Pharmaceutical composition having JAK inhibitory effect

Abstract

Disclosed is a pharmaceutical composition, which comprises the following components as active ingredients: (A) a JAK inhibitor or a pharmaceutically acceptable salt thereof; and (B) cedrol. Also disclosed is a formulation, which comprises the pharmaceutical composition and one or more pharmaceutically acceptable excipient. Further disclosed is a method for treating skin diseases dominated by a JAK pathway, which comprises orally or topically administering the pharmaceutical composition or the formulation. The pharmaceutical composition can synergistically enhance the curative effect on alopecia areata diseases dominated by the JAK pathway, while reducing the dose of prescription, thereby improving the curative effect and medication safety of the composition.

Description

Pharmaceutical compositions with JAK inhibitory activity Technical Field

[0001] This invention relates to the fields of chemical compatibility technology and pharmaceutical technology. Specifically, this invention relates to a method for preparing a pharmaceutical composition for the prevention and treatment of JAK-dominated skin diseases (such as alopecia areata) and its uses. Background Technology

[0002] JAKs are a class of intracellular non-receptor tyrosine kinases that play a pivotal role in signal transduction mediated by many cytokine receptors, participating in important physiological processes such as proliferation, differentiation, apoptosis, and immune regulation of various cell types in organisms. Dysregulation of JAK targets is often associated with various skin diseases, such as alopecia areata (AA). Therefore, this target has gradually become one of the most attractive targets for the treatment of AA, and targeting and inhibiting JAK targets is a novel strategy for AA treatment.

[0003] Baricitinib (BT) is an oral JAK inhibitor that inhibits all JAK receptors and has shown good efficacy in the treatment of alopecia areata. In June 2022, it was approved by the FDA for the treatment of alopecia areata, becoming the first systemic treatment for alopecia areata approved by the FDA. However, BT has certain limitations in treating alopecia areata. Currently, BT is only available in oral formulations, and long-term use increases the risk of systemic immunosuppression.

[0004] Furthermore, although JAK inhibitors have shown promising promise in the treatment of atopic dermatitis (AA), some patients have developed resistance to these drugs, making treatment difficult. Therefore, scientists are increasingly focusing on combination therapy to enhance treatment efficacy.

[0005] There is still a need to develop drug compositions that are effective in treating skin diseases, especially AA, and that reduce JAK inhibitor resistance and side effects. Summary of the Invention

[0006] To address the above needs, the present invention provides a pharmaceutical composition with JAK inhibitory activity, which can prevent and / or treat various skin diseases dominated by the JAK pathway, especially AA disease.

[0007] Therefore, a first aspect of this application provides a pharmaceutical composition comprising the following components as active ingredients:

[0008] (A) A JAK inhibitor or a pharmaceutically acceptable salt thereof;

[0009] (B) Cephalotaxol (CE for short).

[0010] A second aspect of this application provides a formulation comprising the pharmaceutical composition described in the first aspect of this application and one or more pharmaceutically acceptable excipients.

[0011] The third aspect of this application provides the use of the pharmaceutical composition described in the first aspect or the formulation described in the second aspect of this application in the prevention and / or treatment of skin diseases dominated by the JAK pathway, particularly AA diseases.

[0012] The third aspect of this application provides a method for treating skin diseases dominated by the JAK pathway, comprising oral or topical administration of the pharmaceutical composition described in the first aspect or the preparation described in the second aspect of this application.

[0013] The fourth aspect of this application provides the use of a pharmaceutical composition in the preparation of a medicament for treating skin diseases dominated by the JAK pathway, especially AA disease.

[0014] To treat various skin diseases primarily involving the JAK pathway, especially AA, this invention discloses a novel pharmaceutical composition that achieves the following effects by scientifically combining a JAK inhibitor with CE:

[0015] (1) It acts synergistically on the JAK signaling pathway, enhances the efficacy of treatment for AA diseases dominated by the JAK pathway, and achieves a synergistic therapeutic effect.

[0016] (2) The prescription dosage of each active ingredient was reduced, which reduced toxic side effects and drug resistance, and improved the efficacy and safety of the composition.

[0017] The compositions or formulations provided by this invention can be taken orally or applied topically, have clearly defined targets, significant therapeutic effects, and are safe to use, providing a new treatment option for patients with AA diseases dominated by the JAK pathway. Attached Figure Description

[0018] Figure 1 shows the hair growth in mice 9 days after administration in Example 1. Figure a shows images of the backs of mice taken on days 0 and 9, and Figure b shows a bar chart of fur scores for each group of mice. The fur scores were obtained by analyzing the hairless areas in the images using IMAGE J software (National Institutes of Health, USA).

[0019] Figure 2 shows the results of taking images of the backs of mice in each group on day 9 (day 0 after CP injection), day 14 (day 5 after CP injection), and day 16 (day 7 after CP injection) in Example 1.

[0020] Figure 3 shows the effects of CE, BT, and CE+BT on hair length and hair weight in mice with cyclophosphamide-induced alopecia areata after the last administration. It is a bar chart plotted based on the data in Table 2 of Example 1. Figure A is a bar chart of hair length in each group of mice after the last administration, and Figure B is a bar chart of hair weight in each group of mice after the last administration.

[0021] Figure 4 shows the effects of CE, BT, and CE+BT on hair follicle length and number in the skin tissue of mice with cyclophosphamide-induced alopecia areata. Figure a shows HE-stained microscopic images of longitudinal sections of mouse skin tissue for observing hair follicle length. Figure b shows HE-stained microscopic images of transverse sections of mouse skin tissue for observing hair follicle number. Figure c is a bar chart showing the quantification of hair follicle length in each group of mice using IMAGE J software. Figure d is a bar chart showing the quantification of hair follicle number in each group of mice using IMAGE J software.

[0022] Figure 5 shows the effects of CE, BT, and CE+BT on JAK3 / STAT3 protein expression in the skin tissue of mice with alopecia areata. The relative activity is represented by a bar chart. The relative activity was calculated by measuring the protein expression of p-JAK3, JAK3, p-STAT3, STAT3, and β-actin in the hairless skin area of ​​the mice using Western blotting, followed by analysis of the band gray values ​​using IMAGE j software.

[0023] The letters above the bars in Figures 1 and 2-5 are marked using a salience marking method. See the Examples section below for details. Detailed Implementation

[0024] The following details some specific embodiments of the present invention.

[0025] JAK inhibitors are immunosuppressants that selectively inhibit JAK kinase and block the JAK / STAT signaling pathway.

[0026] Both JAK inhibitors and cypressin have limitations when used alone to treat various skin diseases, especially AA (articular dermatitis), which are primarily mediated by the JAK pathway. For example, baricitinib (BT), one of the JAK inhibitors, is currently only available in oral formulations. Long-term use carries a high risk of systemic immunosuppression. Compared to oral formulations, topical formulations can avoid the first-pass effect in the liver, reduce the risk of systemic side effects, and prolong the duration of efficacy.

[0027] Cephalosporin (CE), CAS number 77-53-2, has the following structural formula:

[0028] CE is a small molecule sesquiterpene compound widely found in the volatile oils of various plants such as cypress, cedar, and pine, and has a variety of pharmacological activities, such as anti-inflammatory and hair loss relief.

[0029] In the course of their work, the inventors of this application unexpectedly discovered that the combined use of JAK inhibitors, especially BT and CE, can achieve an unexpected synergistic effect in the treatment of various skin diseases dominated by the JAK pathway, especially AA disease.

[0030] Therefore, the present invention provides a pharmaceutical composition characterized in that it comprises or uses the following components as active ingredients:

[0031] (A) A JAK inhibitor or a pharmaceutically acceptable salt thereof;

[0032] (B)CE.

[0033] In some embodiments, the pharmaceutical composition of the present invention comprises only components (A) and (B) as active ingredients.

[0034] In some embodiments, the JAK inhibitors include baricitinib (BT, CAS No. 1187594-09-7), litecitinib (RT, CAS No. 1792180-81-4), tofacitinib (CAS No. 477600-75-2), olatinib (CAS No. 1208319-26-9), dicitinib (CAS No. 944842-54-0), and perfetinib (CAS No. 9). 44118-01-8), filogrinib (CAS No. 1206161-97-8), filatatinib (CAS No. 936091-26-8), lintatinib (CAS No. 111358-88-4), paclinib (CAS No. 937272-79-2), utpatinib (CAS No. 1310726-60-3), molotinib (CAS No. 1056634-68-4), etc., or combinations thereof.

[0035] In some embodiments, the JAK inhibitor is selected from baricitinib (BT), litexcitinib (RT), tofacitinib, olatinib, deuterated ruxolitinib, jaktinib, dicitinib, perfetinib, frigidinib, fertatinib, lintatinib, paktinib, utpatinib, molotinib, and combinations thereof.

[0036] The pharmaceutical composition of this application may include one or more JAK inhibitors.

[0037] In some preferred embodiments, the JAK inhibitor is BT, RT, or a combination thereof.

[0038] In some further preferred embodiments, the pharmaceutical composition includes BT and CE.

[0039] In some further preferred embodiments, the pharmaceutical composition includes RT and CE.

[0040] The molar ratio of JAK inhibitor to CE can be, for example, at least about 1:1000, at least about 1:900, at least about 1:800, at least about 1:700, at least about 1:600, at least about 1:500, at least about 1:400, at least about 1:300, at least about 1:200, at least about 1:100, at least about 1:90, at least about 1:80, at least about 1:70, at least about 1:60, at least about 1:50, at least about 1:40, at least about 1:30, at least about 1:20, at least about 1:10, at least about 1:5, at least about 1:4, at least about 1:3, at least about 1:1.

[0041] The molar ratio of JAK inhibitor to CE can also be, for example, up to approximately 1000:1, up to approximately 900:1, up to approximately 800:1, up to approximately 700:1, up to approximately 600:1, up to approximately 500:1, up to approximately 400:1, up to approximately 300:1, up to approximately 200:1, up to approximately 100:1, up to approximately 90:1, up to approximately 80:1, up to approximately 70:1, up to approximately 60... :1, at most about 50:1, at most about 40:1, at most about 30:1, at most about 20:1, at most about 10:1, at most about 5:1, at most about 1:1, at most about 1:2, at most about 1:3, at most about 1:4, at most about 1:5, at most about 1:10, at most about 1:15, at most about 1:20, at most about 1:25, at most about 1:30, at most about 1:35, at most about 1:40.

[0042] The molar ratio of JAK inhibitor to CE can be any combination of any of the above ranges. For example, the molar ratio of JAK inhibitor to CE can be in the range of at least about 1:3 and at most about 3:1, at least about 1:1 and at most about 3:1, or at least about 1:3 and at most about 1:1.

[0043] Given the toxicity of JAK inhibitors, the molar ratio of JAK inhibitor to CE is preferably less than 1:1, more preferably less than 1:2, more preferably less than 1:3, more preferably less than 1:4, more preferably less than 1:5, more preferably less than 1:6, more preferably less than 1:7, more preferably less than 1:8, more preferably less than 1:9, and more preferably less than 1:10.

[0044] In some preferred embodiments, the JAK inhibitor is BT, and the molar ratio of BT to CE is 1:(1-100), preferably 1:(20-80), more preferably 1:(30-70), and most preferably 1:(40-60).

[0045] The compositions of the present invention can be used in combination with other drugs. Therefore, in some embodiments, the pharmaceutical composition further comprises one or more other active ingredients that can be used to prevent or treat skin diseases, especially for hair growth, such as minoxidil (CAS No.: 38304-91-5), finasteride (CAS No.: 98319-26-7), etc.

[0046] The pharmaceutical compositions of this invention can be formulated with pharmaceutically acceptable carriers or excipients into clinically acceptable oral or topical formulations. Therefore, this invention also provides a formulation comprising the pharmaceutical composition of this application and one or more pharmaceutically acceptable carriers or excipients.

[0047] In some preferred embodiments, the formulations of the present invention are oral formulations, such as solid dosage forms such as tablets, pills, powders, granules, capsules, or lozenges.

[0048] In some preferred embodiments, the formulations of the present invention are topical formulations, such as glycerin, tinctures, liniments, film-forming agents, ointments, creams, patches, microneedles, gels, liposomes, liposomes, or nanoparticles.

[0049] All formulations involved in this application have the conventional meaning in the art and can be prepared using conventional techniques in the art.

[0050] In some preferred embodiments, the pharmaceutical composition in the formulation of the present invention includes BT and CE, and is an oral solid dosage form.

[0051] In some preferred embodiments, the pharmaceutical composition in the formulation of the present invention includes RT and CE, and is an oral solid dosage form.

[0052] In some preferred embodiments, the pharmaceutical composition in the formulation of the present invention includes BT and CE, and it is a topical formulation.

[0053] In some preferred embodiments, the pharmaceutical composition in the formulation of the present invention includes RT and CE, and it is a topical formulation.

[0054] The present invention also provides the use of the pharmaceutical composition and the formulation in the prevention and / or treatment of skin diseases dominated by the JAK pathway, particularly AA disease.

[0055] The present invention further provides a method for preventing and / or treating skin diseases dominated by the JAK pathway, comprising orally administering the pharmaceutical composition or formulation described in this application.

[0056] The present invention further provides a method for preventing and / or treating skin diseases dominated by the JAK pathway, comprising applying the pharmaceutical composition or formulation described in this application topically.

[0057] Considering the toxicity of some JAK inhibitors, this application preferably uses the pharmaceutical composition topically or formulates it into a topical preparation.

[0058] In this application, topical preparations refer to preparations that can be applied to the surface of the skin.

[0059] This application also provides the use of pharmaceutical compositions in the preparation of remedies for treating skin diseases dominated by the JAK pathway, especially AA diseases, particularly oral and topical remedies.

[0060] The invention will be further described below with examples, but the invention is not limited thereto.

[0061] Example

[0062] The key material information involved in the embodiments is as follows:

[0063] CE, an abbreviation for cypressin, was purchased from Zhejiang Hangzhou Green Biotechnology Co., Ltd., product number P10-0198;

[0064] BT, an abbreviation for baricitinib, was purchased from Jiangsu Aikon Biotechnology, product number K900933.

[0065] CP, an abbreviation for cyclophosphamide, National Drug Approval Number HJ20160467; specification 0.2g; purchased from Baxter Oncology GmbH, specification 0.2g.

[0066] Example 1

[0067] Effects of combined administration of CE and BT on hair growth in mice (C57 / BL-6 mice)

[0068] Sixty male C57 / BL-6 mice aged 6-8 weeks were randomly divided into 6 groups, with 10 mice in each group. The groups were: control group, model group, CE (15 mg / kg) group, CE (30 mg / kg) group, BT (1 mg / kg) group, and CE+BT (15 mg + 0.5 mg / kg) group. After one week of normal feeding, the fur on the back of the mice was trimmed using a hair removal device (2×3 cm). 2 Then, hair removal cream was applied to the prepared back area to remove hair from the mice's backs. The mice were administered the drug via gavage the day after hair removal, once daily. The drugs, concentrations, and dosages administered to each group of mice are shown in Table 1.

[0069] Table 1

[0070] The drug delivery substance is prepared as follows:

[0071] 0.5% Sodium Carboxymethyl Cellulose Aqueous Solution: First, add 0.5g of sodium carboxymethyl cellulose powder to 100mL of distilled water, then place it in a water bath at 60-70℃ and heat it, stirring constantly and adding distilled water to bring the total volume to 100mL during heating. After heating for 5 hours, the 0.5% sodium carboxymethyl cellulose aqueous solution is ready.

[0072] Solutions of CE in 0.5% sodium carboxymethyl cellulose aqueous solution, solutions of BT in 0.5% sodium carboxymethyl cellulose aqueous solution, and solutions of CE and BT together in 0.5% sodium carboxymethyl cellulose aqueous solution:

[0073] Appropriate amounts of CE, BT, and the CE+BT mixture were weighed into a glass mortar and ground into a fine powder using a grinding rod. Then, a 0.5% sodium carboxymethyl cellulose aqueous solution was gradually added to the mortar while continuously stirring to dissolve the powder. The process was stopped when no obvious particulate matter formed in the solution. The drug substance solutions listed in the table are prepared in this way. The specific concentrations are shown in Table 1.

[0074] When preparing the CE+BT (15+0.5mg / kg) group of drugs, CE and BT are prepared together in the same solution and administered simultaneously.

[0075] Images of the mouse backs were taken at key hair growth times (day 0 and day 9). The results are shown in Figure 1a. Furthermore, grayscale analysis of the bald areas on the mouse backs in Figure 1a was performed using IMAGE J (National Institutes of Health, USA) software to obtain a fur score. The fur score in Figure 1b represents the quantified grayscale values ​​of the skin and hair on the mouse backs.

[0076] The experimental results showed that in the first 9 days after hair removal in mice, significant hair growth was observed in the CE (15 mg / kg), CE (30 mg / kg), and CE+BT (15 + 0.5 mg / kg) groups. The CE+BT (15 + 0.5 mg / kg) group showed the best hair growth effect, with a significant difference compared to the CE group.

[0077] The preventive effects of CE, BT, and CE+BT on cyclophosphamide-induced alopecia areata in mice

[0078] On day 9 of the aforementioned experiment, mice in the model group (without CE and / or BT), CE (15 mg / kg), CE (30 mg / kg), BT (1 mg / kg), and CE+BT (15 + 0.5 mg / kg) group were intraperitoneally injected with cyclophosphamide (CP) to establish the alopecia areata model. The model establishment method is described in the literature: “Effect of CeO2 nanozyme on hair growth in cyclophosphamide-induced alopecia areata mice,” Peng Xinxin et al., J Shanxi Med Univ, Feb. 2024, Vol 55, No 2, p. 192.

[0079] The establishment of the model was determined by observing hair loss in each group of mice. If hair loss occurred, it indicated that the model had been established.

[0080] The control group mice were injected with saline on the ninth day after drug administration. Saline did not cause hair loss in the mice, so their hair grew normally.

[0081] Images of the backs of mice were taken on day 9 (day 0 after CP injection), day 14 (day 5 after CP injection), and day 16 (day 7 after CP injection). The results are shown in Figure 2. In Figure 2, the methods of administration of CE and / or BT to mice in the CE (15 mg / kg), CE (30 mg / kg), BT (1 mg / kg), and CE+BT (15 + 0.5 mg / kg) groups were as follows; the methods of administration of CP to mice in the model groups CE (15 mg / kg), CE (30 mg / kg), BT (1 mg / kg), and CE+BT (15 + 0.5 mg / kg) groups were as follows.

[0082] According to the above model, mice experienced hair loss on their backs within one week after CP injection.

[0083] As shown in Figure 2, no hair loss was observed in mice on day 5 after CP injection, indicating that the CP had not yet taken effect. On day 7 after CP injection, hair regrowth in the previously bald areas of the model group mice relapsed, indicating cyclophosphamide-induced alopecia (AA). While hair loss did occur in the CE (15 mg / kg), CE (30 mg / kg), and CE+BT (15 + 0.5 mg / kg) groups, the effect was significantly better than in the model group. This suggests that CE and CE+BT have a certain preventive effect on hair loss in mice. Figure 2 also shows that CE+BT had the best anti-hair loss effect, showing a significant difference compared to the CE group.

[0084] Effects of CE, BT, and CE+BT on hair length and weight in cyclophosphamide-induced alopecia areata mice

[0085] After the last administration (on day 28), the length of the hair on the back of each group of mice was measured using calipers. Then, the hair in the previously shaved areas was removed using a hair removal device, and the weight of the mouse hair was measured using a balance. The results are shown in Table 2 below.

[0086] Table 2. Effects of CE, BT, and CE+BT on hair length and hair weight in cyclophosphamide-induced alopecia areata mice.

[0087] Figure 3 is a bar chart plotted using the data in Table 2. Figure A is a bar chart of hair length in each group of mice after the last administration, and Figure B is a bar chart of hair weight in each group of mice after the last administration. These two figures allow for a more direct comparison of hair length and hair weight in each group of mice.

[0088] In Table 2 and Figure 3, the control group mice had normal hair growth and had not experienced alopecia areata, therefore their hair length and weight showed the best results. The model group mice only experienced alopecia areata and did not receive CE or BT, resulting in the shortest hair length and lightest hair weight. The hair length and weight of the CE (15 mg / kg), CE (30 mg / kg), and BT (1 mg / kg) groups were significantly better than the model group. In the CE+BT (15 + 0.5 mg / kg) group, the CE dosage was half that of the CE (30 mg / kg) group, and the BT dosage was half that of the BT (1 mg / kg) group, yet the hair length and weight were significantly better than these two groups. This indicates that combined administration of BT and CE, with reduced dosage, significantly enhances the efficacy of hair regrowth promotion, exhibiting a synergistic effect, and can be used to treat alopecia areata. Although BT is a first-line drug for alopecia areata treatment, it has certain in vivo toxicity; reducing the BT dosage can reduce drug toxicity and side effects, and improve drug safety.

[0089] Example 2

[0090] Effects of CE+BT combination therapy on hair follicle length and number in mouse skin tissue

[0091] The mice were grouped and administered the same medication as in Example 1. After the last administration, blood was collected from the mice via ocular sampling, and the mice were then euthanized by cervical dislocation. The hair on the backs of the mice was removed using a small animal shaver and collected. The skin on the backs of the mice was lifted with surgical forceps, and the skin was cut along the shaved area using surgical scissors. The cut skin was then split along the midline and used for different purposes. One half of the cut skin was placed in 4% paraformaldehyde solution for subsequent HE staining to observe the length and weight of mouse hair follicles, while the other half was frozen at -80°C for Western blotting experiments to detect the expression of p-JAK3 / JAK3 and p-STAT3 / STA3 proteins.

[0092] Half of the skin previously fixed in 4% paraformaldehyde was removed, embedded, sectioned in paraffin, and then stained using the HE staining method.

[0093] The specific staining steps are as follows:

[0094] Mouse skin sections were sequentially immersed in environmentally friendly dewaxing solution I for 20 min, environmentally friendly dewaxing solution II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, and then rinsed with tap water. Then, hematoxylin and eosin (H&E) high-resolution constant staining kit was used for HE staining according to the instructions. Specifically, the sections were pretreated in the high-resolution constant staining pretreatment solution for 1 min. Afterward, the sections were stained with hematoxylin for 3-5 min, rinsed with tap water, differentiated with differentiation solution, rinsed with tap water, and then blued with blueing solution, and rinsed with running water. The sections were dehydrated in 95% ethanol for 1 min, and then stained with eosin for 15 s. Then, the sections were sequentially immersed in anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min, anhydrous ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, xylene I for 2 min, and xylene II for 2 min, and mounted with neutral resin. Images were acquired and analyzed using a microscope. The results are shown in Figure 4.

[0095] The following are some of the instruments and reagents used in the test:

[0096] Upright optical microscope, manufacturer: Nikon Japan, part number: NIKON ECLIPSE E100;

[0097] Imaging system, manufacturer: Nikon Japan, part number: NIKON DS-U3;

[0098] Environmentally friendly dewaxing solution, manufacturer: Wuhan Saiweier Biotechnology Co., Ltd. (hereinafter referred to as "Saiweier"), product number: G1128

[0099] General-purpose tissue fixative, manufacturer: Xavier, product number: G1101;

[0100] Hematoxylin-Eosin (H&E) High-Resolution Constant Staining Kit, Manufacturer: Xavier, Catalog No.: G1076.

[0101] Figure 4 shows the effects of CE, BT, and CE+BT on hair follicle length and number in the skin tissue of mice with cyclophosphamide-induced alopecia areata.

[0102] Figure 4 shows a microscope image of mouse skin tissue stained with hematoxylin and eosin (HE) for longitudinal section observation of mouse hair follicle length. Figure 4 shows a microscope image of mouse skin tissue stained with HE for transverse section observation of mouse hair follicle number. Figure 4 shows a bar chart quantifying hair follicle length in each group of mice using IMAGE J software. Figure 4 shows a bar chart quantifying hair follicle number in each group of mice using IMAGE J software.

[0103] The significance of hair length and weight in each group of mice was analyzed using a letter-labeled significance method. Specifically, each group was first assigned a letter, af; for example, the control group was assigned a, the model group a, the CE (15 mg / kg) group a, the CE (30 mg / kg) group a, the BT (1 mg / kg) group a, and the CE+BT (15+0.5 mg / kg) group a, f. When the difference between groups was not significant, the same letter (i.e., the previously assigned letter) was marked above each group's bar chart (p > 0.05). If the difference between the control group and the model group was not significant, the model group's bar chart was marked with its own letter b and the same letter a (for the insignificant difference). If the difference between the control group and the CE+BT (15+0.5 mg / kg) group was not significant, the CE+BT (15+0.5 mg / kg) group's bar chart was marked with its own letter f and the letter a (for the group with no significant difference). The significance labeling method on the bar charts in Figures 1 and 3-5 was as described above.

[0104] The results above show that the CE+BT combination at half the dose resulted in longer hair follicle length and a greater number of hair follicles in mouse skin HE staining, significantly superior to the CE and BT groups, and also superior to the control group that did not undergo alopecia areata modeling. This indicates that the combination of CE and BT drugs exhibits a synergistic effect.

[0105] Example 3

[0106] Effects of CE+BT combination therapy on the JAK3 / STAT3 signaling pathway in mouse skin tissue

[0107] The expression of p-JAK3 / JAK3 and p-STAT3 / STAT3 proteins was detected by Western blot experiment using half of the skin that was frozen at -80°C as described in Example 2.

[0108] p-JAK3: Purchased from Absin, product number abs139988;

[0109] JAK3: Purchased from Absin, product number abs115170;

[0110] p-STAT3: Purchased from Absin, product number abs130919;

[0111] STAT3 (purchased from Absin, catalog number abs155916) protein expression.

[0112] The four antibodies mentioned above are referred to as "primary antibodies" in the following tests.

[0113] The specific testing methods are briefly described below:

[0114] Preparation of supernatant

[0115] First, mouse skin stored at -80℃ was removed, and 50 mg of mouse skin tissue was surgically cut into 2 mL EP tubes. 500 μL of RIPA lysis buffer (from Seville, catalog number G2002-100 mL) containing 5 μL of protease inhibitor PMSF (from Seville, catalog number G2008-1 mL) was added to the EP tubes. The skin was then broken up using a cell homogenizer. The tubes were then incubated at 4℃ for 15 min, followed by centrifugation (12000 rpm, 15 min). After centrifugation, the supernatant was collected and used for the following total protein concentration determination and protein denaturation.

[0116] Total protein concentration determination

[0117] The total protein concentration in the supernatant was determined using a BCA kit (G2026-200T, Xavier). See the instruction manual for detailed operating procedures.

[0118] protein denaturation

[0119] Mix the supernatant with 5X reduced protein loading buffer (from Sewell, catalog number G2075-100ML) at a ratio of 4:1. Denature the mixture in a 95°C water bath for 10 min. After cooling, proceed directly to the following protein electrophoresis steps.

[0120] Protein electrophoresis

[0121] Clean the glass plates for gel preparation; first, move the clamping plates on both sides of the gel preparer to the bottom, fully open the pressure caps on both sides, and insert the concave glass and flat glass from the upper side in sequence until they are fully inserted, with the upper part of the glass locking into the two side slots; flip up the pressure caps on both sides, and simultaneously pinch the left side of the pressure cap with your hands, then pull the left side clamping plate upwards until it is locked at the top; then simultaneously pinch the right side of the pressure cap, and pull the right side clamping plate upwards until it is locked at the top; after confirming that the electrophoresis glass is locked and aligned, unscrew the knobs on both sides of the gel preparation base, then place the electrophoresis support in the middle of the gel preparation base and lock it in place, then press the main support with your hands and tighten the knobs on both sides until they are rotated to their limit.

[0122] Preparation of separating and stacking gels: The separating gel (10%) and stacking gel (10%) for electrophoresis were prepared using the one-step PAGE Color Gel Ultra-Rapid Preparation Kit (catalog number G2177-50T, from Xavier). See the kit instructions for preparation methods. Prepare four sets of separating and stacking gels.

[0123] Electrophoresis: Clamp the prepared separating gel and stacking gel onto the gel casting unit, with the stacking gel on top and the separating gel below. Place them into the electrophoresis tank, then fill the inner side with electrophoresis buffer and the outer side with 1 / 3 of the buffer. Use a pipette to add the denatured sample obtained in the protein denaturation step into the casting well of the stacking gel. Electrophoresis at a constant voltage of 200V for 30 minutes, until the blue indicator of the 5X reduced protein loading buffer (the solution used in the protein denaturation step) reaches approximately 1 cm from the bottom, at which point the electrophoresis is terminated. Remove the electrophoresis tank, then disassemble the gel casting unit and place it in transfer buffer (catalog number G2028-1L, from Savill) to prepare for the transfer operation.

[0124] Transfer membrane

[0125] Prepare 24 thin 7×9cm transfer filter papers and 4 5×8cm PVDF membranes. The PVDF membranes should be activated with ethanol for 2 minutes before use. Place a transfer clamp, two sponges, filter papers, and the activated PVDF membranes in a container containing transfer buffer (the same transfer buffer used in the previous step with the gel casting apparatus). Unfold the transfer clamp, red on the left and black on the right. Place a sponge and three sheets of filter paper on each side. Carefully peel the separating gel from the gel casting apparatus and place it on the filter paper (gum on the black side of the transfer clamp). Rinse the gel with transfer buffer to remove air bubbles. Slowly apply the PVDF membrane onto the separating gel, being careful to avoid air bubbles. Then, attach the transfer filter paper and sponge in sequence. Perform the transfer, transferring the sample from the separating gel to the PVDF membrane. Transfer conditions (wet transfer): constant current 300mA, 30 minutes. Repeat the above operation four times. Obtain four PVDF membranes with the sample attached.

[0126] Closed

[0127] The PVDF membranes containing the samples obtained during the transfer step were placed into four 1-cell whole-membrane antibody incubation boxes containing TBST (catalog number G2150-1L, from Sewell) (catalog number G9055-1, from Sewell), quickly rinsed once, and then 5 ml of 5% skim milk (5 mg skim milk powder in 100 ml of water) was added. The boxes were placed on a decolorizing shaker and blocked at room temperature for 30 min.

[0128] Primary Antibody Incubation

[0129] Dilute each antibody according to the instructions for p-JAK3, JAK3, p-STAT3, and STAT3 (these are primary antibodies). Discard the blocking solution in the antibody incubation chamber, add the diluted primary antibody, and incubate overnight at 4°C on a shaker (with slow shaking).

[0130] Washing film

[0131] Discard the solution in the antibody incubation box, then quickly rinse the membrane three times (1 min, 3 times) with TBST, then add TBST, place on a decolorizing shaker for rapid elution, 5 min each time, for a total of three washes;

[0132] Secondary antibody incubation

[0133] The secondary antibody (HRP-labeled goat anti-rabbit IgG, catalog number GB23303, from Xavier) was diluted with TBST at a volume ratio of 1:5000, and then added to the antibody incubation box mentioned above. The box was placed on a shaker and incubated at room temperature for 30 minutes.

[0134] Washing the mask:

[0135] After incubating the secondary antibody, discard the secondary antibody solution in the antibody incubation cassette. Quickly wash the membrane three times with TBST, then add TBST again and place on a decolorizing shaker for rapid elution, 5 minutes each time, for a total of three washes. The washed membrane is then subjected to the next step of chemiluminescence.

[0136] Imaging and Data Analysis

[0137] Chemiluminescence imaging was performed using a chemiluminescence imaging system (manufacturer: Wuhan Saiwell Biotechnology Co., Ltd., catalog number SGG-W2000). After exposure, the original image was saved as a TIFF file. The saved TIFF image was then analyzed using WB analysis software (manufacturer: Wuhan Saiwell Biotechnology Co., Ltd., catalog number AIWBwell). TM Data analysis was performed to obtain protein bands. Then, the gray values ​​of the bands were analyzed using IMAGE j software to calculate the relative activities of p-JAK3 / JAK3 and p-STAT3 / STAT3. The results are shown in Figure 5.

[0138] Figure 5 shows the effects of CE, BT, and CE+BT on the expression of JAK3 / STAT3 protein in the skin tissue of mice with alopecia areata.

[0139] As shown in Figure 5, firstly, the relative vitality of mice in the CE (15 mg / kg) and CE (30 mg / kg) groups differed significantly from that in the model group. Secondly, when the dosages of CE and BT were halved and used in combination, they further promoted the relative vitality of the mice. This indicates that the combination of CE and BT has a synergistic effect and can be used to treat alopecia areata.

[0140] in conclusion

[0141] In the prevention and treatment of alopecia areata dominated by the JAK pathway, the combination of CE and JAK inhibitors showed better intervention effects compared with CE or JAK inhibitors alone, and had a synergistic effect on hair length, weight and JAK protein expression in mice.

Claims

1. A pharmaceutical composition, characterized by, comprising as effective ingredients: (A) a JAK inhibitor or a pharmaceutically acceptable salt thereof; and (B) cembrene.

2. The pharmaceutical composition of claim 1, wherein, The JAK inhibitor includes baricitinib, ruxolitinib, tofacitinib, oclacitinib, deuterated ruxolitinib, upadacitinib, decernotinib, febuxostat, filgotinib, fedratinib, lestaurtinib, pacritinib, upatinib, or momelotinib, and combinations thereof.

3. The pharmaceutical composition of claim 1, wherein, The JAK inhibitor is baricitinib, ruxolitinib, or a combination thereof.

4. The pharmaceutical composition of claim 1, wherein, The pharmaceutical composition includes baricitinib and cembrene.

5. The pharmaceutical composition of claim 1, wherein, The pharmaceutical composition includes ruxolitinib and cembrene.

6. The pharmaceutical composition of claim 1, wherein, The JAK inhibitor is baricitinib, and the molar ratio of baricitinib to cembrene is 1 : (1-100), more preferably 1 : (30-70), most preferably 1 : (40-60).

7. The pharmaceutical composition of claim 1, wherein, The pharmaceutical composition further comprises one or more other active ingredients useful for preventing or treating skin diseases, especially hair growth.

8. A preparation comprising the pharmaceutical composition of any one of claims 1-7 and one or more pharmaceutically acceptable carriers or excipients.

9. The preparation according to claim 8, characterized in that The preparation is an oral preparation, which is a tablet, a pill, a powder, a granule, a capsule, or a lozenge.

10. The formulation of claim 8, wherein, The preparation is an external preparation, which is a glycerin agent, a tincture, a liniment, a film, an ointment, a cream, a patch, a microneedle, a gel, a liposome, an ethosome, or a nanoparticle.

11. Use of the pharmaceutical composition of any one of claims 1-7 or the preparation of any one of claims 8-10 in preventing and / or treating a skin disease dominated by JAK pathway, especially alopecia areata disease.

12. A method for treating a skin disease dominated by JAK pathway, especially alopecia areata disease, comprising orally or externally administering the pharmaceutical composition of any one of claims 1-7 or the preparation of any one of claims 8-10.

13. Use of the pharmaceutical composition of any one of claims 1-7 in the preparation of a medicament, especially an oral medicament or an external medicament, for treating a skin disease dominated by JAK pathway, especially alopecia areata disease.

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