Application of polylactic acid and polylactic acid derivative combined drug in promotion of hair growth

Abstract

The invention belongs to the field of biological medicines, and particularly relates to application of polylactic acid and derivatives thereof in preparation of a medicine for promoting hair growth. The polylactic acid and derivatives thereof can be combined with therapeutic drugs to promote hair growth. Experiments prove that an experimental group containing polylactic acid has a promoting effect on mouse hair growth, and hair growth is rapid; the invention provides a new application of the polylactic acid in promoting hair growth, and the active ingredients of the polylactic acid are high in safety and small in toxic and side effects, so that the polylactic acid has wide application prospects in the fields of pharmaceutical preparations, cosmetics, care products, beauty products and the like for preventing, nursing and treating less hair and alopecia and promoting hair health and growth.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, and particularly relates to application of polylactic acid and its derivatives in promoting hair growth. BACKGROUND

[0002] Hair loss is a disease that has a significant impact on the psychosocial functioning of patients, and is clinically manifested as hair thinning caused by abnormal or excessive hair shedding. Most patients will experience loss of self-esteem, affecting social interaction and overall quality of life. The instinctive reactions resulting therefrom include shame, anger, embarrassment, disgust, vulnerability and sadness, etc. Even when receiving medical treatment for hair loss as a known consequence, patients often list hair loss as the primary side effect they want to treat. Therefore, hair loss disease is a disease that seriously endangers the physical and mental health of patients, and the prevention and treatment of hair loss have become an important field of attention in the medical industry.

[0003] Androgenetic alopecia (AGA), also known as male pattern hair loss or female pattern hair loss, is the most common form of hair loss in the world, characterized by progressive terminal hair loss after puberty. At least 80% of men and 50% of women have this disease by the age of 70, and the incidence increases with age. Androgenetic alopecia presents a unique gender-specific distribution. Male pattern hair loss is usually manifested as reduced hair density and thickness on the temples (bitemporal), vertex (radial progression) and mid-frontal scalp. Female pattern hair loss is characterized by diffuse hair loss and thinning, affecting the frontal hairline and vertex of the scalp. However, the frontal hairline is usually not affected. The mechanism of androgenetic alopecia is rooted in an overreaction to androgens. The typical pattern of male and female hair loss is caused by the distribution of androgen receptors in the scalp.

[0004] Although the prevalence of androgenetic alopecia is high, it can be somewhat challenging to treat androgenetic alopecia due to its chronic nature and the interaction of genetic and environmental factors. Currently, there are only two drugs approved by the US Food and Drug Administration (FDA) for treating androgenetic alopecia, namely topical minoxidil and oral finasteride. Among them, minoxidil, as an effective vasodilator, induces cell proliferation by activating potassium channels on peripheral arterial smooth muscle, and promotes hair growth by increasing vascular endothelial growth factor (VEGF) in dermal papilla cells. In addition, minoxidil can effectively activate the WNT / β-catenin signaling pathway of hair follicle cells to accelerate hair growth. Finasteride is a drug known as a 5α-reductase inhibitor that prevents hair loss by blocking the conversion of testosterone to dihydrotestosterone through 5α-reductase. However, the therapeutic effect of such drugs is limited, and patients need to be dosed frequently and repeatedly, which can easily cause skin allergies, skin inflammation and other side effects. In order to overcome the above-mentioned defects in the prior art, a new emerging treatment method that is non-surgical and has a rapid therapeutic effect is gradually used to improve scalp and hair problems. SUMMARY

[0005] The present application is directed to the problems of poor effect of existing hair growth products and the need for frequent administration, and provides a preparation method of polylactic acid and its derivative microspheres combined with therapeutic drugs. The present application solves the problem of poor therapeutic effect of androgenetic alopecia in the prior art by using polylactic acid and its derivatives and therapeutic drugs in a synergistic manner, has the advantages of safety, high efficiency, low price, etc., and not only can promote hair growth and prevent hair loss, but also can delay scalp aging and continuously exert therapeutic effect.

[0006] Specifically, the present application provides the following technical solutions:

[0007] In a first aspect of the present application, a polylactic acid and its copolymer, polylactic acid microspheres, polylactic acid copolymer microspheres combined with drugs are provided for use in the preparation of a drug and / or cosmetic for promoting hair growth, wherein the molecular weight of the polylactic acid and its copolymer is 400 Da-300 kDa.

[0008] The polylactic acid and its derivatives are selected from the group consisting of poly-L-lactic acid, poly-D-lactic acid, and any copolymer comprising L-lactic acid but not D-lactic acid. The polylactic acid comprises one or more of poly-L-lactic acid, poly-D-lactic acid; preferably poly-L-lactic acid, and the polylactic acid copolymer comprises one or more of poly-lactic-co-glycolic acid, poly-lactic-co- polyethylene glycol, polyethylene glycol-co-poly-lactic-co-glycolic acid, poly-lactic-co- chitosan, poly-lactide-caprolactone; preferably poly-lactic-co-glycolic acid. In certain embodiments, the polylactic acid is poly-L-lactic acid, any copolymer comprising L-lactic acid and ethylene glycol, any copolymer comprising L-lactic acid, ethylene glycol, and glycolic acid, any copolymer comprising L-lactic acid and taurine, any copolymer comprising L-lactic acid and chitosan, and any copolymer comprising L-lactic acid and epsilon-caprolactone; for example, poly-L-lactic acid and any copolymer consisting of L-lactic acid and ethylene glycol.

[0009] The polylactic acid and its copolymers, polylactic acid microspheres, polylactic acid copolymer microspheres are used in the preparation of a medicament for promoting hair growth, wherein the molecular weight of the polylactic acid and its copolymers is between 400 Da and 300 kDa.

[0010] Preferably, the molecular weight of the polylactic acid and its copolymers is between 1000 Da and 100 kDa; for example, 5000 Da.

[0011] In a particular embodiment, the composition comprises particles of the L-lactic acid polymer. Different types of particles can be formulated in the composition. Without being limited by theory, the inventors envision the use of multiple types of particles in the compositions disclosed herein. Thus, in certain embodiments, the particles are selected from the group consisting of micelles, nanoparticles, and microspheres; in other embodiments, the particles are selected from the group consisting of nanoparticles and microspheres; in a particular embodiment, the particles are microspheres. As shown by the inventors in the examples, compositions comprising microspheres of the L-lactic acid polymer are particularly beneficial in the disclosed treatments. Particles of the L-lactic acid polymer provide sustained release of the active agent over time relative to a solution, thus requiring less frequent dosing to achieve the desired therapeutic effect. The present invention relates to the medicinal properties of polylactic acid and its derivatives, which, as described in this patent, produce the therapeutic effects described below.

[0012] In certain embodiments, the polymer has a molecular weight of at least about 400 Da, such as at least about 500 Da, such as at least about 1 kDa, such as at least about 2 kDa, such as at least about 5 kDa, such as at least about 10 kDa, such as at least about 20 kDa, such as at least about 30 kDa, such as at least about 40 kDa, such as at least about 50 kDa, such as at least about 100 kDa, such as at least about 150 kDa, such as at least about 200 kDa, such as at least about 250 kDa, such as at least about 300 kDa. In certain embodiments, the polymer has a molecular weight of no more than about 300 kDa, such as no more than about 275 kDa, such as no more than about 250 kDa, such as no more than about 225 kDa, such as no more than about 200 kDa, such as no more than about 175 kDa, such as no more than about 150 kDa, such as no more than about 125 kDa, such as no more than about 100 kDa. The inventors have found that certain ranges of molecular weight of the polymer can be advantageous, as polymers having a molecular weight of less than 400 Da can degrade too quickly, and polymers having a molecular weight of more than 300 kDa can be difficult to degrade. Thus, in one embodiment, the polymer has a molecular weight ranging from about 400 Da to about 300 kDa, such as from about 500 Da to about 300 kDa, such as from about 1000 Da to about 300 kDa, such as from about 1500 Da to about 275 kDa, such as from about 2000 Da to about 250 kDa, such as from about 2500 Da to about 225 kDa, such as from about 3000 Da to about 200 kDa, such as from about 3500 Da to about 175 kDa, such as from about 4000 Da to about 150 kDa, such as from about 4500 Da to about 125 kDa, such as from about 5000 Da to about 100 kDa. In one embodiment, the polymer has a molecular weight ranging from about 1 kDa to about 100 kDa, such as from about 5 kDa to about 90 kDa, such as from about 10 kDa to about 80 kDa, such as from about 15 kDa to about 70 kDa, such as from about 20 kDa to about 60 kDa, such as from about 25 kDa to about 50 kDa, such as from about 30 kDa to about 40 kDa.

[0013] In a particular embodiment, the composition includes particles of the L-polylactic acid polymer. Different types of particles can be formulated in the composition. Without being limited by theory, the inventors envision using multiple types of particles in the compositions disclosed herein. Thus, in certain embodiments, the particles are selected from the group consisting of micelles, nanoparticles, and microspheres; in other embodiments, the particles are selected from the group consisting of nanoparticles and microspheres; in a particular embodiment, the particles are microspheres. As shown by the inventors in the examples, compositions including microspheres of the L-polylactic acid polymer are particularly beneficial in the disclosed treatments. It is envisioned that particles of the L-polylactic acid polymer can provide sustained release of the active agent over time, thus requiring less frequent dosing to achieve the desired therapeutic effect, relative to, for example, a solution thereof. The present invention relates to the medicinal properties of polylactic acid and its derivatives, which, as described in the present patent, produce the therapeutic effects described below.

[0014] In certain embodiments, the particles have a particle size of at least about 10 microns, at least about 15 microns, at least about 20 microns, at least about 25 microns, at least about 30 microns, at least about 35 microns, at least about 40 microns, at least about 45 microns, at least about 50 microns, at least about 55 microns, at least about 60 microns; and / or a particle size of about 1 to 100 microns, such as about 2 to 90 microns, about 5 to 85 microns, about 10 to 80 microns, about 15 to 75 microns, about 20 to 70 microns, about 25 to 65 microns, about 30 to 60 microns; and / or a particle size of about 45 microns ± 15 microns, such as a particle size of about 45 microns. In an embodiment, the particles have a particle size of about 10 to 100 microns, such as about 15 to 90 microns, such as about 20 to 80 microns, such as about 25 to 70 microns, such as about 30 to 65 microns, such as about 40 to 65 microns. In another aspect, the particles can have any particle size within the indicated range of particle sizes in any distribution pattern in the composition. Alternatively, the particles exhibit a normal distribution within the indicated range of particle sizes in the composition. Thus, in a particular embodiment, the particles exhibit a normal distribution of particle sizes of about 1 to 100 microns in the composition. For the sake of clarity and to avoid any doubt, it is understood that in embodiments in which the particles are microspheres, the particle size corresponds to the size of the microspheres.

[0015] Without being limited by theory, the inventors envision that the smooth surface of the microspheres or other particles disclosed herein is associated with a lower inflammatory response in the patient being treated, as compared to particles having a more uneven and rough structure.

[0016] A composition of poly-L-lactic acid and / or its copolymer for promoting hair growth is provided in the present disclosure, wherein the poly-L-lactic acid and / or its copolymer is as defined in any embodiment of the present disclosure; and the composition comprises at least one pharmaceutically acceptable carrier and / or excipient.

[0017] In certain embodiments, the content of L-lactic acid polymer in the composition is about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%. In certain embodiments, the content of L-lactic acid polymer in the composition is about 1% to 100%, about 5% to 95%, about 10% to 90%, about 15% to 85%, about 20% to 80%, about 25% to 75%, about 30% to 70%, about 35% to 65%, about 40% to 60%, about 45% to 55%. In one embodiment, the composition comprises at least about 3% of the poly-L-lactic acid, for example about 3% of the poly-L-lactic acid. It should be understood that the percentage corresponds to the concentration ratio of weight and volume (weight / volume; w / v).

[0018] In one embodiment, the method for preparing poly-lactic acid microspheres or poly-lactic acid copolymer microspheres comprises the following steps:

[0019] 1) mixing poly-lactic acid or poly-lactic acid copolymer with a solvent to obtain a poly-lactic acid solution or a poly-lactic acid copolymer solution;

[0020] 2) mixing the poly-lactic acid solution or the poly-lactic acid copolymer solution with an aqueous polyvinyl alcohol solution, high-speed emulsifying, stirring to remove the solvent, and freeze-drying to obtain poly-lactic acid microspheres.

[0021] In a preferred embodiment, the solvent is any one of dichloromethane, chloroform, ethyl acetate, acetone or toluene, or a mixture of any two or more thereof.

[0022] In a preferred embodiment, in step 1) above, the mass-volume ratio of poly-lactic acid or poly-lactic acid copolymer to solvent is 1 g: 1 mL ~ 1 g: 50 mL, and in a more preferred embodiment, the mass-volume ratio of poly-lactic acid or poly-lactic acid copolymer to solvent is 1 g: 10 mL ~ 1 g: 40 mL, for example 1 g: 30 mL.

[0023] And / or, in step 2), the mass concentration of the aqueous polyvinyl alcohol solution is 0.05% ~ 5.0%, and in a preferred embodiment, the mass concentration of the aqueous polyvinyl alcohol solution is 0.1% ~ 2.0%, for example 1.5%.

[0024] and / or, in step 2), the volume ratio of the polylactic acid solution or the polylactic acid copolymer solution to the polyvinyl alcohol aqueous solution is 1:5-1:30, in a preferred embodiment, the volume ratio of the polylactic acid solution or the polylactic acid copolymer solution to the polyvinyl alcohol aqueous solution is 1:10-1:20, for example, 1:13, 1:15;

[0025] and / or, in step 2), the rotation speed of the high-speed emulsification is 1000 rpm / min-5000 rpm / min, for example, 3000 rpm / min;

[0026] and / or, in step 2), the emulsification time is 5-20 minutes, for example, 10 minutes.

[0027] In an embodiment, the mass concentration of the polylactic acid and its copolymer, the polylactic acid microspheres, and the polylactic acid copolymer microspheres in the drug or the cosmetic is 0.1%-80%, in a preferred embodiment, the mass concentration of the polylactic acid and its copolymer, the polylactic acid microspheres, and the polylactic acid copolymer microspheres is 1%-5%, for example, 1%, 3%, 5%.

[0028] In an embodiment, the drug and / or the cosmetic can further comprise an excipient;

[0029] In a preferred embodiment, the excipient comprises at least one of a stabilizer, a filler, a binder, and a surfactant;

[0030] In a more preferred embodiment, the surfactant is selected from one or more of polyethylene glycol, sodium dodecyl sulfate, Tween, Span;

[0031] In a more preferred embodiment, the stabilizer is selected from one or both of carboxymethyl cellulose and mannitol;

[0032] In a more preferred embodiment, the filler is selected from one or more of lactose, mannitol, cyclodextrin, sorbitol;

[0033] In a more preferred embodiment, the binder is selected from one or more of hydroxypropyl cellulose, methyl cellulose, sodium hyaluronate, collagen, polyvinylpyrrolidone;

[0034] In an embodiment, the drug can be optionally further combined with other therapeutic drugs, which include but are not limited to the following categories:

[0035] 1. vasodilator drugs, such as minoxidil, minoxidil sulfate, etc.;

[0036] 2.5 alpha-reductase inhibitors, such as finasteride, dutasteride, etc.;

[0037] 3. Androgen receptor antagonists, such as spironolactone, cyproterone acetate, ketoconazole, flutamide, etc.;

[0038] 4. Prostaglandin analogues, such as latanoprost, bimatoprost, etc.;

[0039] 5. Traditional Chinese medicine extracts, such as forsythoside, red ginseng, saw palmetto, baicalin, ginkgo biloba, radix scrophulariae, etc., and active ingredients thereof.

[0040] In one embodiment, the polylactic acid and its copolymer materials and the therapeutic drugs are dissolved in a solvent;

[0041] In one embodiment, the administration of the drug includes, but is not limited to, injection, smearing, etc.

[0042] In one embodiment, the dosage form of the drug is injection, ointment, etc.

[0043] Compared with the prior art, the present application has the following significant advantages:

[0044] The technical scheme of the present application solves the problem of poor treatment effect of androgenetic alopecia in the prior art by using polylactic acid and its derivatives and therapeutic drugs in a synergistic manner, and has the advantages of safety, high efficiency, low price, etc. It not only promotes hair growth and prevents hair loss, but also delays scalp aging and continuously exerts therapeutic effect. BRIEF DESCRIPTION OF DRAWINGS

[0045] The accompanying drawings are used to provide a further understanding of the present application, and constitute a part of the specification, together with the embodiments of the present application, to explain the present application, and do not constitute a limitation of the present application. In the drawings:

[0046] Figure 1 The effect diagram of each group of mice after administration of different concentrations of polylactic acid microspheres in the embodiments of the present application.

[0047] Figure 2 The effect diagram of each group of mice after administration of polylactic acid microspheres in the embodiments of the present application.

[0048] Figure 3 The effect diagram of each group of mice after administration of polylactic acid microspheres in the embodiments of the present application. DETAILED DESCRIPTION

[0049] The preferred embodiments of the present application are described below in conjunction with the accompanying drawings, and it should be understood that the preferred embodiments described herein are only used to illustrate and explain the present application, and do not limit the present application.

[0050] Example 1: Preparation method of polylactic acid (PLLA) microspheres

[0051] 10 g of polylactic acid (PLLA) (molecular weight 10 kDa) was dissolved in 200 mL of dichloromethane. Then, the polymer solution was added to 2000 mL of 1.0% polyvinyl alcohol aqueous solution. After emulsification at 5000 rpm / min for 15 min, the mixture was stirred for 6 hours to remove the dichloromethane, resulting in PLLA microspheres. The prepared microspheres had a smooth surface and a particle size of 3–55 μm.

[0052] Example 2: Preparation method of polylactic acid (PLLA) drug-loaded microspheres

[0053] 10 g of polylactic acid (PLLA) (molecular weight 10 kDa) was dissolved in 200 mL of dichloromethane. 3.33 g of the therapeutic agent was added to the solution and stirred until completely dissolved. Then, 2000 mL of a 1.0% (w / w) aqueous solution of polyvinyl alcohol was added to the polymer and therapeutic agent mixture. Emulsification was carried out at 5000 rpm for 15 min, followed by stirring for 6 hours to remove the dichloromethane, yielding PLLA drug-loaded microspheres. The prepared microspheres had a smooth surface and a particle size of 3–55 μm.

[0054] Example 3: Preparation method of polylactic acid-glycolic acid copolymer microspheres

[0055] 10g of polylactic acid-glycolic acid copolymer (PLGA) (molecular weight 10kDa) was dissolved in 200mL of dichloromethane. Then, the polymer solution was added to 2000mL of 1% polyvinyl alcohol aqueous solution. After emulsification at 5000rpm / min for 15 minutes, the mixture was stirred for 6 hours to remove the dichloromethane. After drying, polylactic acid-glycolic acid copolymer microspheres were obtained. The microspheres had a smooth surface and a particle size of 3-55μm.

[0056] Example 4: Preparation method of polylactic-glycolic acid copolymer (PLGA) drug-loaded microspheres

[0057] 10 g of polylactic acid-glycolic acid copolymer (PLGA) (molecular weight 10 kDa) was dissolved in 100 mL of dichloromethane. 3.33 g of the therapeutic agent was added to the solution and stirred until completely dissolved. Then, 2000 mL of a 1.0% (w / w) aqueous solution of polyvinyl alcohol was added to the polymer-drug mixture. The mixture was emulsified at 5000 rpm for 15 min, followed by stirring for 6 hours to remove the dichloromethane, yielding PLGA drug-loaded microspheres. The prepared microspheres had a smooth surface and a particle size of 3–55 μm.

[0058] Example 4: Establishment of androgen-induced alopecia mouse model and screening of appropriate ratios

[0059] 1. Experimental Grouping

[0060] Grouping Group Number of mice Administration method Administration dose Group A Normal group 8 Smearing Corresponding volume of normal saline Group B Androgen alopecia model group 8 Intraperitoneal injection 100 μL Group C Concentration 1% PLLA microsphere group 8 Smearing 100 μL Group D Concentration 3% PLLA microsphere group 8 Smearing 100 μL Group E Concentration 5% PLLA microsphere group 8 Smearing 100 μL

[0061] Note: The polylactic acid microspheres used in groups A, B, and C are poly-L-lactic acid microspheres prepared according to the method described in Example 1. All concentrations mentioned herein are mass concentrations.

[0062] Except for the normal group mice which did not receive subcutaneous injection of dihydrotestosterone (DHT), all mice underwent daily intraperitoneal injections of DHT to establish an androgenetic alopecia model. The specific modeling methods and steps are as follows:

[0063] 1) C57BL / 6 mice were placed in a constant temperature room (24±2℃) with a 12-hour dark-light cycle for 7 days for acclimatization. 2) Establishment of the androgenic alopecia mouse model (AGA model): Dihydrotestosterone was dissolved in corn oil (10mg / ml, i.e., 1%).

[0064] Except for the normal group, all other groups of mice were injected intraperitoneally with 0.1 ml for 15 consecutive days, starting 4 days before hair removal, in order to establish the AGA model.

[0065] 3) Hair removal in experimental animals: After mice were anesthetized with isoflurane, rosin and paraffin were mixed in a 1:1 volume ratio and heated to melt. After the temperature dropped, the mixture was applied to the back of the mice. After solidification, the hair on the back of the mice was removed. The hair removal area was 2cm × 3cm. The mice were then fed under the original conditions after hair removal treatment, and each group continued to receive the corresponding pre-hair removal intervention for the specified number of days.

[0066] 2. Mice in each group were administered the drug for 15 days, and photographs were taken on days 1, 8, 11, and 15 after administration. Figure 1 ), and observe the hair growth of mice.

[0067] 3. On day 15 of drug administration, 10 newly grown hairs were randomly selected from the experimental area, and their lengths were measured and recorded using an electronic digital caliper.

[0068] 4. On day 15, mice were sacrificed to assess model establishment and hair growth. Skin samples from the backs of mice were fixed with paraformaldehyde and stained with hematoxylin and eosin (HE) to observe hair follicle cell growth. The total number of hair follicle cells was counted in cross-sections of the skin at the same level.

[0069] 1) Dewaxing paraffin sections: First place them in xylene I for 20 min, then in xylene II for 20 min, then in a series of ethanol solutions for 5 min each, and finally rinse with tap water.

[0070] 2) Hematoxylin staining: Immerse the sections in the staining solution for 3-5 minutes, then rinse with tap water, then differentiate with differentiation solution, and finally rinse with tap water.

[0071] 3) Eosin staining: The sections were dehydrated in 85% and 95% graded ethanol solutions for 5 min, and then immersed in eosin staining solution for 5 min.

[0072] 4) Dehydration and sealing: The sections were sequentially immersed in a gradient of ethanol for 5 min each, then xylene I for 5 min, followed by xylene II for 5 min, and then sealed with transparent neutral glue.

[0073] 5) Microscopic observation, image acquisition and analysis.

[0074] 6) Statistical Analysis Methods: All data were analyzed using SPSS blotting, and all results are expressed as mean ± standard deviation. Each experiment was repeated three times, with three parallel samples. For the normal group compared to the androgenetic alopecia model group, * indicates p < 0.05, indicating a statistically significant difference; ** indicates p < 0.01, indicating a statistically significant difference. For the treatment group compared to the androgenetic alopecia model group, # indicates p < 0.05, indicating a statistically significant difference; ## indicates p < 0.01, indicating a statistically significant difference.

[0075] 5. Experimental Results

[0076] The hair growth in mice 15 days after drug administration is shown in the table below:

[0077] Table 1: Effects on the length of new hair regrowth in androgenetic alopecia mice

[0078]

[0079] Note: Compared with the androgenetic alopecia model group, * indicates p<0.05, which is statistically significant, and ** indicates p<0.01, which is statistically significant; compared with the androgenetic alopecia model group, # indicates p<0.05, which is statistically significant, and ## indicates p<0.01, which is statistically significant.

[0080] Table 2: Effects on the number of hair follicles in the anagen phase on the dorsal skin of mice

[0081]

[0082] Note: Compared with the androgenetic alopecia model group, * indicates p<0.05, which is statistically significant, and ** indicates p<0.01, which is statistically significant; compared with the androgenetic alopecia model group, # indicates p<0.05, which is statistically significant, and ## indicates p<0.01, which is statistically significant.

[0083] from Figure 1As shown in Tables 1 and 2, the results indicate that intraperitoneal injection of dihydrotestosterone (DHT) slows down the growth of new hair in mice. After 15 days of treatment, all groups of poly-L-lactic acid microspheres accelerated the rate of new hair growth and the number of hair follicles in the anagen phase in mice with androgenetic alopecia. Among them, the 3% poly-L-lactic acid microsphere group showed the best effect in new hair growth.

[0084] Example 5: Verification of the effects of polylactic acid and its copolymers

[0085] 1. Experimental Grouping

[0086] Grouping Group Number of mice Administration method Administration dose Group A Normal group 8 Smearing Corresponding volume of normal saline Group B Androgen alopecia model group 8 Intraperitoneal injection 100 μL Group C Concentration 3% PLLA microsphere group 8 Smearing 100 μL Group D Concentration 3% PLGA microsphere group 8 Smearing 100 μL

[0087] Note: The polylactic acid used in Group C was poly-L-lactic acid microspheres prepared according to the method described in Example 1. The particle size of the prepared poly-L-lactic acid microspheres was 3–55 μm. The polylactic acid-glycolic acid copolymer microspheres used in Group D were prepared according to the method described in Example 2. The molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer microspheres was 80:20. A microsphere concentration of 3% was chosen to achieve the effects described in the examples. Only one concentration in the examples is used as an example for detailed explanation; other concentrations are not described here.

[0088] Except for the normal group mice which did not receive subcutaneous injection of dihydrotestosterone (DHT), all mice underwent daily intraperitoneal injections of DHT to establish an androgenetic alopecia model. The specific modeling methods and steps are as follows:

[0089] 1) Select C57BL / 6 mice and place them in a constant temperature room (24±2℃) with a dark-light cycle of 12 hours for 7 days for acclimatization.

[0090] 2) Establishment of androgenic alopecia mouse model (AGA model): Dihydrotestosterone was dissolved in corn oil (10 mg / ml, i.e., 1%). Except for the normal group, mice in other groups were injected intraperitoneally with 0.1 ml for 15 consecutive days, starting 4 days before hair removal, in order to establish the AGA model.

[0091] 3) Hair removal in experimental animals: After mice were anesthetized with isoflurane, rosin and paraffin were mixed in a 1:1 volume ratio and heated to melt. After the temperature dropped, the mixture was applied to the back of the mice. After solidification, the hair on the back of the mice was removed. The hair removal area was 2cm × 3cm. The mice were then fed under the original conditions after hair removal treatment, and each group continued to receive the corresponding pre-hair removal intervention for the specified number of days.

[0092] 4) The specific dosing regimens for each group are as follows:

[0093] Group A received daily application of saline solution.

[0094] Group B received daily intraperitoneal injections of dihydrotestosterone (1%).

[0095] Group C received daily application of poly-L-lactic acid microspheres (3%).

[0096] Group D received daily application of polylactic acid-glycolic acid copolymer microspheres (3%).

[0097] 2. Mice in each group were administered the drug for 15 days, and photographs were taken on days 1, 8, 11, and 15 after administration. Figure 2 ), and observe the hair growth of mice.

[0098] 3. On day 15 of drug administration, 10 newly grown hairs were randomly selected from the experimental area, and their lengths were measured and recorded using an electronic digital caliper.

[0099] 4. On day 15, mice were sacrificed to assess model establishment and hair growth. Skin samples from the backs of mice were fixed with paraformaldehyde and stained with hematoxylin and eosin (HE) to observe hair follicle cell growth. The total number of hair follicle cells was counted in cross-sections of the skin at the same level.

[0100] 1) Dewaxing paraffin sections: First place them in xylene I for 20 min, then in xylene II for 20 min, then in a series of ethanol solutions for 5 min each, and finally rinse with tap water.

[0101] 2) Hematoxylin staining: Immerse the sections in the staining solution for 3-5 minutes, then rinse with tap water, then differentiate with differentiation solution, and finally rinse with tap water.

[0102] 3) Eosin staining: The sections were dehydrated in 85% and 95% graded ethanol solutions for 5 min, and then immersed in eosin staining solution for 5 min.

[0103] 4) Dehydration and sealing: The sections were sequentially immersed in a gradient of ethanol for 5 min each, then xylene I for 5 min, followed by xylene II for 5 min, and then sealed with transparent neutral glue.

[0104] 5) Microscopic observation, image acquisition and analysis.

[0105] 6) Statistical Analysis Methods: All data were analyzed using SPSS blotting, and all results are expressed as mean ± standard deviation. Each experiment was repeated three times, with three parallel samples. For the normal group compared to the androgenetic alopecia model group, * indicates p < 0.05, indicating a statistically significant difference; ** indicates p < 0.01, indicating a statistically significant difference. For the treatment group compared to the androgenetic alopecia model group, # indicates p < 0.05, indicating a statistically significant difference; ## indicates p < 0.01, indicating a statistically significant difference.

[0106] Table 3: Effects on the length of new hair regrowth in androgenetic alopecia mice

[0107]

[0108] Note: Compared with the androgenetic alopecia model group, * indicates p<0.05, which is statistically significant, and ** indicates p<0.01, which is statistically significant; compared with the androgenetic alopecia model group, # indicates p<0.05, which is statistically significant, and ## indicates p<0.01, which is statistically significant.

[0109] Table 4: Effects on the number of hair follicles in the anagen phase on the dorsal skin of mice

[0110]

[0111] Note: Compared with the androgenetic alopecia model group, * indicates p<0.05, which is statistically significant, and ** indicates p<0.01, which is statistically significant; compared with the androgenetic alopecia model group, # indicates p<0.05, which is statistically significant, and ## indicates p<0.01, which is statistically significant.

[0112] The experimental results in Tables 3 and 4 show that intraperitoneal injection of dihydrotestosterone can effectively alleviate the growth of new hair in mice. The hair growth in the PLLA microsphere group was better than that in the polylactic acid-glycolic acid copolymer microsphere group, which may be related to the content of L-lactic acid in the polymer microspheres.

[0113] Example 6: Efficacy Verification of the Therapeutic Drug

[0114] 1. Experimental Grouping

[0115]

[0116]

[0117] Except for the normal group mice which did not receive subcutaneous injection of dihydrotestosterone (DHT), all mice underwent daily intraperitoneal injections of DHT to establish an androgenetic alopecia model. The specific modeling methods and steps are as follows:

[0118] 1) Select C57BL / 6 mice and place them in a constant temperature room (24±2℃) with a dark-light cycle of 12 hours for 7 days for acclimatization.

[0119] 2) Establishment of androgenic alopecia mouse model (AGA model): Dihydrotestosterone was dissolved in corn oil (10 mg / ml, i.e., 1%). Except for the normal group, mice in other groups were injected intraperitoneally with 0.1 ml for 15 consecutive days, starting 4 days before hair removal, in order to establish the AGA model.

[0120] 3) Hair removal in experimental animals: After mice were anesthetized with isoflurane, rosin and paraffin were mixed in a 1:1 volume ratio and heated to melt. After the temperature dropped, the mixture was applied to the back of the mice. After solidification, the hair on the back of the mice was removed. The hair removal area was 2cm × 3cm. The mice were then fed under the original conditions after hair removal treatment, and each group continued to receive the corresponding pre-hair removal intervention for the specified number of days.

[0121] 4) The specific dosing regimens for each group are as follows:

[0122] Group A received daily application of saline solution.

[0123] Group B received daily intraperitoneal injections of dihydrotestosterone (1%).

[0124] Group C applied minoxidil (3%) daily.

[0125] Group D applied finasteride (3%) daily.

[0126] Group E applied spironolactone (3%) daily.

[0127] Group F applied latanoprost (3%) daily.

[0128] 2. On day 15 of drug administration, 10 newly grown hairs were randomly selected from the experimental area, and their lengths were measured and recorded using an electronic digital vernier caliper.

[0129] 3. On day 15, mice were sacrificed to assess the model establishment and hair growth. Skin samples from the backs of mice were fixed with paraformaldehyde and stained with hematoxylin and eosin (HE) to observe the growth of hair follicle cells. The total number of hair follicle cells was counted in cross-sections of the skin at the same level.

[0130] 1) Dewaxing paraffin sections: First place them in xylene I for 20 min, then in xylene II for 20 min, then in a series of ethanol solutions for 5 min each, and finally rinse with tap water.

[0131] 2) Hematoxylin staining: Immerse the sections in the staining solution for 3-5 minutes, then rinse with tap water, then differentiate with differentiation solution, and finally rinse with tap water.

[0132] 3) Eosin staining: The sections were dehydrated in 85% and 95% graded ethanol solutions for 5 min, and then immersed in eosin staining solution for 5 min.

[0133] 4) Dehydration and sealing: The sections were sequentially immersed in a gradient of ethanol for 5 min each, then xylene I for 5 min, followed by xylene II for 5 min, and then sealed with transparent neutral glue.

[0134] 5) Microscopic observation, image acquisition and analysis.

[0135] 6) Statistical Analysis Methods: All data were analyzed using SPSS blotting, and all results are expressed as mean ± standard deviation. Each experiment was repeated three times, with three parallel samples. For the normal group compared to the androgenetic alopecia model group, * indicates p < 0.05, indicating a statistically significant difference; ** indicates p < 0.01, indicating a statistically significant difference. For the treatment group compared to the androgenetic alopecia model group, # indicates p < 0.05, indicating a statistically significant difference; ## indicates p < 0.01, indicating a statistically significant difference.

[0136] Table 5: Effects on the length of new hair regrowth in androgenetic alopecia mice

[0137]

[0138] Note: Compared with the androgenetic alopecia model group, * indicates p<0.05, which is statistically significant, and ** indicates p<0.01, which is statistically significant; compared with the androgenetic alopecia model group, # indicates p<0.05, which is statistically significant, and ## indicates p<0.01, which is statistically significant.

[0139] Table 6: Effects on the number of hair follicles in the anagen phase on the dorsal skin of mice

[0140]

[0141]

[0142] Note: Compared with the androgenetic alopecia model group, * indicates p<0.05, which is statistically significant, and ** indicates p<0.01, which is statistically significant; compared with the androgenetic alopecia model group, # indicates p<0.05, which is statistically significant, and ## indicates p<0.01, which is statistically significant.

[0143] The experimental results in Tables 5 and 6 show that the treatment groups all increased the hair growth rate and the number of hair follicles in the anagen phase in mice with androgenetic alopecia, with the minoxidil group showing the best treatment effect.

[0144] Example 7: Efficacy verification of polylactic acid in combination with other therapeutic drugs

[0145] 1. Experimental Grouping

[0146] Grouping Group Number of mice Administration method Administration dose Group A Normal group 8 Smearing Corresponding volume of normal saline Group B Androgen alopecia model group 8 Intraperitoneal injection 100 μL Group C PLLA microsphere group 8 Smearing 100 μL Group D PLLA microsphere + minoxidil group 8 Smearing 100 μL Group E PLLA microsphere + finasteride group 8 Smearing 100 μL Group F PLLA microsphere + spironolactone group 8 Smearing 100 μL Group G PLLA microsphere + latanoprost group 8 Smearing 100 μL

[0147] Note: The polylactic acid used in groups C, D, E, F, and G was poly-L-lactic acid microspheres prepared according to the method described in Example 1. The particle size of the prepared poly-L-lactic acid microspheres was 3–55 μm. The polylactic acid-glycolic acid copolymer microspheres used in group D were prepared according to the method described in Example 2. The molar ratio of lactic acid to glycolic acid in the polylactic acid-polyglycolic acid copolymer microspheres was 80:20. A microsphere concentration of 3% was chosen to achieve the effects described in the examples. Only one concentration in the examples is used as an example for detailed explanation; other concentrations are not described here.

[0148] Except for the normal group mice which did not receive subcutaneous injection of dihydrotestosterone (DHT), all mice underwent daily intraperitoneal injections of DHT to establish an androgenetic alopecia model. The specific modeling methods and steps are as follows:

[0149] 1) Select C57BL / 6 mice and place them in a constant temperature room (24±2℃) with a dark-light cycle of 12 hours for 7 days for acclimatization.

[0150] 2) Establishment of androgenic alopecia mouse model (AGA model): Dihydrotestosterone was dissolved in corn oil (10 mg / ml, i.e., 1%). Except for the normal group, mice in other groups were injected intraperitoneally with 0.1 ml for 15 consecutive days, starting 4 days before hair removal, in order to establish the AGA model.

[0151] 3) Hair removal in experimental animals: After mice were anesthetized with isoflurane, rosin and paraffin were mixed in a 1:1 volume ratio and heated to melt. After the temperature dropped, the mixture was applied to the back of the mice. After solidification, the hair on the back of the mice was removed. The hair removal area was 2cm × 3cm. The mice were then fed under the original conditions after hair removal treatment, and each group continued to receive the corresponding pre-hair removal intervention for the specified number of days.

[0152] 4) The specific dosing regimens for each group are as follows:

[0153] Group A received daily application of saline solution.

[0154] Group B received daily intraperitoneal injections of dihydrotestosterone (1%).

[0155] Group C received daily application of poly-L-lactic acid microspheres (3%).

[0156] Group D received daily application of a combination of poly-L-lactic acid microspheres (3%) and minoxidil (3%).

[0157] Group E received a daily application of a combination of poly-L-lactic acid microspheres (3%) and finasteride (3%).

[0158] Group F received daily application of a combination of poly-L-lactic acid microspheres (3%) and spironolactone (3%).

[0159] Group G received daily application of a combination of poly-L-lactic acid microspheres (3%) and latanoprost (3%).

[0160] 2. Mice in each group were administered the drug for 15 days, and photographs were taken on days 1, 8, 11, and 15 after administration. Figure 3 ), and observe the hair growth of mice.

[0161] 3. On day 15 of drug administration, 10 newly grown hairs were randomly selected from the experimental area, and their lengths were measured and recorded using an electronic digital caliper.

[0162] 4. On day 15, mice were sacrificed to assess model establishment and hair growth. Skin samples from the backs of mice were fixed with paraformaldehyde and stained with hematoxylin and eosin (HE) to observe hair follicle cell growth. The total number of hair follicle cells was counted in cross-sections of the skin at the same level.

[0163] 7) Dewaxing paraffin sections: First place them in xylene I for 20 min, then in xylene II for 20 min, then in a series of ethanol solutions for 5 min each, and finally rinse with tap water.

[0164] 8) Hematoxylin staining: Immerse the sections in the staining solution for 3-5 minutes, then rinse with tap water, then differentiate with differentiation solution, and finally rinse with tap water.

[0165] 9) Eosin staining: The sections were dehydrated in 85% and 95% graded ethanol solutions for 5 min, and then immersed in eosin staining solution for 5 min.

[0166] 10) Dehydration and sealing: The sections were sequentially immersed in a gradient of ethanol for 5 min each, then xylene I for 5 min, followed by xylene II for 5 min, and then sealed with transparent neutral glue;

[0167] 11) Microscopic observation, image acquisition and analysis.

[0168] 12) Statistical Analysis Methods: All data were analyzed using SPSS, and all results are expressed as mean ± standard deviation. Each experiment was repeated three times, with three parallel samples. For the normal group compared to the androgenetic alopecia model group, * indicates p < 0.05, indicating a statistically significant difference; ** indicates p < 0.01, indicating a statistically significant difference. For the treatment group compared to the androgenetic alopecia model group, # indicates p < 0.05, indicating a statistically significant difference; ## indicates p < 0.01, indicating a statistically significant difference.

[0169] Table 7: Effects on the length of new hair regrowth in androgenetic alopecia mice

[0170]

[0171] Note: Compared with the androgenetic alopecia model group, * indicates p<0.05, which is statistically significant, and ** indicates p<0.01, which is statistically significant; compared with the androgenetic alopecia model group, # indicates p<0.05, which is statistically significant, and ## indicates p<0.01, which is statistically significant.

[0172] Table 8: Effects on the number of hair follicles in the anagen phase on the dorsal skin of mice

[0173]

[0174] Note: Compared with the androgenetic alopecia model group, * indicates p<0.05, which is statistically significant, and ** indicates p<0.01, which is statistically significant; compared with the androgenetic alopecia model group, # indicates p<0.05, which is statistically significant, and ## indicates p<0.01, which is statistically significant.

[0175] from Figure 3 The experimental results in Tables 7 and 8 show that poly-L-lactic acid and other treatment drugs increased the hair growth rate and the number of hair follicles in the anagen phase in mice with androgenetic alopecia, indicating that this combined treatment is effective.

[0176] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of a combined drug of polylactic acid and its copolymer microspheres in the preparation of a drug and / or cosmetic for promoting hair growth, wherein, The molecular weight of the polylactic acid and the copolymer thereof is 400 Da-300 kDa.

2. Use according to claim 1, wherein The polylactic acid comprises one or more of levorotatory polylactic acid and racemic polylactic acid; preferably, the polylactic acid is levorotatory polylactic acid; the polylactic acid copolymer comprises one or more of polylactic acid-glycolic acid copolymer, polylactic acid-polyethylene glycol copolymer, polyethylene glycol-polylactic acid-glycolic acid copolymer, polylactic acid-chitosan copolymer and lactide-caprolactone copolymer; preferably, the polylactic acid copolymer is polylactic acid-glycolic acid copolymer.

3. Use according to any one of claims 1-2, characterized in that, The preparation method of the polylactic acid microspheres or the polylactic acid copolymer microspheres comprises the following steps: 1) mixing the polylactic acid or the polylactic acid copolymer with a solvent to obtain a polylactic acid solution or a polylactic acid copolymer solution; 2) mixing the polylactic acid solution or the polylactic acid copolymer solution with an aqueous polyvinyl alcohol solution, high-speed emulsifying, stirring to remove the solvent, and freeze-drying to obtain the polylactic acid microspheres.

4. Use according to claim 3, wherein the compound is ###0002### The solvent is any one of dichloromethane, chloroform, ethyl acetate, acetone or toluene, or a mixture of any two or more thereof.

5. The use according to claim 3, wherein In step 1), the mass-volume ratio of the polylactic acid or the polylactic acid copolymer to the solvent is 1 g:5 mL-1 g:30 mL, preferably 1 g:10 mL-1 g:20 mL, for example 1 g:15 mL; and / or, in step 2), the mass concentration of the aqueous polyvinyl alcohol solution is 0.05%-5.0%; and / or, in step 2), the volume ratio of the polylactic acid solution or the polylactic acid copolymer solution to the aqueous polyvinyl alcohol solution is 1:5-1:30; and / or, in step 2), the rotation speed of high-speed emulsification is 1000 rpm / min-5000 rpm / min; and / or, in step 2), the emulsification time is 5-20 minutes.

6. Use according to any one of claims 1 to 5, wherein When administered, the mass concentration of the polylactic acid and the copolymer thereof, the polylactic acid microspheres or the polylactic acid copolymer microspheres is 0.1%-80%, preferably 1%-5%, for example 1%, 3%, 5%.

7. The use according to claim 1, wherein The medicine and / or the cosmetic optionally further comprises an auxiliary material, which comprises at least one of a stabilizer, a filler, a binder and a surfactant.

8. The use according to claim 1, wherein The medicine and / or the cosmetic optionally further comprises other medicines, which have the effect of promoting hair growth.

9. Use according to claim 8, wherein the compound is ###0002### The other medicines include but are not limited to vasodilator medicines, 5α-reductase inhibitor medicines, androgen receptor antagonist medicines, prostaglandin analogue medicines and / or traditional Chinese medicine extracts.

10. Use according to any one of claims 1 to 9, wherein The administration mode of the medicine includes but is not limited to intravenous injection, in-situ injection, subcutaneous injection, oral administration, smearing, etc.

11. Use according to any one of claims 1 to 9, wherein The dosage form of the medicine is injection, ointment, etc.

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