Composition, pharmaceutical preparation and application thereof

Abstract

The invention discloses a composition, a pharmaceutical preparation and application thereof. Based on a stress-induced alopecia resting model, it is proved for the first time that the main component trans-stilbene glucoside (TSG) of polygonum multiflorum and the main components indirubin and indigo blue of indigo naturalis have a remarkable drug effect on stress-induced alopecia resting model mice, and the components are easy to extract and have high safety. A stable and reliable animal model and an evaluation method are provided for treatment strategies and new drug research and development of stress-induced alopecia in the resting period.

Description

Technical Field

[0001] This disclosure generally relates to the field of medicine, and particularly to a composition, a pharmaceutical preparation, and their use in the preparation of a drug for treating and / or preventing alopecia or hypotrichosis. Background Art

[0002] Hair loss has always been a stubborn disease that plagues the general public. Clinically common types of hair loss include androgenetic alopecia (AGA, also known as seborrheic alopecia), telogen effluvium (TE), and alopecia areata (AA, which is mostly related to autoimmune abnormalities), physical or chemical damage-induced hair loss, etc. Among them, TE is the second most common type of hair loss clinically after AGA, and it refers to hair loss caused by an increased proportion of telogen hairs due to acute or chronic stress (such as mental stress, metabolic stress, hormonal changes, or drugs). Chronic stress is an important cause of TE, known as stress-induced telogen effluvium (Str-TE, also known as stress-induced hair loss). Clinically, there are already effective drugs on the market for most types of hair loss. For example, finasteride is used to treat androgenetic alopecia; minoxidil topical application has a certain effect on some hair loss patients; in 2022, the JAK inhibitors baricitinib and ritlecitinib were approved for the treatment of severe alopecia areata. However, the above drugs mainly treat the local hair follicle microenvironment, and the overall efficacy is limited. In recent years, with the continuous increase in social life stress, the number of people with stress-induced hair loss is increasing rapidly. According to statistics, the average hair loss incidence rate at all ages at home and abroad reaches 20%-30%, and about 1 / 4 of them are stress-induced hair loss. In particular, stress-induced hair loss often induces or coexists with other types of hair loss. Therefore, the number of people with stress-induced hair loss is extremely large. It is worth noting that although hair loss caused by short-term stress is reversible and can be relieved by relieving the stress source, long-term chronic stress can lead to the senescence of hair follicle stem cells through various pathways, making stress-induced hair loss enter an irreversible stage and requiring drug intervention treatment. However, there is currently a lack of effective drugs for stress-induced telogen effluvium caused by complex inducements in clinical practice.

[0003] At present, finasteride, minoxidil, and baricitinib are first-line drugs for treating hair loss. However, the main indications of the above drugs are male pattern hair loss or alopecia areata, and they have low clinical cure rates, many adverse reactions, as well as problems such as easy drug resistance and induced or aggravated hair loss. Pfizer has recently developed and launched a new drug for treating hair loss, ritlecitinib, which is indicated for alopecia areata in adolescents and adults aged 12 years and above. The clinical evidence-based research on other hair loss treatment products is insufficient, and they have been repeatedly reported for illegally adding hormone components such as progesterone and minoxidil. However, with the continuous increase in social competition pressure, the population with stress-induced telogen effluvium is increasing rapidly. The phenomenon of "hairline recession" among people born in the 1970s, 1980s, and even 1990s has become a unique social problem at present, and the treatment demand is extremely high. According to statistics, the market size of the hair loss treatment-related industry in China has increased from 7.8 billion yuan in 2016 to 18.4 billion yuan in 2020. Thus, although the population with stress-induced telogen effluvium is large, there is still a lack of safe and effective treatment drugs for stress-induced telogen effluvium at home and abroad. Therefore, it is urgent to research and develop new drugs for the safe and effective treatment of stress-induced telogen effluvium. Summary of the Invention

[0004] Based on this, the present application provides a composition for treating and / or preventing alopecia or hypotrichosis, comprising: trans-resveratrol glucoside or a pharmaceutically acceptable salt thereof, indirubin or a pharmaceutically acceptable salt thereof, and indigo or a pharmaceutically acceptable salt thereof.

[0005] On the other hand, the present application also provides a pharmaceutical preparation, wherein the pharmaceutical preparation comprises the composition described herein.

[0006] On the other hand, the present application also provides the use of the composition described herein and the pharmaceutical preparation described herein in the preparation of a drug for treating and / or preventing alopecia or hypotrichosis.

[0007] Based on the stress-induced telogen effluvium model, the present application has first demonstrated that trans-resveratrol glucoside (TSG), the main component of Polygonum multiflorum Thunb., combined with indirubin and indigo, the main components of Indigo Naturalis, has a significant pharmacological effect on mice with stress-induced telogen effluvium model, and these components are easy to extract and have high safety. It provides a stable and reliable animal model and evaluation method for the treatment strategy and new drug research and development of stress-induced telogen effluvium.

[0008] Other features and advantages of the present application will be described in the following specification, and, in part, will become apparent from the specification, or will be understood by implementing the present application. Other advantages of the present application can be achieved and obtained through the solutions described in the specification and the drawings. Brief Description of the Drawings

[0009] The accompanying drawings are used to provide an understanding of the technical solution of the present application and form a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation to the technical solution of the present application.

[0010] Figure 1 This shows the growth status of the back hair of mice in each group in Example 1 of the present application (n = 6).

[0011] Figure 2 This shows the PAH concentration in the serum of mice in each group in Example 1 of the present application. Among them, * indicates the significant difference between each group and the TSG group.

[0012] Figure 3 This shows the PAH accumulation in the kidney tissues of mice in each group in Example 1 of the present application. Among them, * indicates the significant difference between each group and the TSG group.

[0013] Figure 4 This shows the growth status of the back hair of mice in each group after adrenalectomy in Example 2 of the present application (n = 6). Detailed implementation mode

[0014] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs. When expressing a quantity, concentration, or other value or parameter in the form of a range, a preferred range, or a preferred upper limit of a value and a preferred lower limit of a value, it should be understood that any range is equivalently disclosed by combining any upper limit of the range or preferred value with any lower limit of the range or preferred value, regardless of whether the range is specifically disclosed. Unless otherwise specified, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range.

[0015] The terms "about" and "approximately", when used in combination with numerical variables, generally refer to the value of the variable and all values of the variable within the experimental error (e.g., within the 95% confidence interval of the mean value) or within ±10% of the specified value, or within a wider range.

[0016] The expression "comprising" or similar expressions synonymous therewith, such as "including", "containing", and "having", etc., are open-ended and do not exclude additional unlisted elements, steps, or components. The expression "consisting of" excludes any unstated element, step, or component. The expression "consisting essentially of" refers to a range limited to the specified elements, steps, or components, plus optionally existing elements, steps, or components that do not substantially affect the basic and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of".

[0017] The expression "at least one" or "one or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0018] Irreversible hair loss caused by long-term chronic stress is related to both abnormal regulation of the local microenvironment of hair follicles and abnormal distal regulation of adrenal-stress hormones. Therefore, for the treatment of stress-induced telogen effluvium, it is difficult to achieve good therapeutic effects only by local action, and it is necessary to "attack on two fronts" and treat both the adrenal distal pathway and the local microenvironment of hair follicles simultaneously. At this time, the characteristics and advantages of traditional Chinese medicine with multiple targets and multiple effects are highlighted.

[0019] Based on the understanding in the traditional Chinese medicine theory "Plain Questions: Six-Factor Explanation of the Manifestations of the Zang-Fu Organs": "The kidneys,... their brilliance manifests in the hair", traditional Chinese medicine syndrome differentiation of "kidney deficiency" may be one of the important causes of hair loss. Long-term stress can cause "kidney deficiency" through the HPA axis (Hypothalamic-Pituitary-Adrenal Axis), so traditional Chinese medicines for tonifying the kidney such as Polygonum multiflorum are commonly used to treat hair loss. The applicant has been engaged in the toxicity / efficacy research of the traditional Chinese medicine for tonifying the kidney, Polygonum multiflorum, for a long time, and for the first time, distinguished the toxic components and efficacy components of Polygonum multiflorum. Previous experiments have first confirmed that the main efficacy component of Polygonum multiflorum, trans-diphenylethylene glycoside (TSG), has good efficacy on a mouse model of stress-induced telogen effluvium, and a novel action pathway based on adrenal distal regulation has been discovered. At the same time, in order to further improve the efficacy of TSG in treating stress-induced telogen effluvium, the research group screened small molecules of traditional Chinese medicine acting on renal organic anion transporters (OATs), and found that indirubin and indigo, when combined with TSG (TSG:indirubin:indigo = 1:2:2), can significantly improve the efficacy of TSG in treating stress-induced telogen effluvium compared with the separate use of TSG, indirubin, and indigo. For this reason, the applicant took the combined application of TSG with indirubin and indigo as the research object, and carried out research on the efficacy and mechanism of multi-pathway integration for the treatment of stress-induced telogen effluvium targeting the adrenal distal and the local microenvironment of hair follicles. More importantly, so far, there is still a lack of safe and effective therapeutic drugs for stress-induced telogen effluvium.

[0020] In one aspect, the present application provides a composition for treating and / or preventing alopecia or hypotrichosis, comprising: trans-diphenylethylene glycoside or a pharmaceutically acceptable salt thereof, indirubin or a pharmaceutically acceptable salt thereof, and indigo or a pharmaceutically acceptable salt thereof.

[0021] In some embodiments, the molar ratio between trans-diphenylethylene glycoside or a pharmaceutically acceptable salt thereof, indirubin or a pharmaceutically acceptable salt thereof, and indigo or a pharmaceutically acceptable salt thereof is about 0.7-1.3:1.5-2.5:1.5-2.5.

[0022] In some embodiments, the molar ratio between trans - resveratrol glucoside or a pharmaceutically acceptable salt thereof, indirubin or a pharmaceutically acceptable salt thereof, and indigo or a pharmaceutically acceptable salt thereof is about 0.7:1.5:1.5, 0.7:1.5:1.7, 0.7:1.5:1.9, 0.7:1.5:2.1, 0.7:1.5:2.3, 0.7:1.5:2.5, 0.7:1.7:1.5, 0.7:1.7:1.7, 0.7:1.7:1.9, 0.7:1.7:2.1, 0.8:1.7:1.5, 0.8:1.7:1.7, 0.8:1.7:1.9, 0.8:1.7:2.1, 0.8:1.7:2.3, 0.8:1.7:2.5, 0.8:1.9:1.5, 0.8:1.9:1.7, 0.8:1.9:1.9, 0.8:1.9:2.1, 0.9:1.9:1.5, 0.9:1.9:1.7, 0.9:1.9:1.9, 0.9:1.9:2.1, 0.9:1.9:2.3, 0.9:1.9:2.5, 0.9:2.1:1.5, 0.9:2.1:1.7, 0.9:2.1:1.9, 0.9:2.1:2.1, 1.0:2.1:1.5, 1.0:2.1:1.7, 1.0:2.1:1.9, 1.0:2.1:2.1, 1.0:2.1:2.3, 1.0:2.1:2.5, 1.0:2.3:1.5, 1.0:2.3:1.7, 1.0:2.3:1.9, 1.0:2.3:2.1, 1.1:2.3:1.5, 1.1:2.3:1.7, 1.1:2.3:1.9, 1.1:2.3:2.1, 1.1:2.3:2.3, 1.1:2.3:2.5, 1.1:2.5:1.5, 1.1:2.5:1.7, 1.1:2.5:1.9, 1.1:2.5:2.1, 1.2:1.5:1.5, 1.2:1.5:1.7, 1.2:1.5:1.9, 1.2:1.5:2.1, 1.2:1.5:2.3, 1.2:1.5:2.5, 1.2:1.7:1.5, 1.2:1.7:1.7, 1.2:1.7:1.9, 1.2:1.7:2.1, 1.3:1.5:1.5, 1.3:1.5:1.7, 1.3:1.5:1.9, 1.3:1.5:2.1, 1.3:1.5:2.3, 1.3:1.5:2.5, 1.3:1.7:1.5, 1.3:1.7:1.7, 1.3:1.7:1.9 or 1.3:1.7:2.1.

[0023] In some embodiments, the molar ratio between trans-stilbene glucoside or a pharmaceutically acceptable salt thereof, indirubin or a pharmaceutically acceptable salt thereof, and indigo or a pharmaceutically acceptable salt thereof is about 1:2:2.

[0024] Trans-stilbene glucoside (TSG) is a polyhydroxystilbene compound and belongs to a configuration of stilbene glucosides. In nature, trans-stilbene glucoside mainly exists in natural plants in the form of aglycone or glycoside, especially in the Polygonaceae plant Polygonum multiflorum Thunb. Its chemical name is 2,3,5,4'-tetrahydroxy-stilbene-2-O-β-D-glucoside, and its molecular formula is C 20 H 22 O 9 with a molecular weight of 406.383. This compound is unstable in aqueous solution under high temperature conditions (such as 80 °C) and is also unstable in acidic solutions. The CAS registry number of stilbene glucoside is 82373-94-2.

[0025] The molecular formula of indirubin is C 16 H 10 N 2 O 2 and its chemical name is 2-(2-oxo-1H-indol-3-ylidene)-1H-indol-3-one, with a molecular weight of 262.2628, which is an isomer of indigo.

[0026] The chemical structure of indirubin contains two indole rings and mainly exists in the traditional Chinese medicine Indigo Naturalis. It is a dark red needle-like crystal with a density of 1.417 g / cm 3 and a melting point of about 350 °C. In terms of solubility, indirubin is soluble in ethyl acetate, acetone, chloroform, and ether, slightly soluble in ethanol, but insoluble in water.

[0027] Indigo is a natural pigment with a molecular formula of C 16 H 10 N 2 O 2 .

[0028] Indigo was initially extracted from plants such as Indigofera tinctoria and some other plants and belongs to indole compounds. Its molecular structure consists of two indole rings, having a unique conjugated double bond system, which makes it appear dark blue.

[0029] On the other hand, the present application also provides a pharmaceutical preparation, wherein the pharmaceutical preparation contains the composition described herein.

[0030] In some embodiments, the pharmaceutical preparation further comprises one or more of the following auxiliary active ingredients: steroids, indole compounds, antifungal agents, anti-inflammatory agents, coolants, and antipruritics.

[0031] It has been reported that reducing the production of androgens, estrogens, and epidermal growth factor (EDG) can be used for treating or preventing hair loss. Thus, steroids that can block or inhibit hormones or factors related to hair loss can be optionally added to the pharmaceutical preparations described herein. For example, 5α-reductase inhibitors block the conversion of testosterone to the active 5α-dihydrotestosterone (DHT) form and have been used in the treatment of hair loss disorders. Steroids for reducing the production of these compounds and factors or inhibiting them include testolactone, pregnenolone, dehydroepiandrosterone (DHEA), diosgenin, spironolactone, finasteride, and tamoxifen.

[0032] Indole compounds such as indole, melatonin, N-[2-(5-methoxy-1H-indol-3-yl)ethyl]acetamide, skatole, and indole-3-carbinol; certain bioflavonoids such as quercetin, methylchalcone; and anti-inflammatory fatty acids such as TESTRIOLATE or PX-13 have also been demonstrated to be useful for treating hair loss, and thus these compounds can be added to the pharmaceutical preparations of the present application.

[0033] Antifungal substances have also been shown to be useful for treating hair loss. Thus, the pharmaceutical preparations of the present application can comprise antifungal substances. Suitable antifungal substances include azole antifungal agents such as ketoconazole, miconazole, clotrimazole, econazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, and terconazole; polyene antifungal agents such as natamycin, rimocidin, filipin, nystatin, amphotericin B, and candicidin; allylamines such as terbinafine, amorolfine, naftifine, and butenafine; echinocandin antifungal agents such as anidulafungin, caspofungin, micafungin; and other known antifungal agents such as combinations of benzoic acid with a stratum corneum separating agent, ciclopirox, flucytosine, griseofulvin, gentian violet, haloprogin, tolnaftate, undecylenic acid, and tea tree oil.

[0034] Anti-inflammatory drugs are effective in reducing inflammation and pain, and also have an incidental effect on hair loss disorders. Anti-inflammatory drugs suitable for the pharmaceutical preparations of the present application include, but are not limited to, 2-arylpropionic acid (profens), salicylates, arylalkanoic acids, N-arylanthranilic acids (fenamates), pyrazolidine derivatives, oxicams, COX-2 inhibitors, sulfonanilides, and other known anti-inflammatory drugs such as liquiritigenin and ω-3 fatty acids. Ibuprofen is most preferably included as the anti-inflammatory drug.

[0035] The antipruritic agent or ingredient is a mild analgesic, which can be added to the pharmaceutical preparations of the present application to reduce related side effects such as irritation or itching. Such ingredients include, but are not limited to, zinc oxide (calamine), menthol, phenol, and camphor.

[0036] In some embodiments, the pharmaceutical preparation further comprises a carrier or diluent. Suitable carriers or diluents can be water-soluble and / or alcohol-soluble, and can include a viscous matrix to keep the pharmaceutical preparations described herein in place during use. Suitable diluents used as carriers to form lotions include water and lower alcohols or polyols, such as methanol, ethanol, isopropanol, glycerol, or propylene glycol. Paraffin components and emulsion matrices can be used to form creams or ointments. The carrier can include other conventional carriers or diluents, such as glucose, lactose, corn starch, starch paste, gum arabic, gelatin, mannitol, magnesium trisilicate, potato starch, urea, keratin, or colloidal silica.

[0037] In some embodiments, the dosage form of the pharmaceutical preparation is selected from one or more of capsules, tablets, pills, liquids, powders, granules, fine granules, film coatings, pills, lozenges, sublinguals, collodions, buccal preparations, pastes, syrups, suspensions, elixirs, emulsions, coatings, ointments, plasters, cataplasms, transdermal preparations, lotions, inhalants, aerosols, injections, or suppositories.

[0038] In some embodiments, the pharmaceutical preparation is formulated for administration by one or more routes selected from: oral, intravenous, intradermal, transdermal, intrathecal, intraarterial, intraperitoneal, intranasal, rectal, topical, intramuscular, subcutaneous, mucosal, inhalation, injection, infusion, or any combination thereof. In some embodiments, the pharmaceutical preparation is formulated for oral administration. In some embodiments, the pharmaceutical preparation is formulated for transdermal or intradermal administration.

[0039] In some embodiments, the pharmaceutical preparation may further comprise at least one ingredient selected from the group consisting of: mineral supplements, vitamin supplements, essential oils, fragrances, pigments, preservatives, and skin absorption enhancers.

[0040] In some embodiments, suitable mineral supplements include, but are not limited to, iron, zinc, copper, magnesium, and calcium, and combinations thereof. In some embodiments, suitable vitamin supplements include, but are not limited to, vitamin A (retinol), B vitamins such as vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine), B7 (biotin), B9 (folic acid), and B12 (cobalamin), vitamin C (ascorbic acid), vitamin D (calciferol), vitamin E (tocopherol), and vitamin K (phyllochinone), and combinations thereof. In some embodiments, suitable essential oils include, but are not limited to, jasmine oil, tea tree oil, and lemon oil. In some embodiments, suitable fragrances include menthol, benzyl alcohol, eugenol, phenoxyethanol, isopropyl palmitate, isopropyl myristate, benzyl salicylate, phenethyl salicylate, thymol, isopentyl salicylate, polyethylene glycol octylphenyl ether surfactant (Triton X-100 surfactant), benzoic acid, benzyl benzoate, methyl salicylate, phenol, oleic acid, caproic acid, or carbaryl. In some embodiments, suitable conventional pigments include, but are not limited to, tartrazine, quinoline yellow, sunset yellow, amaranth, carmine, erythrosine, red 2G, allura red AC, patent blue V, indigo carmine, brilliant blue FCF, fast green FCF, green S, and iron oxide. In some embodiments, suitable preservatives include, but are not limited to, parabens such as methylparaben and propylparaben, sorbic acid, potassium sorbate, quaternium-15, methylchloroisothiazolinone, and iodopropynyl butylcarbamate (IPBC), and natural preservatives such as lemon oil. In some embodiments, suitable skin penetration enhancers include 1,5-pentanediol, N-dodecyl-2-pyrrolidone and its acetate analogs, fatty acids such as oleic acid, terpenes, esters such as isopropyl myristate, furanocoumarins and furanocoumarin analogs, methyl nicotinate, MSM-decyl methyl sulfoxide, diethylene glycol, citric acid, pyruvic acid, phenoxyethanol, diethylene glycol monoethyl ether (Transcutol), phosphatidylcholine, medium-chain triglyceride oil (MCT oil), and water.

[0041] In another aspect, the present application also provides the use of the compositions described herein and the pharmaceutical formulations described herein in the preparation of a medicament for treating and / or preventing alopecia or hypotrichosis.

[0042] The composition or pharmaceutical formulation can be administered to the affected area of the patient in any manner that delivers an effective amount of the composition or pharmaceutical formulation described herein to the affected area of the patient. Preferably, the composition or pharmaceutical formulation is topically administered to the affected area of the patient, which can be the scalp or another body surface. Preferably, the composition or pharmaceutical formulation is administered to the affected area 1-3 times per day, most preferably twice a day. Optionally, the composition or pharmaceutical formulation in the form of a shampoo can also be used alone daily for treating the condition, or in combination with topical administration.

[0043] In some embodiments, the composition or pharmaceutical formulation described herein is used for treating diseases or conditions that can cause hair loss, including genetic and congenital alopecia, hypotrichosis, trichotillomania, tinea capitis, telogen effluvium, anagen effluvium, monilethrix, trichorrhexis nodosa, infections involving the hair or scalp such as fungal infections, infestations involving the hair or scalp, endocrine disorders such as hyperthyroidism, hypothyroidism and diabetes, lupus, hormonal imbalances such as as a result of polycystic ovary syndrome, and sebaceous cysts.

[0044] In some embodiments, the composition or pharmaceutical formulation described herein is used for treating diseases of hair loss, which include but are not limited to alopecia areata, alopecia areata universalis or alopecia areata totalis, male or female pattern alopecia, androgenetic alopecia (AGA), alopecia totalis, alopecia universalis, alopecia partialis, alopecia barbae, alopecia cicatricial, alopecia areata ophiasis, alopecia areata diffusa, reticular alopecia areata, sisaipho alopecia areata, congenital alopecia, drug-induced alopecia, mucinotic alopecia, marginal alopecia, androgenetic alopecia, traction alopecia and syphilitic alopecia. In some embodiments, the composition or pharmaceutical formulation described herein is used for treating telogen effluvium. In some embodiments, the composition or pharmaceutical formulation described herein is used for treating stress-induced telogen effluvium (abbreviated as stress alopecia).

[0045] In some embodiments, alopecia or hypotrichosis is telogen effluvium; preferably, the telogen effluvium is stress-induced telogen effluvium. Existing research on the treatment of hair loss has mostly focused on male pattern baldness, alopecia areata, etc., and stress-induced telogen effluvium has not been taken as the core consideration factor. Based on the applicant's previous work and the characteristics of stress-induced telogen effluvium, the present research method established an animal model of stress-induced telogen effluvium and found that the combined application of trans-resveratrol glucoside, indirubin, and indigo (TSG: indirubin: indigo = 1:2:2) can treat chronic stress-induced hair loss. The combined use of these traditional Chinese medicine small molecules delays the renal excretion of TSG and enhances the efficacy of treating stress-induced telogen effluvium.

[0046] This application describes multiple embodiments, but the description is exemplary rather than restrictive, and it will be apparent to those of ordinary skill in the art that there can be more embodiments and implementation solutions within the scope of the embodiments described in this application. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically restricted, any feature or element of any embodiment can be combined with any other feature or element in any other embodiment, or can replace any other feature or element in any other embodiment.

[0047] This application includes and contemplates combinations with features and elements known to those of ordinary skill in the art. The embodiments, features, and elements disclosed in this application can also be combined with any conventional features or elements to form a unique inventive solution. Any feature or element of any embodiment can also be combined with features or elements from other inventive solutions to form another unique inventive solution. Therefore, it should be understood that any feature shown and / or discussed in this application can be implemented alone or in any suitable combination. Therefore, the embodiments are not limited except as defined by the appended claims and their equivalents. In addition, various modifications and changes can be made within the scope of the appended claims.

[0048] In addition, when describing representative embodiments, the specification may have presented the method and / or process as a specific sequence of steps. However, to the extent that the method or process does not depend on the specific order of the steps described herein, the method or process should not be limited to the specific order of steps described. As will be understood by those of ordinary skill in the art, other step orders are possible. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation on the claims. In addition, the claims directed to the method and / or process should not be limited to performing their steps in the order written, and those skilled in the art can easily understand that these orders can be varied and still remain within the spirit and scope of the embodiments of this application.

[0049] In the following examples, the experimental methods without specific conditions are generally determined according to national standards. The experimental materials without sources indicated in the following examples are all commercially available raw materials. The equipment used in each step of the following examples is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer. Unless otherwise defined or stated, all professional and scientific terms used in this application have the same meaning as those familiar to persons skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the methods of this application.

[0050] Example

[0051] The relevant materials used in the examples are as follows:

[0052] 1. Reagents, materials

[0053] The drugs used in the experiment include TSG, indirubin, and indigo, which are used to evaluate their therapeutic effects on stress-induced alopecia. Hormonal drugs include prednisone to simulate the stress state. Probenecid (0.05 g / kg) is used as a positive control drug, and para-aminohippuric acid (PAH, 0.03 g / kg, intravenous injection (iv)) is used for renal function detection.

[0054] The experiment involves solvents and additives such as 0.5% sodium carboxymethylcellulose (CMC-Na) solution, dextrin, and sucrose. C57BL / 6 mice and NIH mice are used in animal experiments, and surgical instruments, serum separation tubes, tissue homogenizers, RNA extraction kits, etc. are equipped.

[0055] 2. Instruments

[0056] The main instruments include an animal restraint device (for model establishment), a small animal anesthesia machine (for surgery), an electronic balance (for drug weighing), a centrifuge (for sample processing), a PCR instrument (for gene expression analysis), a spectrophotometer / HPLC (for PAH concentration determination), a -80 °C refrigerator (for sample preservation), etc.

[0057] In this application, Example 1 is based on a stress-induced telogen effluvium model to evaluate the pharmacological effects of TSG, indirubin, indigo, and their combined formulations (the ratio of different drugs is the molar ratio) on stress-induced telogen effluvium model mice and their effects on renal organic anion transporters; Example 2 is based on an adrenalectomy and stress-induced telogen effluvium (STR) model to further evaluate the pharmacological effects of TSG, indirubin, indigo, and their combined formulations on STR model mice and their effects on renal organic anion transporters.

[0058] Example 1.

[0059] 1) Construction of a stress-induced telogen effluvium mouse model

[0060] The mice were placed in a state of chronic stress restraint. During the experiment, they were continuously restrained for 6 hours every day (from 9:00 to 15:00). On the 8th day after the start of the experiment, the back hair of the experimental animals was depilated. According to the hair growth cycle of the back of the mice, the experiment lasted for 25 days. This model simulated the characteristics of patients with stress-induced telogen effluvium (also known as stress alopecia) in clinical practice, and the pharmacodynamic effects of TSG, indirubin, indigo and their combined formulations could be evaluated.

[0061] 2) Administration and evaluation of pharmacodynamic effects

[0062] On the stress alopecia mouse (STR) model induced by chronic stress restraint, TSG, TSG + indirubin (TSG:indirubin = 1:2), TSG + indigo (TSG:indigo = 1:2), TSG + indirubin + indigo (TSG:indirubin:indigo = 1:2:2) were administered by gavage. The ratio of each component was the molar ratio. The dose of TSG was 118 mg / kg, twice a day for 5 consecutive days. Evaluate their pharmacodynamic effects. The evaluation indicators mainly included the overall hair growth of the back of the mice, etc.

[0063] The results are as Figure 1 shown. It can be seen from the figure that the hair growth effect of each group of mice was macroscopically evaluated by the degree of darkening in the depilated area (indicating the induction of the mid-growth phase). After 24 days of treatment, compared with the untreated STR group of mice, the mice in the TSG + indirubin + indigo (TSG:indirubin:indigo = 1:2:2) group had obvious hair regeneration, and the depilated skin on the back was covered with black hair, while most of the depilated skin of the STR group of mice did not fully grow hair. The results showed that under stress conditions, TSG + indirubin + indigo (TSG:indirubin:indigo = 1:2:2) had an obvious promoting effect on hair growth.

[0064] 3) Evaluation of the effect of administration on renal organic anion transporters

[0065] NIH mice were intragastrically administered with TSG, TSG + indirubin (TSG:indirubin = 1:2), TSG + indigo (TSG:indigo = 1:2), and TSG + indirubin + indigo (TSG:indirubin:indigo = 1:2:2). The ratio of each component was the molar ratio. The dose of TSG was 118 mg / kg, twice a day for 5 consecutive days. At the same time, a positive control group of probenecid (0.05 g / kg / day), two control groups of two solvents (pure water and 0.5% sodium carboxymethylcellulose (CMC-Na) aqueous solution), and an additive group of dextrin plus sucrose (1.5 g / kg / day each) were set up. After the last administration, the para-aminohippuric acid (PAH, iv, 0.03 g / kg) clearance experiment was carried out. That is, at 1, 2.5, 5, 7.5, 10, and 20 min after iv PAH, 10 mice were taken from each group, euthanized, whole blood was collected to prepare serum, and the double kidneys were quickly removed. The right kidney was homogenized and the PAH accumulation was measured, and the left kidney tissue was used to extract mRNA. The Kiguchi method was used to measure the PAH concentration in serum and kidney tissue homogenate. The main pharmacokinetic parameters of PAH in serum and kidney tissue were calculated using pharmacokinetic software (DAS 2.0). The expression of Oat1, Oat2, and Oat3 mRNA in mouse kidney tissue was measured by real-time quantitative PCR method.

[0066] The results are as Figure 2 and Figure 3 shown. Figure 2 The PAH concentration in the serum of mice in each group is shown. Figure 3 The PAH accumulation in the kidney tissue of mice in each group is shown. It can be seen from the figure that compared with the group only given TSG, the PAH concentration in the TSG + indirubin + indigo (TSG:indirubin:indigo = 1:2:2) group and the positive control group was significantly decreased, while the PAH accumulation was significantly increased. This phenomenon is closely related to the functional changes of organic anion transporters (OATs) in the kidney. As the main transporter protein in the kidney, OATs is responsible for transporting PAH and its metabolites in the blood into renal tubular cells and then excreting them through the kidney. When the drug intervention of the administration group and the positive drug group occurs, the drug can change the transport mode of PAH by regulating the expression or activity of OATs.

[0067] The mRNA expression results of Oat1, Oat2, and Oat3 are shown in Table 1 below. It can be seen from Table 1 that the mRNA expression levels of Oat1, Oat2, and Oat3 in the TSG + indirubin + indigo (TSG:indirubin:indigo = 1:2:2) group and the positive control group are significantly higher than those in the group given only TSG. The significant increase in the mRNA expression of Oat1, Oat2, and Oat3 indicates that drug intervention can enhance the expression of OATs by activating transcription factors or signal pathways, regulate the transport function of OATs, and enhance the excretion and transport of organic anions such as PAH. This mechanism explains, to a certain extent, the decrease in PAH concentration and the increase in accumulation.

[0068] Table 1: mRNA expression levels of Oat1, Oat2, and Oat3

[0069]

[0070] Note: a, b, c indicate significant differences between each group and the pure water control group.

[0071] In summary, the research results show that in the TSG + indirubin + indigo (TSG:indirubin:indigo = 1:2:2) group and the positive control group, the concentration of PAH is significantly decreased, while the accumulation of PAH is significantly increased. At the same time, the mRNA expression of Oat1, Oat2, and Oat3 is significantly increased. These results can reflect that the drug affects the transport, metabolism, and excretion processes of PAH and other organic anions by regulating the function and expression of renal organic anion transporters (OATs).

[0072] Example 2.

[0073] 1) Construction of a stress-induced telogen effluvium mouse model with adrenalectomy

[0074] Further, based on the stress-induced alopecia mouse model, a bilateral adrenalectomy experiment was performed on mice to clarify whether the adrenal gland is an important target organ for the treatment of stress-induced alopecia with TSG or TSG + indirubin + indigo (TSG:indirubin:indigo = 1:2:2). The adrenalectomy mouse model was established by removing the adrenal glands of mice on both sides before the establishment of the stress-induced telogen effluvium mouse model, and the remaining experimental steps were the same as before.

[0075] 2) Drug administration and evaluation of pharmacodynamic effects

[0076] After adrenalectomy in C57BL / 6 mice, they were intragastrically administered with TSG, TSG + indirubin + indigo (TSG: indirubin: indigo = 1:2:2) twice a day for 5 consecutive days. At the same time, a positive control group with probenecid (0.05 g / kg), two control groups with two solvents (pure water and 0.5% sodium carboxymethylcellulose (CMC-Na) aqueous solution), and an additive group with dextrin plus sucrose (1.5 g / kg each) were set up. After the last administration, the para-aminohippuric acid (PAH, iv, 0.03 g / kg) clearance experiment was carried out. That is, at 1, 2.5, 5, 7.5, 10, and 20 min after iv PAH, 10 mice in each group were respectively sacrificed, whole blood was collected to prepare serum, and both kidneys were quickly removed. After homogenizing the right kidney tissue, the PAH accumulation was measured, and the left kidney tissue was used to extract mRNA. The Kiguchi method was used to measure the PAH concentration in serum and kidney tissue homogenate. The main pharmacokinetic parameters of PAH in serum and kidney tissue were calculated using pharmacokinetic software (DAS 2.0). The mRNA expressions of Oat1, Oat2, and Oat3 in mouse kidney tissue were measured by real-time quantitative PCR method.

[0077] The hair growth status on the back of mice in each group after adrenalectomy was as Figure 4 shown. It can be seen that in mice with adrenalectomy (ADX), hair growth was not significantly affected, indicating that the absence of the adrenal gland had limited direct impact on hair growth. Especially in the case without stress stimulation, the hair on the back skin of mice showed normal growth. Further observation found that there was no obvious improvement in hair growth in all the administered groups of mice after adrenalectomy, suggesting that the absence of the adrenal gland might weaken the therapeutic effect, but overall the hair of mice was not severely damaged.

[0078] In mice with adrenalectomy and stress stimulation (ADX + STR), the back skin still maintained relatively normal hair coverage and did not show obvious hair thinning. This result indicates that in the case of adrenal gland absence, the HPA axis cannot be activated, and the typical stress response cannot be triggered, thus protecting hair follicles from stress-induced growth inhibition. At the same time, there was no significant change in hair density after administration, indicating that in the case of adrenal gland absence, the effect of TSG + indirubin + indigo (TSG: indirubin: indigo = 1:2:2) might be limited and could not further promote hair growth.

[0079] The results of the PAH clearance experiment in each group of mice are shown in Table 2 below:

[0080] Table 2. Results of PAH clearance experiment

[0081]

[0082] Note: a, b, c indicate significant differences between each group and the pure water control group.

[0083] As can be seen from Table 2, the PAH clearance rates of the TSG + indirubin + indigo (TSG: indirubin: indigo = 1:2:2) group and the positive control group were significantly higher than those of the pure water control group. This may be closely related to the increased sensitivity of the kidneys to drug intervention after adrenalectomy and the enhanced transport function of OATs. This result suggests that the drug significantly promoted the renal clearance of PAH by regulating the activity or expression of OATs, providing an important basis for further studying the regulatory effect of the drug on OATs.

[0084] The experimental results of the PAH accumulation in the renal tissue homogenates of mice in each group are shown in Table 3 below:

[0085] Table 3. PAH accumulation in renal tissue homogenates

[0086]

[0087] Note: * indicates the significant difference between each group and the pure water control group.

[0088] As can be seen from Table 3, the PAH accumulation levels in the TSG + indirubin + indigo (TSG: indirubin: indigo = 1:2:2) group and the TSG group were both significantly higher than those of the pure water control group. This result indicates that drug intervention may lead to an increase in the PAH accumulation in renal tissues by regulating the PAH transport mechanism in the kidneys, possibly related to the enhanced transport function of OATs by drug intervention, the alteration of the renal metabolism and excretion mechanisms of PAH, and the more sensitive response of the kidneys to these changes in the adrenalectomized environment. The mRNA expression results of Oat1, Oat2, and Oat3 are shown in Table 4 below:

[0089] Table 4. mRNA expression of Oat1, Oat2, and Oat3 in renal tissues

[0090]

[0091] Note: a, b, and c indicate the significant differences between each group and the pure water control group.

[0092] As can be seen from Table 4, compared with the pure water control group, the TSG + indirubin + indigo (TSG: indirubin: indigo = 1:2:2) group and the positive control group both showed significantly increased mRNA expression levels. This indicates that drug intervention significantly promoted the expression of these organic anion transporters (OATs). This may be closely related to the changes in hormone levels after adrenalectomy and the upregulation effect of the drug on OATs. This result provides a theoretical basis for further studying how the drug promotes the clearance of metabolites and drugs by regulating renal organic anion transporters.

[0093] The pharmacokinetic parameter results are shown in Table 5 below:

[0094] Table 5. Pharmacokinetic parameters

[0095]

[0096] Note: a, b, and c represent significant differences between each group and the pure water control group.

[0097] It can be seen from Table 5 that the pharmacokinetic parameters in the TSG + indirubin + indigo (TSG: indirubin: indigo = 1:2:2) group and the positive control group were significantly increased compared with those in the pure water control group. The adrenal glands had been removed before the experiment, resulting in changes in the renal hormonal environment, which might increase the sensitivity of the kidneys to drug intervention. The drug enhanced the function of organic anion transporters by upregulating the expression of OAT1, OAT2, and OAT3, and accelerated the drug clearance process. Therefore, the significant increase in the pharmacokinetic parameters in the TSG + indirubin + indigo group and the positive control group reflected the regulatory effect of the drug on renal organic anion transporters.

[0098] In summary, the significant increase in the PAH clearance rate indicated that drug intervention accelerated the clearance of PAH. The significant increase in the mRNA expression of OAT1, OAT2, and OAT3 in renal tissues further supported the role of the drug in enhancing PAH clearance by upregulating the expression of organic anion transporters. In addition, the accumulation of PAH in renal tissue homogenates was also significantly increased, indicating that the drug might increase the uptake and accumulation of PAH in the kidneys by regulating the function of these transporters. Overall, drug intervention significantly promoted the expression and function of renal organic anion transporters, improved the clearance efficiency of PAH, and improved the pharmacokinetic parameters.

[0099] It can be seen from Example 1 and Example 2 that under pressure conditions, TSG + indirubin + indigo (TSG: indirubin: indigo = 1:2:2) had an obvious promoting effect on hair growth, and this ratio was significantly superior to that of using TSG alone, TSG + indirubin (TSG: indirubin = 1:2), and TSG + indigo (TSG: indigo = 1:2) groups, indicating that this ratio could exert the best drug efficacy. In the presence of the adrenal glands, drug intervention could significantly increase the PAH clearance rate and enhance the expression of OAT1, OAT2, and OAT3 in the kidneys, thereby accelerating the clearance and metabolism of PAH. After adrenalectomy, the hormonal environment of the kidneys changed, and the effect of drug intervention on renal organic anion transporters was more significant, further promoting the accumulation and clearance of PAH. These results showed that the combined use of these traditional Chinese medicine small molecules delayed the renal excretion of TSG and enhanced the drug efficacy in treating stress-induced telogen effluvium.

[0100] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present application. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present application.

Claims

1. A composition comprising: trans-stilbene glycoside or a pharmaceutically acceptable salt thereof, indirubin or a pharmaceutically acceptable salt thereof, and indigo or a pharmaceutically acceptable salt thereof.

2. The composition according to claim 1, wherein The molar ratio between the trans-stilbene glycoside or a pharmaceutically acceptable salt thereof, the indirubin or a pharmaceutically acceptable salt thereof, and the indigo or a pharmaceutically acceptable salt thereof is about 0.7-1.3:1.5-2.5:1.5-2.

5.

3. The composition according to claim 2, wherein The molar ratio between the trans-stilbene glycoside or a pharmaceutically acceptable salt thereof, the indirubin or a pharmaceutically acceptable salt thereof, and the indigo or a pharmaceutically acceptable salt thereof is about 1:2:

2.

4. A pharmaceutical preparation, wherein The pharmaceutical preparation comprises the composition according to any one of claims 1 to 3.

5. The pharmaceutical preparation according to claim 4, wherein The pharmaceutical preparation may further comprise one or more of the following auxiliary active ingredients: steroids, indole compounds, antifungal drugs, anti-inflammatory drugs, cooling agents and antipruritic drugs.

6. The pharmaceutical preparation according to claim 4 or 5, wherein The pharmaceutical preparation further comprises a carrier or a diluent.

7. The pharmaceutical preparation according to any one of claims 4 to 6, wherein The dosage form of the pharmaceutical preparation is selected from one or more of capsules, tablets, pills, liquids, powders, granules, fine granules, film coatings, pills, lozenges, sublingual agents, peptizers, buccal preparations, pastes, syrups, suspensions, elixirs, emulsions, coatings, ointments, plasters, poultices, transdermal preparations, lotions, inhalants, aerosols, injections or suppositories.

8. The pharmaceutical preparation according to any one of claims 4 to 7, wherein The pharmaceutical preparation is formulated for administration by one or more routes selected from the group consisting of oral, intravenous, intradermal, transdermal, intrathecal, intraarterial, intraperitoneal, intranasal, intrarectal, topical, intramuscular, subcutaneous, mucosal, inhalation, injection, infusion, or any combination thereof.

9. Use of the composition according to any one of claims 1 to 3 or the pharmaceutical preparation according to any one of claims 4 to 8 in the preparation of a medicament for treating and / or preventing alopecia or hypotrichosis.

10. The use according to claim 9, wherein The alopecia or hypotrichosis is telogen effluvium; preferably, the telogen effluvium is stress-induced telogen effluvium.

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