Hair growth medicine and application thereof in preparation of medicine for preventing and treating male hormone alopecia

Abstract

The invention relates to a hair growth medicine and application thereof in preparation of a medicine for preventing and treating male hormone alopecia, and belongs to the technical field of biological medicines. The invention discloses an application of menadione in preparation of a hair growth medicament. According to the present invention, the drugs capable of effectively treating male hormone alopecia are screened by using the high throughput screening method, comprise menthone, menadione and caffeic acid, and can significantly increase the hair follicle number, the hair follicle length and the corium layer thickness of the skin, the composition is used for treating male hormone alopecia, effectively promotes hair growth, and has no obvious side effects, and the expression quantity of the hair growth promoting gene is increased, and the expression quantity of the hair growth inhibiting gene is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a hair growth medicine and application of the same in preparation of a medicine for preventing and treating androgen alopecia. BACKGROUND

[0002] Current drugs for treating androgen alopecia (AGA) mainly include: (1) Minoxidil. It is the first drug approved by the US Food and Drug Administration for treatment, and is also used for many other hair loss diseases. Minoxidil is converted into its active metabolite minoxidil sulfate in vivo, and acts as a potent arteriole vasodilator, binds to adenosine triphosphate (ATP) sensitive potassium channels and relaxes the surrounding smooth muscle, activates potassium channels on peripheral arterial smooth muscle, and local application has been shown to stimulate skin blood flow within 10-15 min, and induce cell proliferation. Minoxidil has the greatest effect in the parietal and frontal regions of the scalp, and minoxidil can increase vascular endothelial growth factor (VEGF) in human dermal papilla cells (DPCs) and stimulate the production of prostaglandin E2, thus can slow down the rate of hair loss by prolonging the growth phase, and promote hair regeneration by increasing hair diameter and density. The effect of minoxidil is mainly on the hair follicle, and the conversion rate of its active metabolite in the hair follicle is higher than that in the surrounding skin. In men, 5% minoxidil formulation is significantly more effective than 2% solution, while in women, both 2% and 5% formulations have a tendency to increase hair growth. (2) Finasteride. Finasteride is a drug for treating AGA and benign prostatic hyperplasia (Benign prostatichyperplasia, BPH). It blocks the expression of 3-oxo 5α steroid 4 dehydrogenase 2 (SRD5A2), thereby blocking the conversion of testosterone into its active form dihydrotestosterone (DHT). DHT plays a key role in androgenetic alopecia, and finasteride can significantly reduce plasma and scalp DHT levels, slow down the process of hair loss, and even possibly promote the growth of new hair. Therefore, finasteride is used to treat male androgenetic alopecia, especially hair loss in the crown and central scalp area.

[0003] Side effects can occur with topical minoxidil use on the scalp, including irritation and allergic contact dermatitis, itching, scalp irritation, and facial hirsutism. Minoxidil has a low treatment efficiency in clinical applications. Finasteride can cause some short-term and long-term side effects. Male patients who commit suicide due to sexual side effects after continuous use of finasteride are known as post-finasteride syndrome. Side effects of finasteride include, but are not limited to, decreased libido, erectile dysfunction, and ejaculation problems. Although these side effects usually disappear after stopping the drug, some men can experience persistent sexual dysfunction. Finasteride can be harmful to women, especially pregnant women, as it can affect the development of the male fetus's sexual organs. In further studies, potential side effects include skin erythema and contact dermatitis, increased liver enzymes, testicular pain, etc. Therefore, when using finasteride for treatment, regular doctor check-ups are recommended to monitor the effects of the drug and any possible side effects.

[0004] At present, there are few drugs for treating androgenic alopecia. It is urgent to find an effective and less side effect drug for treating androgenic alopecia. SUMMARY

[0005] The present application aims to overcome the deficiencies of the prior art and provide a hair growth drug and its application in the preparation of a drug for preventing and treating androgenic alopecia.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] In a first aspect, the present application provides the use of tetraenemenaquinone in the preparation of a hair growth drug.

[0008] The present application screens out the effective androgenic alopecia treatment drug tetraenemenaquinone through high-throughput screening, which can significantly increase the expression of hair follicle cell hair growth promoting genes and significantly reduce the expression of hair growth inhibiting genes, and promote skin hair growth.

[0009] Further, the hair growth drug is a drug for preventing and treating androgenic alopecia.

[0010] Still further, the hair growth drug increases the expression amount of hair growth promoting genes and / or reduces the expression amount of hair growth inhibiting genes.

[0011] Still further, the hair growth promoting genes include at least one of IGF1, VEGFA, FGF2, and CTNNB1.

[0012] Still further, the hair growth inhibiting genes include at least one of AR, TGFB2, DKK1, IL-6, and TP53.

[0013] Still further, the hair growth drug promotes hair follicle growth and / or increases dermal layer thickness.

[0014] Further, the hair growth medicine increases the number of hair follicles and / or increases the length of hair follicles.

[0015] In a second aspect, the present application provides a composition containing menthone, menadione and caffeic acid.

[0016] Further, the composition contains 3-5% menthone in volume concentration, 1-2 mg / mL menadione and 20-40 mg / mL caffeic acid.

[0017] Preferably, the composition contains 5% menthone in volume concentration, 2 mg / mL menadione and 40 mg / mL caffeic acid.

[0018] In a third aspect, the present application provides use of the composition in the preparation of a hair growth medicine.

[0019] Further, the hair growth medicine is a medicine for preventing and treating androgenetic alopecia.

[0020] Further, the hair growth medicine increases the number of hair follicles and / or increases the length of hair follicles.

[0021] Further, the hair growth promoting gene includes at least one of IGF1, VEGFA, FGF2 and CTNNB1.

[0022] Further, the hair growth inhibiting gene includes at least one of AR, TGFB2, DKK1, IL-6 and TP53.

[0023] Further, the hair growth medicine promotes the growth of hair follicles and / or increases the thickness of the dermal layer.

[0024] Further, the hair growth medicine increases the number of hair follicles and / or increases the length of hair follicles.

[0025] In a fourth aspect, the present application provides a hair growth medicine containing menadione.

[0026] Further, the hair growth medicine contains the composition.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] The present application screens drugs that can be effectively used for treating androgenetic alopecia by a method of high-throughput screening of drugs for treating androgenetic alopecia, including menthone, menadione and caffeic acid, which can significantly increase the number of hair follicles, the length of hair follicles and the thickness of the dermal layer, increase the expression amount of hair growth promoting genes and decrease the expression amount of hair growth inhibiting genes, are used for treating androgenetic alopecia, effectively promote hair growth and have no obvious side effects. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 For ssGSEA analysis results.

[0030] Figure 2 For the effects of 30 drugs on hair growth promoting genes. A is CTNNB1; B is IGF1; C is VEGFA. NC is negative control; minoxidil is minoxidil, which is a positive control; ns is no significant difference; * is p<0.05; ** is p<0.01; *** is p<0.005; **** is p<0.001.

[0031] Figure 3 For the effects of 30 drugs on hair growth inhibiting genes. A is AR; B is DKK1; C is TGFB2; D is TP53. NC is negative control; minoxidil is minoxidil, which is a positive control; ns is no significant difference; * is p<0.05; ** is p<0.01; *** is p<0.005; **** is p<0.001.

[0032] Figure 4 For the effects of drugs No. 5, No. 7, No. 12, No. 23, No. 27 and No. 28 on hair growth related genes in 3D culture method of inverted culture. A is hair growth promoting gene; B is hair growth inhibiting gene. NC is negative control; minoxidil is minoxidil, which is a positive control; ns is no significant difference; * is p<0.05; ** is p<0.01; *** is p<0.005; **** is p<0.001.

[0033] Figure 5 For the experimental process of mice.

[0034] Figure 6 For the hair growth results of the first cycle of the menthone mouse experiment.

[0035] Figure 7 For the hair growth results of the second cycle of the menthone mouse experiment.

[0036] Figure 8 For the hair growth results of the third cycle of the menthone mouse experiment.

[0037] Figure 9 For the HE staining pictures of the longitudinal section of the skin of the menthone mouse.

[0038] Figure 10 For the HE staining pictures of the cross section of the skin of the menthone mouse.

[0039] Figure 11The effects of menthone on the hair follicles and dermis of mice in the third cycle of the experiment. A represents the number of hair follicles; B represents the length of hair follicles; and C represents the thickness of the dermis.

[0040] Figure 12 The hair growth results of menadione in the first cycle of the mouse experiment.

[0041] Figure 13 The hair growth results of caffeic acid in the first cycle of the mouse experiment.

[0042] Figure 14 The effects of hair growth promoting genes by hair growth composition and other drugs.

[0043] Figure 15 The effects of hair growth inhibiting genes by hair growth composition and other drugs.

[0044] Figure 16 The fold change of the effects of hair growth inhibiting genes by hair growth composition compared with other drugs. A represents menthone; B represents caffeic acid; and C represents menadione.

[0045] Figure 17 The hair growth results of hair growth composition and other drugs in the second cycle of the mouse experiment. DETAILED DESCRIPTION

[0046] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific examples. Unless otherwise specified, other materials and reagents used in the examples can be obtained from commercial channels.

[0047] Example 1 cell data

[0048] 300 natural product drugs were selected to treat DPCs, and RNA sequencing analysis data of the treated DPCs were obtained. Minoxidil was used as a positive control, and DMSO was used as a negative control. The differences in gene expression levels between the natural product drug group and the negative control group were obtained. Through single-sample gene set enrichment analysis (ssGSEA) method, the signal pathways and genes closely related to AGA treatment of 300 natural product drugs were sorted out and arranged into a special hair growth promoting gene set. Subsequently, ssGSEA analysis score was performed on whether 300 natural product drugs had AGA treatment potential. The top 10 drugs were marked in red, and the 10th-20th drugs were marked in blue. Figure 1) and inhibit the expression of AR, DKK1, TGFB2 and TP53 (Fig. Figure 3 A, Figure 3 B, Figure 3 C and Figure 3 D), which have potential hair growth promotion effect. Among them, the 5th, 7th, 12th, 23rd, 27th and 28th drugs were determined as the key research objects, and these drugs were verified by the inverted culture 3D culture method, and the expression of AGA treatment related genes was the most significant (Fig. Figure 4 A and Figure 4 B).

[0049] The inverted culture 3D culture method was used to further verify the influence of different drugs on hair growth related genes. The culture medium (F12 culture medium added with Hyclone serum, the volume ratio of Hyclone serum to F12 culture medium was 1:9) and each drug to be tested (2 μM) were mixed to suspend the DPCs cells, so that the final concentration of DPCs cells was 300 cells / μL, and a DPCs cell suspension was obtained. The DPCs cell suspension was dropped on the cover of the culture dish, inverted, and under the action of gravity, droplets similar to hair papilla structure were formed, and the volume of each droplet was 10 μL, which was cultured in a cell incubator for 36 h. This culture method makes the cells form a more three-dimensional structure similar to the interaction between the bottom and the upper surface during the culture process, thereby helping to maintain the biological activity and function of DPCs.

[0050] II. Experimental results

[0051] Among the 30 drugs, most of them can effectively up-regulate the expression of CTNNB1, IGF1 and VEGFA genes (Fig. Figure 2 A, Figure 2 B and Figure 2 C), and inhibit the expression of AR, DKK1, TGFB2 and TP53 (Fig. Figure 3 A, Figure 3 B, Figure 3 C and Figure 3 D), which have potential hair growth promotion effect. Among them, the 5th, 7th, 12th, 23rd, 27th and 28th drugs were determined as the key research objects, and these drugs were verified by the inverted culture 3D culture method, and the expression of AGA treatment related genes was the most significant (Fig. Figure 4 A and Figure 4 B).

[0052] ​​​​​​​​​​​​​​​​​​Both drug 12 and drug 28 significantly up-regulated the expression of two key genes, IGF1 and VEGAF; drug 23 significantly up-regulated the expression of the hair growth promoting gene IGF1. Further, as shown, drug 12 down-regulated two hair growth inhibiting genes, TGFB2 and TP53; drug 23 down-regulated the hair growth inhibiting gene TGFB2; and drug 28 down-regulated three hair growth inhibiting genes, AR, DKK1 and TP53. In view of the above results, drug 12, drug 23 and drug 28 show potential therapeutic value and are worthy of further research and verification.

[0053] Drug 12 is menthone, with the chemical name 2-isopropyl-5-methylcyclohexanone, the molecular formula C 10 H 18 O, the molecular weight of 154.25 g / mol, and the CAS number of 10458-14-7 (standard mixture, DL type) or 89-80-5 (L-menthone), which is a monoterpene ketone compound with a significant mint characteristic aroma, and is currently mainly used in the fields of perfumes, food industry and pharmaceutical excipients, etc.

[0054] Drug 23 is caffeic acid, with the chemical name 3,4-dihydroxycinnamic acid, the molecular formula C9H8O4, the molecular weight of 180.16 g / mol, and the CAS number of 331-39-5, which is currently mainly used in the fields of medicine and health (antioxidant and anti-inflammatory, neuroprotection), cosmetics industry (whitening, ultraviolet absorption), food industry (natural antioxidant) and textile industry.

[0055] Drug 28 is menaquinone-4 (MK-4), with the molecular formula C 31 H 40 O2, the molecular weight of 444.65 g / mol, and the CAS number of 863-61-6, which is currently mainly used in anti-hemorrhagic drugs, osteoporosis treatment, and nutritional supplements.

[0056] Example 2 Mouse Experiment

[0057] I. Experimental Method

[0058] As Figure 5 shown, 7-week-old C57 / BL6 male mice were shaved and treated with depilatory cream on the back hair in one piece, and divided into four groups, namely the negative control group (solvent), the positive control group (5% minoxidil, mass concentration), and two groups of experimental drug groups (menthone, volume concentration of 3% or 5%), and according to the regularity of mouse hair growth, three cycles of drug administration experiment were carried out, with a frequency of 1 time / 1 day, and the mouse back hair growth was recorded.

[0059] To more accurately evaluate the effect of menthone on the growth of mouse hair follicles, HE staining analysis was performed on the skin of mice after administration.

[0060] Menthol was replaced with 2 mg / mL tetraenemenaquinone, 20 mg / mL caffeic acid and 40 mg / mL caffeic acid, respectively, and the other animal experiment methods were the same to evaluate the effect of tetraenemenaquinone and caffeic acid on mouse hair growth.

[0061] II. Experimental results

[0062] As shown in Figure 6 , Figure 7 and Figure 8 , after administration in each cycle, the mice treated with menthone showed hair growth, and the hair growth was more uniform than the positive control group. The hair growth of the control group was slower and uneven. At the same time, the body weight of the mice was recorded continuously, and it was found that the application of the drug did not affect the body weight of the mice, indicating that menthone had no obvious side effects on the body weight of the mice at the dose and frequency used.

[0063] As shown in Figure 9 , Figure 10 , Figure 11 A, Figure 11 B and Figure 11 C, according to the HE staining analysis and statistical results, it was found that menthone could significantly increase the number of hair follicles, the length of hair follicles and the thickness of the dermis of mice after long-term administration.

[0064] As shown in Figure 12 and Figure 13 , tetraenemenaquinone and caffeic acid also had a promoting effect on hair growth in mice.

[0065] Example 3 Mixture of menthone, tetraenemenaquinone and caffeic acid

[0066] In order to achieve better therapeutic effect on androgenetic alopecia, the mixture of menthone, tetraenemenaquinone and caffeic acid was combined to obtain a hair growth composition. The solvent used to prepare the hair growth composition was composed of 70% (v / v) ethanol and 30% (v / v) glycerol, the concentration of menthone was 5%, the concentration of tetraenemenaquinone was 2 mg / mL, and the concentration of caffeic acid was 40 mg / mL.

[0067] The effect of the hair growth composition, 5% menthone, 2 mg / mL tetraenemenaquinone and 40 mg / mL caffeic acid on the expression of genes commonly seen in AGA treatment was verified by the inverted culture method of 3D culture in Example 1.

[0068] The effects of the hair growth composition, 5% menthone, 2 mg / mL tetraene-menaquinone, and 40 mg / mL caffeic acid on hair growth in mice were verified using the method described in Example 2.

[0069] II. Experimental Results

[0070] like Figure 14 and Figure 15 As shown, the combination of the three drugs significantly increased the expression of hair growth-promoting genes CTNNB1 and FGF2 in dermal papilla cells compared with the use of the three drugs alone, and significantly decreased the expression of hair growth-inhibiting genes AR, TGFB2, DKK1 and IL-6.

[0071] like Figure 16 As shown in Figure A, the combination of the three drugs upregulated CTNNB1 by 2.9 times, IGF-1 by 11.5 times, and FGF2 by 2.6 times, and downregulated AR by 0.4 times, DKK1 by 0.6 times, and IL-6 by 0.5 times compared to using menthone alone.

[0072] like Figure 16 As shown in B, the combination of the three drugs upregulated CTNNB1 by 2.5 times, upregulated FGF2 by 2 times, downregulated AR by 0.4 times, downregulated DKK1 by 0.3 times, and downregulated IL-6 by 0.4 times compared with caffeic acid alone.

[0073] like Figure 16 As shown in C, the combination of the three drugs upregulated CTNNB1 by 2.5 times, upregulated FGF2 by 1.5 times, downregulated AR by 0.4 times, downregulated TGFB2 by 0.3 times, downregulated DKK1 by 0.4 times, and downregulated IL-6 by 0.5 times compared with the use of tetraene-naphthoquinone alone.

[0074] like Figure 17 As shown, all three drug combinations promoted hair growth on the backs of mice compared to the negative control. The hair growth combination resulted in faster hair growth, with mice clearly entering the anagen phase by day 7. By day 14, hair growth was faster and more uniform than the positive control. Therefore, this hair growth combination can be effectively used to treat androgenetic alopecia.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of tetraene-menaquinone in the preparation of hair growth drugs.

2. The application as described in claim 1, characterized in that, The hair growth drug mentioned is a drug for preventing androgenetic alopecia.

3. The application as described in claim 2, characterized in that, The hair growth drug increases the expression level of hair growth promoting genes and / or decreases the expression level of hair growth inhibiting genes.

4. The application as described in claim 3, characterized in that, The hair growth promoting genes include at least one of IGF1, VEGFA, FGF2, and CTNNB1.

5. The application as described in claim 3, characterized in that, The germinal growth inhibitory gene includes at least one of AR, TGFB2, DKK1, IL-6, and TP53.

6. A composition, characterized in that, The composition contains menthone, tetraene-menaquinone, and caffeic acid.

7. The composition according to claim 6, characterized in that, The composition contains 3-5% menthone, 1-2 mg / mL tetraene-menaquinone, and 20-40 mg / mL caffeic acid by volume.

8. The composition according to claim 7, characterized in that, It contains 5% menthone, 2 mg / mL tetraene-menaquinone, and 40 mg / mL caffeic acid by volume.

9. Use of the composition according to any one of claims 6 to 8 in the preparation of a hair growth medicine.

10. A hair growth drug, characterized in that, The hair growth drug contains tetraene-naphthoquinone.

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