleanolic acid on the proliferation activity of HDPC are as follows: Fig.10 As shown. Among them, the concentrations in the first row of the horizontal axis represent the three concentrations of co-amorphous, the concentrations in the second row represent the three concentrations of arginine, and the concentrations in the third row represent the three concentrations of oleanolic acid. ＊ Compared with the control group, there was a statistically significant difference (P<0.05). ＊＊ Compared with the control group, there was a statistically significant difference (P<0.01). ＊＊＊ Compared with the control group, there was a statistically significant difference (P<0.001). ## It indicated that there was a statistical difference compared with the co-amorphous product (P<0.01).

[0169] Depend on Fig.10 It can be seen that when the concentration of the co-amorphous substance is 0.40 μg / mL, it has a significant promoting effect on the proliferation of HDPC cells. Arginine (0.17 μg / mL) and oleanolic acid (0.23 μg / mL) at the corresponding concentrations also have a certain promoting effect on proliferation, but the proliferation effect is not as good as the co-amorphous substance, indicating that preparing arginine and oleanolic acid into a co-amorphous form not only does not affect the performance of their respective efficacy, but also makes the proliferation effect of the composition better than that of arginine and oleanolic acid when used alone. When the concentration of the co-amorphous substance is 8.00 μg / mL, oleanolic acid (4.6 μg / mL) at the corresponding concentration has a certain toxic effect on cells, while the co-amorphous substance does not, indicating that the co-amorphous substance can reduce the toxicity of oleanolic acid, further proving the advantage of the combination of the two drugs.

[0170] (2) Cell PCR experiment

[0171] In order to investigate whether arginine-oleanolic acid co-amorphous products can regulate the expression of hair growth-related signaling pathways Wnt / β-catenin and growth factors after acting on HDPC cells, dihydrotestosterone (DHT) was used to model HDPC cells, and real-time quantitative PCR (qRT-PCR) experiments were performed after administration to investigate the mRNA expressions of CTNNBIP1, DKK1, TGFB1 and IGF1.

[0172] The specific method is as follows: HDPC cells in the logarithmic growth phase were digested and inoculated in a 12-well plate at a density of 3×10 5 / well, cultured overnight to allow cells to adhere to the wall, then removed the original culture medium, added DMEM basal culture medium to the control group, added 1 μM DHT solution to the model group, and added drug solution and 1 μM DHT solution to the other drug groups, respectively, and incubated for 24 hours; the drug groups were arginine, oleanolic acid and co-amorphous drugs, the dosage of co-amorphous drugs was 0.40 μg / mL, and the dosage of single drugs and corresponding drugs in co-amorphous was consistent (arginine (0.17 μg / mL) and oleanolic acid (0.23 μg / mL)); then the cells were treated according to the instructions of the PCR kit, and fluorescence quantification was performed using a real-time fluorescence quantitative PCR instrument.

[0173] The relative expression of CTNNBIP1 mRNA in human dermal follicle papilla cells is shown in Fig.11 As shown, ＊ Compared with the model group, there was a statistically significant difference (P<0.05). ＊＊＊ Compared with the model group, there was a statistically significant difference (P<0.001). # Compared with the amorphous group, there was a statistically significant difference (P<0.05). ##It indicated that there was a statistical difference compared with the co-amorphous group (P<0.01).

[0174] The relative expression of DKK1 mRNA in human dermal follicle papilla cells is shown in Fig.12 As shown. Among them, ＊＊＊ Compared with the model group, there was a statistically significant difference (P<0.001). ## Compared with the amorphous group, there was a statistically significant difference (P<0.01). ### It indicated that there was a statistical difference compared with the co-amorphous group (P<0.001).

[0175] The relative expression of TGFB1 mRNA in human dermal hair follicle papilla cells is as follows Fig.13 As shown. Among them, ＊ Compared with the model group, there was a statistically significant difference (P<0.05). ＊＊＊ Compared with the model group, there was a statistically significant difference (P<0.001). ## It indicated that there was a statistical difference compared with the co-amorphous group (P<0.01).

[0176] The relative expression of IGF1 mRNA in human dermal follicle papilla cells is as follows Fig.14 As shown. Among them, ＊＊ Compared with the model group, there was a statistically significant difference (P<0.01). ＊＊＊ Compared with the model group, there was a statistically significant difference (P<0.001). ## Compared with the amorphous group, there was a statistically significant difference (P<0.01). ### It indicated that there was a statistical difference compared with the co-amorphous group (P<0.001).

[0177] Depend on Figure 11-14 It can be seen that the arginine-oleanolic acid co-amorphous compound prepared in Example 1 can reverse the effect of dihydrotestosterone on the expression of various factors in HDPC cells after acting on human dermal hair follicle papilla cells, which is specifically manifested as: up-regulating the mRNA expression of positive factors related to hair regeneration such as CTNNBIP1 and IGF1 in cells, and down-regulating the mRNA expression of negative factors such as DKK1 and TGFB1. In addition, the above-mentioned effects of the arginine-oleanolic acid co-amorphous compound prepared in Example 1 are better than those of the single drug groups (arginine group, oleanolic acid group) at the corresponding concentrations, indicating that the arginine-oleanolic acid co-amorphous compound prepared in Example 1 has a better effect of regulating hair growth.

[0178] (3) Antioxidant test

[0179] Hair loss is closely related to vascular insufficiency and oxidative stress. Oxidative stress is mainly induced by excessive reactive oxygen species (ROS), which leads to apoptosis of human dermal follicle papilla cells and weakens the ability of hair follicles to transform from the resting phase to the growth phase. This experiment investigated the antioxidant capacity of arginine, oleanolic acid and the arginine-oleanolic acid co-amorphous prepared in Example 1 through antioxidant damage experiments and ROS scavenging experiments.

[0180] Antioxidant damage experiment: using H 2 O 2 A cell oxidative damage model was created to simulate the condition of human dermal hair follicle papilla cells under oxidative stress. Specifically, HDPC in the logarithmic growth phase were digested and inoculated in a 96-well plate at a density of 8,000 cells / well. The plate was then placed in an incubator for overnight culture. After the cells adhered to the wall, the original culture medium was removed and different drugs were treated. The control group was only given basic DMEM culture medium without any drugs, and the model group was given 600 μM H 2 O 2 , the drug group was treated with H 2 O 2 On the basis of the above, arginine, oleanolic acid and the arginine-oleanolic acid co-amorphous prepared in Example 1 were added respectively; the dosage of the co-amorphous drug was 0.40 μg / mL, and the dosage of the single drug was consistent with the dosage of the corresponding drug in the co-amorphous (arginine (0.17 μg / mL) and oleanolic acid (0.23 μg / mL)); after 24 hours of drug incubation, the cell survival rate was detected by the MTT method to understand the protective ability of different drugs against cell oxidative damage.

[0181] The survival rate of human dermal hair follicle papilla cells in each treatment group under hydrogen peroxide conditions was as follows Fig.15 As shown. Among them, ＊＊＊ Compared with the model group, there was a statistically significant difference (P<0.001). ### It indicated that there was a statistical difference compared with the co-amorphous group (P<0.001).

[0182] Depend on Fig.15 It can be seen that the cell survival rate of the model group was greatly reduced, indicating that the cell viability decreased under oxidative stress conditions. Compared with the model group, arginine, oleanolic acid and co-amorphous groups can improve the cell survival rate, indicating that all three have the ability to resist oxidative damage. Among them, the arginine-oleanolic acid co-amorphous group had the best protective effect on human dermal hair follicle papilla cells, and the cell survival rate was equivalent to that of the control group. This shows that arginine and oleanolic acid prepared into arginine-oleanolic acid co-amorphous drugs can improve the survival rate of human dermal hair follicle papilla cells, improve antioxidant capacity, and thus improve the prevention and treatment of hair loss.

[0183] ROS scavenging experiment: HDPC cells in the logarithmic growth phase were digested and inoculated in 12-well plates at a density of 1×10 5 The cells were placed in an incubator overnight and cultured. After the cells adhered to the wall, the original culture medium was removed and different drugs were treated. The control group was only given basic DMEM culture medium without any drugs, and the model group was given 600 μM H 2 O 2 , the drug group was treated with H 2 O 2 On the basis of the above, arginine, oleanolic acid and the arginine-oleanolic acid co-amorphous prepared in Example 1 were added respectively. The dosage of the co-amorphous drug prepared in Example 1 was 0.40 μg / mL, and the dosage of the single drug was consistent with the corresponding drug in the co-amorphous (arginine (0.17 μg / mL) and oleanolic acid (0.23 μg / mL)). After incubation for 24 hours, the original culture medium was removed, the cells were washed with PBS for 3 times, and the diluted ROS fluorescent probe DCFH-DA was incubated with the cells for 1 hour, followed by washing with PBS for 3 times to remove the fluorescent probe that did not enter the cells. Finally, pictures were taken with a fluorescence microscope to evaluate the drug's ability to scavenge ROS.

[0184] The results of ROS fluorescence detection produced by human dermal hair follicle papilla cells in each treatment group under hydrogen peroxide conditions are as follows Fig.16 As shown. Among them, Fig.16 The upper middle row shows the images of cells in each treatment group under a fluorescence microscope. Since reactive oxygen species (ROS) are labeled, the expression of ROS can be visualized; Fig.16 The middle and lower rows are pictures of cells in each treatment group in bright field. In addition, the scale bars of each picture are the same as those in the cell pictures in the amorphous group in bright field, which are all 200 μm.

[0185] Depend on Fig.16 It can be seen that both arginine and oleanolic acid have the activity of clearing excessive ROS produced by cells due to oxidative damage, and the arginine-oleanolic acid co-amorphous compound formed by arginine and oleanolic acid has better ROS scavenging ability than single drugs (arginine or oleanolic acid). This shows that the preparation of arginine-oleanolic acid co-amorphous drugs with arginine and oleanolic acid can improve the scavenging ability of ROS, thereby improving the prevention and treatment effects of hair loss.

[0186] (4) Cell migration assay

[0187] Vascularization around hair follicles is essential for maintaining healthy hair growth. Insufficient vascularization will limit the delivery of nutrients, biological factors, immune cells, etc. to hair follicles, affecting the transformation of hair follicle cycle and the repair of hair follicle damage. Human umbilical vein endothelial cells (HUVEC) are a commonly used model cell for studying angiogenesis. The potential of HUVEC cells to participate in angiogenesis can be evaluated by detecting their proliferation and migration ability. This experiment will investigate the ability of the arginine-oleanolic acid co-amorphous compound prepared in Example 1 to promote the migration of HUVEC cells. The specific method is as follows:

[0188] HUVEC cells were plated at 5 × 10 5 The cells were inoculated at a density of 100 μg / mL in a 6-well plate and cultured overnight in an incubator. A 200 μL sterilized pipette tip was used to draw a line perpendicular to the bottom edge of the 6-well plate, the culture medium was aspirated, and the cells were washed 2-3 times with sterile PBS to remove the scratched cells. Arginine, oleanolic acid, and arginine-oleanolic acid co-amorphous were diluted to concentrations of 0.34 μg / mL, 0.46 μg / mL, and 0.80 μg / mL with incomplete DMEM culture medium, respectively, and incubated with the cells. The group with only basic DMEM culture medium was set as the control group. At 0 h, 24 h, and 48 h after drug incubation, the cell migration was observed under a microscope, and the scratch area at different times was counted using Image J software. The scratch area at 0 h was defined as the initial scratch area, and the cell migration rate was calculated as follows:

[0189] Cell migration rate / %=(initial scratch area-scratch area) / initial scratch area×100%.

[0190] The promoting effect of each treatment group on the migration of human umbilical vein endothelial cells is as follows Fig.17 As shown, the scale bars in each figure are the same as those in the amorphous group incubated for 48 hours.

[0191] The statistical results of the migration area of ​​human umbilical vein endothelial cells in each treatment group are as follows Fig.18 As shown. Among them, ＊ Compared with the control group, there was a statistically significant difference (P<0.05). ＊＊ Compared with the control group, there was a statistically significant difference (P<0.01). ＊＊＊ Compared with the control group, there was a statistically significant difference (P<0.001) ## Compared with the amorphous group, there was a statistically significant difference (P<0.01). ### It indicated that there was a statistical difference compared with the co-amorphous group (P<0.001).

[0192] Depend on Fig.17 and Fig.18It can be seen that compared with the control group, arginine and oleanolic acid have a certain promoting effect on the migration of HUVEC cells, and the arginine-oleanolic acid co-amorphous compound formed by the two has a better promoting effect on migration than the arginine group and oleanolic acid group administered alone. This shows that the use of the co-amorphous compound of the two drugs to treat hair loss can help the formation of blood vessels around the hair follicles and provide nutritional conditions for hair growth.

[0193] In summary, the results of cell experiments show that the co-amorphous compound composed of arginine and oleanolic acid can play a role in treating hair loss by promoting the proliferation of human hair papilla cells, upregulating the Wnt / β-catenin pathway, improving the oxidative stress around the hair follicles and the microenvironment of insufficient angiogenesis. Compared with the use of arginine and oleanolic acid alone, the combined use of drugs will not affect the effect of the drugs, and can play a complementary role in the treatment mechanism.

[0194] 8. In vivo pharmacodynamic evaluation

[0195] A 7-week-old C57BL / 6 male mouse was used to establish an animal model of hair loss. After the back hair of the control group mice was removed, no treatment was performed. The mice in the other groups were topically applied with 0.1 mL of a 0.5% testosterone solution on the hair removal site every day for 2 consecutive weeks. The testosterone solution was prepared with 50% ethanol. Except for the control group, the other mice were randomly divided into 5 groups (n=6). The administration method of each group is as follows: (1) Control group: no treatment; (2) Model group: topically applied with 0.1 mL of a 0.5% testosterone solution every day; (3) Co-amorphous gel group: topically applied with Example 4 hydrogel every 3 days; (4) Blank microneedle group: blank microneedles without any drug were applied once every 3 days; (5) Co-amorphous microneedle group: co-amorphous microneedles in Application Example 1 were applied once every 3 days; (6) 5% minoxidil group: topically applied with 0.1 mL of 5% minoxidil commercial tincture once a day. In the above groups, except for the control group (1), the other groups were topically applied with 0.1 mL of 0.5% testosterone solution every day, and each group (3)-(6) was administered according to the above design 8 hours later. During the administration period, photos were taken on days 0, 3, 6, 9, 12, 14, and 16 to record the hair regeneration of the mice, and the hair coverage of the back skin of the mice was calculated using Image J software. After the experiment, the mice were killed by cervical dislocation, and their back hair and skin were removed, and the length of the regenerated hair of the mice was measured using a vernier caliper.

[0196] The growth of back hair in each group of mice during the drug administration period is shown in the figure Fig.19 shown.

[0197] The statistical chart of the regenerated hair coverage rate of each group of mice after drug administration is as follows Fig. 20 As shown. Among them, ＊＊＊It indicated that there was a statistical difference compared with the amorphous microneedle group (P<0.001).

[0198] The statistical diagram of the regenerated hair length of each group of mice after drug administration is shown in the figure below. Fig.21 As shown. Among them, ＊＊＊ It indicated that there was a statistical difference compared with the amorphous microneedle group (P<0.001).

[0199] Depend on Fig.19 It can be seen that compared with the model group, the four drug-treated groups all had the effect of promoting hair growth in mice. Fig. 20 and Fig.21 The statistics of the regenerated hair coverage rate and regenerated hair length of each group of mice show that the co-amorphous microneedle group and the 5% minoxidil tincture group have comparable effects in promoting hair growth, which are better than the co-amorphous gel group and the blank microneedle group. The blank microneedle group can stimulate the skin after the blank microneedle is applied, which can increase the blood supply to the hair follicles to a certain extent, so it also has the effect of promoting hair growth, but its effect is the worst among all the drug administration groups. In addition, the above-mentioned transdermal experiment studies have shown that the drug delivery efficiency of the co-amorphous gel group and the co-amorphous drug-loaded microneedle group is much higher than that of Comparative Example 1; and since the drug in Comparative Example 1 is in a suspended state, it is difficult for the drug to penetrate into the skin, so the poor treatment effect is foreseeable, so this group was not designed in the animal experiment to avoid waste of experimental animal resources.

[0200] In summary, the arginine and pentacyclic triterpenoid compounds of the present invention have a synergistic effect. Compared with the use of the active ingredients alone, they can play the advantages of complementary therapeutic mechanisms and significantly improve the therapeutic effect of hair loss. In addition, the co-amorphous system formed by arginine and pentacyclic triterpenoid compounds can upregulate the Wnt / β-catenin pathway, improve the microenvironment of oxidative stress and insufficient angiogenesis around the hair follicles, provide favorable conditions for the growth of hair follicles, and effectively prevent and treat hair loss. In addition, after the co-amorphous system obtained by arginine and pentacyclic triterpenoid compounds is further processed into microneedles, the therapeutic effect is enhanced. At the same time, the detachable design of the microneedles allows the drug to be effectively retained in the skin and continuously released, thereby reducing the frequency of administration, providing a novel and effective strategy for the treatment of hair loss.

[0201] The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A composition for treating hair loss, It is characterized in that The invention comprises arginine and pentacyclic triterpenoid compounds; the composition is a co-amorphous substance.

2. The composition according to claim 1, It is characterized in that The pentacyclic triterpenoid compound comprises at least one of oleanolic acid, glycyrrhetinic acid, ursolic acid, asiatic acid, betulinic acid and hawthorn acid.

3. The composition according to claim 1, It is characterized in that The molar ratio of arginine to pentacyclic triterpenoid compound is (0.9-4.5):

1.

4. A method for preparing the composition according to any one of claims 1 to 3, It is characterized in that The following steps are involved: The raw material components are mixed, ultrasonically treated and dried to prepare the composition.

5. The preparation method according to claim 4, It is characterized in that The following steps are involved: (1) mixing arginine and water to obtain an arginine solution; mixing a pentacyclic triterpenoid compound, an alcohol and / or an ether to obtain a dispersion of the pentacyclic triterpenoid compound; (2) The arginine solution obtained in step (1) and the dispersion of pentacyclic triterpenoid compounds are mixed, subjected to ultrasonic treatment, and dried to obtain the composition.

6. A hydrogel, It is characterized in that The invention comprises the composition according to any one of claims 1 to 3.

7. A microneedle, It is characterized in that It comprises a backing layer, a separation layer and a needle tip in sequence; the needle tip is composed of the hydrogel according to claim 6.

8. The microneedle according to claim 7, It is characterized in that The mass ratio of the backing layer, the separation layer and the needle tip is (1.5-4.5):(0.8-1.2):

1.

9. The method for preparing the microneedle according to any one of claims 7 to 8, It is characterized in that The following steps are involved: The needle tip, the separation layer and the backing layer are sequentially molded in a mold to obtain the microneedle.

10. Use of the composition according to any one of claims 1 to 3, the hydrogel according to claim 6 or the microneedle according to any one of claims 7 to 8 in the preparation of a drug for treating hair loss.

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