A compound preparation for treating alopecia areata

Abstract

The present application relates to a compound preparation for treating alopecia areata, active ingredient A is a direct or indirect EPO receptor agonist, active ingredient B is tofacitinib or its pharmaceutically acceptable salt, and the EPO receptor agonist is preferably roxadustat. The beneficial effect is that roxadustat and tofacitinib or its pharmaceutically acceptable salt are both limited when used alone for alopecia areata treatment, but they act on different target points in the pathogenesis of alopecia areata respectively, so they are made into a compound preparation, the key signal pathway in the pathogenesis of alopecia areata is inhibited by roxadustat to compensate for the defect of tofacitinib in insufficient curative effect on moderate and severe patients from the etiology, and the hair repair effect of tofacitinib is improved from the symptom by the good hair growth promoting effect of roxadustat, the curative effects of the two are complementary, the alopecia areata is treated both from the etiology and the symptom, and the side effect risk can be reduced by appropriately reducing the dose of tofacitinib according to the synergistic effect.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, in particular to a compound preparation for treating alopecia areata. Background Art

[0002] Alopecia areata is a common, non-scarring hair loss disease caused by an inflammatory response of the hair follicles. It can be divided into localized patchy alopecia areata, total alopecia and universalis according to its pathological characteristics and severity. The global incidence rate is about 1% to 2%. Most patients are children and young adults. Alopecia areata affects the patient's appearance and mental health. Conventional treatment drugs (including glucocorticoids, compound glycyrrhizin, contact sensitizers, hair growth promoters, etc.) are limited due to side effects, incomplete treatment and only effective for mild alopecia areata. Therefore, new treatment methods need to be developed.

[0003] Alopecia areata is an inflammatory autoimmune disease associated with the destruction of the immune immunity mechanism of hair follicles, in which the inflammatory cytokine signaling pathway, the JAK (Janus kinase)-STAT pathway, plays a key role. In recent years, JAK inhibitors have become a new strategy for the treatment of alopecia areata. Cytokines bind to their receptors, activating the JAK pathway to phosphorylate STAT. STAT dimers are transported to the cell nucleus to activate gene transcription, leading to further production of cytokines and self-antigens, activating inflammation and immune responses; the JAK / STAT signaling pathway is involved in the hair growth cycle, is upregulated during the catagen and telogen phases, and is inhibited during the growth phase. Blocking the relevant JAK-STAT pathway can reduce the production of self-antigens and inflammatory mediators, and prevent the immune response of hair follicles.

[0004] From the above, we can see that JAK inhibitors can block cytokine signaling, which plays a key role in the pathogenesis of alopecia areata. Therefore, compared with other drugs, they are theoretically more effective. Tofacitinib (Pacritinib) is a new oral kinase inhibitor that can specifically inhibit JAK2, IRAK1 and CSF1R. Because it does not inhibit JAK1, it can avoid the potential side effects of inhibiting JAK1.

[0005] A breakdown in hair follicle immune privilege (IP) is a prerequisite for the development of alopecia areata. Because failure of the IP mechanism allows for the development of AA, it is reasonable to anticipate that restoring and / or supplementing IP signaling is a highly effective approach for treating AA and preventing future relapses. Erythropoietin (EPO) is an evolutionarily conserved hormone that has been well documented to promote erythropoiesis by binding to the homodimeric EPO receptor (EPOR) 2. Over the past few decades, evidence has emerged demonstrating that EPO's effects extend far beyond erythropoiesis. By binding to tissue protection receptors (TPRs), EPO inhibits proinflammatory cytokines, protects cells from apoptosis, and promotes wound healing. EPO plays a central role in tissue protection and repair, previously thought to be mediated by inhibiting proinflammatory cytokines and downregulating apoptosis. Recent studies have shown that EPO and its derivatives can also act directly on immune cells, modulating the immune environment surrounding hair follicles to restore IP signaling and thereby achieve therapeutic effects on AA.

[0006] In summary, by inhibiting the JAK signaling pathway to reduce the attack of immune cells on hair follicle tissue and using EPOR agonists to restore the immune immunity of hair follicles, the normal physiological function of hair follicles can be restored from the source. There is currently a lack of sustained, effective or permanent treatment methods to reverse alopecia areata, and there is an urgent need for a compound preparation that can restore the immune immunity of hair follicles. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a compound preparation for treating alopecia areata, which can be administered systemically or topically to overcome the deficiencies in the above-mentioned prior art.

[0008] The technical solution of the present invention to solve the above technical problems is as follows: a compound preparation for treating alopecia areata, comprising: an active ingredient A and an active ingredient B, wherein the active ingredient A is a direct or indirect EPO receptor (Erythropoietin receptor, EPOR) agonist, and the active ingredient B is tofacitinib or a pharmaceutically acceptable salt thereof.

[0009] On the basis of the above technical solution, the present invention can also be improved as follows.

[0010] Furthermore, the compound preparation is a liniment or a gel;

[0011] Calculated as an EPO receptor agonist, the percentage of the active ingredient A in the compound preparation is 0.1 wt% to 10 wt% or 0.1% (w / v) to 10% (w / v);

[0012] The percentage of the active ingredient B in the complex preparation is 0.1 wt% to 10 wt% or 0.1% (w / v) to 10% (w / v) based on tofacitinib.

[0013] Further, the present application is directed to a composition for treating, preventing or alleviating alopecia areata by topical or injectable administration to a subject in need thereof, the composition comprising a therapeutically effective amount of at least one EPO receptor agonist selected from the group consisting of epoetin beta, pegetacar, pegetamoyl, molisast, darpinast, roxastast and combinations thereof, in fact, not limited to epoetin beta, pegetacar, pegetamoyl, molisast, darpinast, roxastast and other common EPO receptor agonist drugs, which have the effect of being combined with tofacitinib.

[0014] Further, the concentration of the EPO receptor agonist is about 0.01% to about 1%, about 1% to about 3%, about 3% to about 5% w / w; from 5% to 10% w / w or from 10% to 20% w / w.

[0015] Further, the EPO receptor agonist is roxastast.

[0016] Further, the complex preparation further comprises a pharmaceutically acceptable excipient.

[0017] The commonly used pharmaceutical excipients for liniments and gels in pharmacy include but are not limited to the following categories:

[0018] Gel base: This is the most important component of the gel, commonly used gel base includes carbomer, xanthan gum, etc., which provides the necessary consistency and stability for the gel.

[0019] pH adjuster: used to adjust the pH value of the gel to ensure the stability of the drug and reduce irritation. Commonly used pH adjusters include citric acid, sodium hydroxide, phosphate buffer, etc.

[0020] Humectant: increase the moisture of the gel, commonly used humectants are glycerol, propylene glycol, hyaluronic acid, etc.

[0021] Preservative: prevent the growth of microorganisms during storage and use of the gel, commonly used preservatives are benzalkonium chloride, parabens, etc.

[0022] Stabilizer: used to maintain the stability of the drug in the gel, commonly used stabilizers include EDTA (used to chelate metal ions), cyclodextrins, etc.

[0023] Cosolvents and solubilizers: help dissolve drugs and improve their solubility and bioavailability. Commonly used cosolvents and solubilizers include polyethylene glycol (PEG), poloxamer, etc.

[0024] Transdermal absorption enhancers: Increase the absorption of drugs through the skin. Commonly used transdermal absorption enhancers include laurocapram (Azone) and the like.

[0025] Other excipients: such as emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, etc. These excipients are also used in ointments and gels, depending on the specific requirements and purpose of the preparation.

[0026] The selection and use of these excipients requires comprehensive consideration based on the drug's properties, the formulation's process requirements, and the expected efficacy and safety. Furthermore, the excipient's incompatibility and stability must be considered to ensure that no adverse reactions occur between the excipient and the drug, potentially impacting the drug's efficacy and safety.

[0027] Furthermore, the "pharmaceutically acceptable salt" includes acid and base addition salts selected from citrate, hydrochloride, oxalate, phosphate, sulfate, fumarate, succinate, tartrate, nitrate or maleate; it should be understood that when a compound or example in the present application is shown as a specific salt, it is intended to cover the corresponding free base and other salts of the corresponding free base (including pharmaceutically acceptable salts of the corresponding free base).

[0028] Furthermore, the "pharmaceutically acceptable acid addition salt" refers to those salts formed with inorganic or organic acids that retain the biological effectiveness and properties of the free base and do not have biological or other undesirable effects and properties, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and the organic acid can be selected from aliphatic, cycloaliphatic, aromatic, aromatic aliphatic, heterocyclic, carboxylic and sulfonic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, pamoic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, salicylic acid.

[0029] Further, the "pharmaceutically acceptable base addition salts" include those derived from inorganic bases of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts; specific base addition salts are ammonium, potassium, sodium, calcium and magnesium salts; salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion-exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, tromethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins; specific organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, tromethamine, dicyclohexylamine, choline and caffeine. The pharmaceutically acceptable excipients can include at least one selected from the group consisting of penetration enhancers, surfactants, crystallization inhibitors, foam adjuvants, thickening agents, pH adjusters, gel bases, antioxidants, metal ion chelating agents, humectants and solvents.

[0030] Further, the content of the solvent is 5wt% to 80wt% of the compound external preparation.

[0031] Further, the liniment includes: active ingredient A, active ingredient B, solvent and pharmaceutically acceptable excipients; the percentage of the active ingredient A in the compound external preparation is 0.1wt% to 10wt% based on the EPO receptor agonist; the percentage of the mass of the active ingredient B to the volume of the compound external preparation is 0.1% (w / v) to 10% (w / v) based on the JAK inhibitor; the solvent is a mixture of water and alcohol; the content of the solvent is 50wt% to 99.8wt% of the liniment; the content of the pharmaceutically acceptable excipients is 0 to 20wt% of the liniment.

[0032] Further, the gel includes: active ingredient A, active ingredient B, solvent and pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include gel bases and other excipients, the other excipients include at least one selected from the group consisting of penetration enhancers, surfactants, crystallization inhibitors, antioxidants, bacteriostatic agents; the percentage of the active ingredient A in the compound external preparation is 0.1wt% to 5wt% based on the EPO receptor agonist; the percentage of the mass of the active ingredient B to the volume of the compound external preparation is 1% (w / v) to 5% (w / v) based on the JAK inhibitor; the solvent is a mixture of water and alcohol; the content of the solvent is 50wt% to 98.8wt% of the compound external preparation; the content of the gel base is 0.1wt% to 30wt% of the compound external preparation, and the content of the other excipients is 0 to 20wt% of the compound external preparation.

[0033] Furthermore, the composition comprises, by weight percentage, 0.5% to 6.0% of active ingredient A, 0.5% to 6.0% of active ingredient B, 0.1% to 1% of thickener, 0.1% to 2.0% of pH regulator, 0.1% to 2.0% of antioxidant, 0.1% to 0.5% of metal chelating agent, 1% to 10% of moisturizing agent, and the remainder being solvent.

[0034] The beneficial effects of the present invention are:

[0035] The EPO receptor agonist roxadustat and tofacitinib or their pharmaceutically acceptable salts both have limitations when used alone to treat alopecia areata, but the two happen to act on different targets in the pathogenesis of alopecia areata, so it can be considered to prepare a compound preparation. Roxadustat can inhibit the key signaling pathways in the pathogenesis of alopecia areata, thereby making up for the lack of efficacy of tofacitinib in moderate and severe patients from the etiology. At the same time, the good hair growth-promoting effect of roxadustat can improve the shortcomings of tofacitinib in hair repair from the symptomatic point of view. The two have complementary efficacy and treat both the symptoms and the root causes. Based on the synergistic effect, the dose of tofacitinib can be appropriately reduced to reduce the risk of side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 To compare the hair growth of each group before and after administration;

[0037] Figure 2 A is a comparison of the skin growth area on the back.

[0038] Figure 2 B is the comparison of the area of ​​the back skin growth area quantified by ImageJ software

[0039] Figure 2 C is a comparison of hair growth scores assessed using a scoring index (0 = no growth, 1 = 0-20% growth, 2 = 20-40%, 3 = 40-60%, 4 = 60-80%, 5 = 80-100%).

[0040] Figure 2 D is the comparison of the number of hair follicles in the back skin.

[0041] Figure 2 E is the A / T ratio of the dorsal skin hair follicles.

[0042] Figure 2 F and 2G are HE staining of the dorsal skin (×100 magnification). DETAILED DESCRIPTION

[0043] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar improvements without violating the scope of the present invention. Therefore, the present invention is not limited to the specific implementation disclosed below.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0045] Experimental Example 1: Gel

[0046] (1) Prescription

[0047]

[0048] (2) Preparation method

[0049] Preparation of aqueous phase mixture: Dissolve butylated hydroxytoluene, glycerol, rosuvastatin, and tofacitinib in odorless ethanol;

[0050] Preparation of oil phase mixture: Dissolve lactic acid and anhydrous citric acid in purified water;

[0051] Mix the above two solutions, add Carbomer 980 to disperse evenly, add triethanolamine and adjust the pH to about 5.0.

[0052] Comparative Example 1: Rosuvastatin Gel

[0053] (1) Prescription

[0054]

[0055] (2) Preparation method

[0056] Preparation of aqueous phase mixture: dissolve butylated hydroxytoluene, glycerol, and rosuvastatin in odorless ethanol; preparation of oil phase mixture: dissolve lactic acid and anhydrous citric acid in purified water;

[0057] Mix the above two solutions, add Carbomer 980 to disperse evenly, add triethanolamine and adjust the pH to about 5.0.

[0058] Comparative Example 2: Tofacitinib-Gel

[0059] (1) Prescription

[0060]

[0061]

[0062] (2) Preparation method

[0063] Preparation of aqueous phase mixture: Dissolve butylated hydroxytoluene, glycerol, and tofacitinib in odorless ethanol;

[0064] Preparation of oil phase mixture: Dissolve lactic acid and anhydrous citric acid in purified water;

[0065] Mix the above two solutions, add Carbomer 980 to disperse evenly, add triethanolamine and adjust the pH to about 5.0.

[0066] Experimental Example 2

[0067] Evaluation of the therapeutic effect of rosuvastatin combined with tofacitinib for alopecia areata

[0068] (1) Animals

[0069] Mouse model of alopecia areata: 6-8 week old C57BL / 6 mice, half male and half female, were lightly anesthetized with ether. A mixture of rosin and paraffin (1:1) was heated and melted and applied to the back. After solidification and hardening, it was removed. After 8-9 days, the growth phase hair follicles were induced in the skin of the mouse back. A single dose of 150 mg / kg of cyclophosphamide was injected intraperitoneally. After 4-5 days, the hair on the back of the mouse began to fall off, and the alopecia areata model was established.

[0070] (2) The test animals were grouped according to the type of drug administered:

[0071] ①Normal group (no medication)

[0072] ②Alopecia areata model group (no medication)

[0073] ③ Roxadustat combined with tofacitinib group (Example 1)

[0074] ④ Rosuvastatin group (Comparative Example 1)

[0075] ⑤ Tofacitinib group (Comparative Example 2)

[0076] Note: Normal mice were depilated before administration to induce hair growth; except for the normal group and the alopecia areata model group

[0077] Except for the mice in group 1 who were not given the drug, the other groups were all given the drug using alopecia areata mice.

[0078] (3) Dosage regimen

[0079] Dosage: 1.5 g / time; 2 times / day; continuous administration for 4 weeks;

[0080] Administration: Apply to the alopecia areata area on the back of mice.

[0081] (4) Evaluate the treatment of alopecia areata in each group of mice according to the following standards

[0082] Before administration, an area of ​​approximately 6 cm was selected on the back of each mouse. 2 The alopecia areata skin of the mice was used as the experimental area. After administration, the hair growth of the mice was observed every day. Five hairs were randomly plucked from each mouse and the length was measured using a vernier caliper. The average value was taken. After 28 days, the 6 cm 2 The experimental area was removed with the skin attached, and the hair was carefully plucked with tweezers and weighed. The skin tissue from the experimental area was then fixed with 10% formaldehyde. Four sections of tissue, each 1.5 cm long, were taken along the longitudinal section of the hair follicles. The tissue was then dehydrated, embedded in paraffin, and stained with hematoxylin and eosin. The skin tissue of the mice was examined under a light microscope to observe indicators related to hair growth, including:

[0083] ① Diameter of superficial dermal blood vessels (μm): Randomly measure the diameters of 5 cross-sectioned capillaries and calculate the average;

[0084] ②Number of superficial dermal blood vessels: Count at 200x magnification in 5 visual fields and calculate the average number;

[0085] ③ Number of newly formed hair follicles in the superficial dermis: Count at 100x magnification for 5 fields of view and calculate the average.

[0086] The mean values ​​of the data in each group were statistically analyzed using t-test, *P<0.05, **P<0.01, compared with the mice in the alopecia areata model group. The results are as follows:

[0087] Table 1. Effects of each drug group on mouse hair growth

[0088]

[0089]

[0090] Table 2. Effects of each drug group on mouse skin capillaries and hair follicles

[0091] Group n Number of hair follicles Number of capillaries Capillary diameter (μm) Normal group 16 9.42±0.34** 7.46±0.73** 7.53±1.15** Alopecia areata model group 16 7.61±1.17 6.47±1.85 6.29±0.73 Example 1 group 16 9.25±0.62** 7.45±1.11** 7.82±1.51** Comparative Example 1 16 8.85±1.66** 7.59±1.56** 8.01±1.83** Comparative Example 2 16 9.28±0.97** 7.35±1.30** 6.99±1.55**

[0092] Analysis of results: It can be seen from the results in Table 1 and Table 2 that the hair growth rate of the group using roxadustat combined with tofacitinib is faster than that of the group using either component alone, and the number of hair follicles, capillaries and capillary diameter are significantly increased, indicating that roxadustat combined with tofacitinib can play a synergistic role.

[0093] Example 3

[0094] Roxadustat combined with tofacitinib promotes hair growth in C3H / HEJ mice

[0095] Male C3H / HEJ mice were selected and imiquimod cream was applied to the back of the mice every three days for two weeks before administration. The hair on the back of the mice was removed one day before administration. A single-dose group of roxadustat (roxadustat concentration of 4 mg / mL, 0.1 mL each time), a single-dose group of tofacitinib (tofacitinib concentration of 10 mg / mL, 0.1 mL each time), and a roxadustat combined with tofacitinib group (roxadustat concentration of 4 mg / mL, 0.1 mL each time, tofacitinib concentration of 10 mg / mL, 0.1 mL each time) were set up. The positive control was minoxidil (minoxidil 50 mg / mL, 0.2 mL). The roxadustat single-dose group, the tofacitinib single-dose group, and the tofacitinib group were locally injected intradermally once a day; minoxidil was applied to the hairless area twice a day. The treatment was continued for 15 consecutive days.

[0096] Hair growth was recorded daily using a digital camera. Changes in hair area were assessed using ImageJ image analysis software. The area under the hair area-time curve was calculated.

[0097] After the administration, the animals were euthanized and the experimental areas of some animals were observed for pathological reasons. Figure 2 Shown are: (A) Comparison of dorsal skin growth areas. (B) Comparison of dorsal skin growth areas quantified using ImageJ software. (C) Comparison of hair growth scores assessed using a scoring index (0 = no growth, 1 = 0%-20% growth, 2 = 20%-40%, 3 = 40%-60%, 4 = 60%-80%, 5 = 80%-100%). (D) Comparison of the number of hair follicles in dorsal skin. (E) A / T ratio of dorsal skin hair follicles. (F, G) HE staining of dorsal skin. (*, p < 0.05; **, p < 0.01; ***, p < 0.001.)

Claims

1. A compound preparation for treating alopecia areata, characterized in that: The active ingredients are: active ingredient A and active ingredient B, wherein the active ingredient A is an EPO receptor agonist, and the active ingredient B is tofacitinib or a pharmaceutically acceptable salt thereof; The EPO receptor agonist is rosuvastatin.

2. A compound preparation for treating alopecia areata according to claim 1, characterized in that: The compound preparation is a liniment or a gel; Calculated as an EPO receptor agonist, the percentage of the active ingredient A in the compound preparation is 0.1 wt% to 10 wt% or 0.1% w / v to 10% w / v; Calculated on the basis of tofacitinib, the percentage of the active ingredient B in the compound preparation is 0.1 wt% to 10 wt% or 0.1% w / v to 10% w / v.

3. A compound preparation for treating alopecia areata according to claim 1, characterized in that The concentration of the EPO receptor agonist is 0.01% to 1%, 1% to 3%, 3% to 5% w / w; from 5% to 10% w / w or from 10% to 20% w / w.

4. A compound preparation for treating alopecia areata according to claim 1, characterized in that The compound preparation also includes pharmaceutically acceptable excipients.

5. The compound preparation according to claim 1, characterized in that The content of the solvent is 5wt% to 80wt% of the compound preparation.

6. The compound preparation according to claim 2, characterized in that The composition of the liniment is: active ingredient A, active ingredient B, solvent and pharmaceutically acceptable excipients; calculated as EPO receptor agonist, the percentage of the active ingredient A in the compound preparation is 0.1wt% to 10wt%; the percentage of the mass of the active ingredient B to the volume of the compound preparation is 0.1%w / v to 10%w / v; the solvent is a mixture of water and alcohol; the content of the solvent is 50wt% to 99.8wt% of the liniment; the content of the pharmaceutically acceptable excipient is 0 to 20wt% of the liniment.

7. The compound preparation according to claim 2, characterized in that The gel preparation comprises: active ingredient A, active ingredient B, a solvent and pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include a gel matrix and other excipients, and the other excipients include at least one selected from a penetration enhancer, a surfactant, a crystal inhibitor, an antioxidant and an antibacterial agent; calculated as an EPO receptor agonist, the percentage of the active ingredient A in the compound preparation is 0.1wt% to 5wt%; the mass of the active ingredient B to the volume of the compound preparation is 1%w / v to 5%w / v; the solvent is a mixture of water and alcohol; the content of the solvent is 50wt% to 98.8wt% of the compound preparation; the content of the gel matrix is ​​0.1wt% to 30wt% of the compound preparation, and the content of the other excipients is 0 to 20wt% of the compound preparation.

8. The compound preparation according to claim 2, characterized in that The gel composition is composed of 0.5% to 6.0% active ingredient A, 0.5% to 6.0% active ingredient B, 0.1% to 1% thickener, 0.1% to 2.0% pH regulator, 0.1% to 2.0% antioxidant, 0.1% to 0.5% metal chelating agent, 1% to 10% moisturizing agent, and the remainder is solvent.

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