Use of compound in preparation of drug for treating psoriasis

Abstract

Use of a compound in the preparation of a drug for treating psoriasis. Particularly, disclosed is new use of lefamulin or retapamulin in the preparation of a drug for treating psoriasis. The experiment proves that lefamulin or retapamulin has significant efficacy in treating psoriasis with rapid onset of action and small toxic and side effects, is a safe, efficient, and stable drug for treating psoriasis with a simple preparation process, is suitable for industrial production, and is easy to popularize, thereby providing a new drug source for treating psoriasis.

Description

Use of a compound in preparing a medicament for treating psoriasis Technical Field

[0001] The present invention belongs to the field of medical technology and relates to the use of a compound in preparing a drug for treating chronic inflammatory skin diseases, and in particular to the use of a compound in preparing a drug for treating psoriasis. Background Art

[0002] Psoriasis is a common chronic inflammatory and proliferative skin disease characterized by erythematous scaling and epidermal thickening. It can affect multiple tissues, including the skin, mucous membranes, and joints. Psoriasis has a long course, is difficult to cure, and is prone to recurring attacks, often lasting a lifetime. The itching and pain it causes have a serious impact on the patient's physical and mental health, and also impose a huge economic burden on the patient's family and society as a whole. The etiology and pathogenesis of psoriasis are complex, with genetic, infectious, immune, metabolic, psychological, and environmental factors all closely related to the disease. These factors interact with each other to form a network, which also leads to the multicentric nature of psoriasis and the complexity of its prevention and treatment.

[0003] There is currently no effective cure for psoriasis. Common clinical treatments can be divided into physical therapy, topical medications, and systemic medications. Physical therapy and topical medications are suitable for patients with milder symptoms, while moderate and severe cases require systemic medications. Traditional topical treatments for psoriasis primarily use topical glucocorticoids. However, long-term use of topical glucocorticoids can cause a series of adverse reactions, including skin atrophy and hirsutism.

[0004] In recent years, progress has been made in the development of monoclonal antibody drugs targeting key inflammatory factors in the pathogenesis of psoriasis, such as tumor necrosis factor alpha (TNF-α) and interleukin 17 (IL-17). However, drugs such as adalimumab (TNF-α monoclonal antibody) and secukinumab (IL-17 monoclonal antibody) have inherent drawbacks, including high cost, inability to be taken orally or topically, and severe side effects with long-term use.

[0005] Therefore, the development of drugs for psoriasis is one of the major issues that need to be addressed urgently in experimental research and clinical practice. Recent studies have shown that small molecule drugs have many advantages, such as low cost and flexible administration methods. Therefore, the search and development of small molecule drugs for psoriasis is a hot topic in current research.

[0006] Retapamulin, molecular formula C 30 H 46NO4S, the structural formula is shown in formula (I),

[0007] It is a topical antibiotic, often used as an external antibacterial drug. Studies on this compound have found that it has strong antibacterial activity against Staphylococcus aureus and Streptococcus pyogenes. There have been no reports on its use in the treatment of psoriasis so far.

[0008] Lefamulin, molecular formula C 28 H 45 NO5S, the structural formula is shown in formula (II),

[0009] It is a pleuromutilin antibiotic. Studies on this compound have found that it is mainly used to treat community-acquired bacterial pneumonia (CABP) caused by sensitive microorganisms. There have been no reports on its use in the treatment of psoriasis.

[0010] Summary of the Invention

[0011] In response to the above problems, one of the objects of the present invention is to provide a class of compounds for use in the preparation of drugs for the treatment of psoriasis. Such compounds are lefamulin or retapamulin or their derivatives. Such compounds are non-hormonal compounds and can be administered externally, significantly reducing side effects and having less impact on the whole body, and can be better used in drugs for the treatment of psoriasis.

[0012] The present invention mainly found that lefamulin and retapamulin have a significant improvement effect on the imiquimod-induced psoriasis-like model in mice. The results of immunoblotting experiments suggest that lefamulin and retapamulin have antagonistic effects on TNF-α and its pathway, so they can be used in the treatment of psoriasis.

[0013] The present invention uses imiquimod (IMQ) to model psoriasis in mice, resulting in psoriatic dermatitis on the back skin of the mice. Retapamulin and lefamulin topical preparations are prepared, and lefamulin and retapamulin topical preparations are used to treat psoriatic dermatitis. It is found that both drugs can treat imiquimod-induced psoriatic dermatitis. In addition, the present invention uses TNF-α to model cell death in the L929 cell line and then conducts experiments. The experimental results show that the administration of the drugs retapamulin and lefamulin can significantly inhibit TNF-α-induced L929 cell death and alleviate IMQ-induced skin erythema and scaling. Chemical proteomics is an important tool for discovering the targets of small molecule drugs. Experiments have found that lefamulin interacts with heterogeneous nuclear ribonucleoprotein U (hnRNP-U). Using siRNA to knock down hnRNP-U can inhibit TNF-α-induced cell death. Immunoblotting results suggest that lefamulin can inhibit hnRNP-U expression in HaCaT cells. Immunohistochemistry revealed high expression of hnRNP-U in psoriatic-like and psoriatic lesions in mice, suggesting a correlation between hnRNP-U and psoriasis disease activity. These results demonstrate that retapamulin and lefamulin can inhibit the TNF-α pathway through hnRNP-U, thereby improving IMQ-induced psoriasis symptoms. Furthermore, these small molecules can be administered topically, resulting in relatively minimal side effects and systemic effects, suggesting their potential application in the treatment of psoriasis.

[0014] To achieve the above objectives, the present invention provides the following technical solutions: On one hand, the present invention provides a use of a compound in the preparation of a drug for treating psoriasis, wherein the compound is retapamulin or lefamulin or a derivative thereof.

[0015] In some embodiments, the derivative is in the form of a salt and / or a solvate.

[0016] In some embodiments, the derivative of lefamulin is in the form of lefamulin acetate or lefamulin hydrochloride.

[0017] In some embodiments, the psoriasis is psoriasis vulgaris.

[0018] In some embodiments, the medicament contains a therapeutically effective amount of lefamulin or retapamulin and a pharmaceutically acceptable carrier.

[0019] In some embodiments, the therapeutically effective amount is 1% to 10%, specifically, the therapeutically effective amount is 1% to 9%, 2% to 9%, 3% to 9%, 4% to 9%, 5% to 9%, 6% to 9%, 7% to 9%, 8% to 9%, 1% to 8%, 2% to 8%, 3% to 8%, 4% to 8%, 5% to 8%, 6% to 8%, 7% to 8%, 1% to 7%, 2% to 7%, 3% to 7%, 4% to 7%, 5% to 7%, 6% to 7%, 1% to 6%, 2% to 6%, 3% to 6%, 4% to 6%, 5% to 6%, 1% to 5%, 2% to 5%, 3% to 5%, 4% to 5%, 1% to 4%, 2% to 4%, 3% to 4%, 1% to 3%, 2% to 3%.

[0020] In some embodiments, the pharmaceutically acceptable carrier includes one or more of a diluent, a solubilizer, a cosolvent, a disintegrant, a dispersant, a lubricant, a flavoring agent, an antioxidant, a binder, an absorbent, a wetting agent, a buffer, and a cross-linking agent.

[0021] In some embodiments, the drug is formulated into a pharmaceutically acceptable dosage form.

[0022] In some embodiments, the dosage form comprises pills, tablets, powders, capsules, granules, powders, pellets, drops, patches, tinctures, pastes, lotions, sprays, injections, suspensions, creams, ointments, gels, and suppositories.

[0023] In some embodiments, the drug is a topical drug.

[0024] In some embodiments, the therapeutically effective amount of lefamulin or retapamulin is 1% to 10%, specifically, the therapeutically effective amount is 1% to 9%, 2% to 9%, 3% to 9%, 4% to 9%, 5% to 9%, 6% to 9%, 7% to 9%, 8% to 9%, 1% to 8%, 2% to 8%, 3% to 8%, 4% to 8%, 5% to 8%, 6% to 8%, 7% to 8%, 1% to 7%, 2% to 7%, 3% to 7%, 4% to 7%, 5% to 7%, 6% to 7%, 1% to 6%, 2% to 6%, 3% to 6%, 4% to 6%, 5% to 6%, 1% to 5%, 2% to 5%, 3% to 5%, 4% to 5%, 1% to 4%, 2% to 4%, 3% to 4%, 1% to 3%, 2% to 3%, specifically, the therapeutically effective amount is 5%.

[0025] In some embodiments, the topical medication is in the form of a spray, an aerosol, a patch, a tincture, a paste, a lotion, a cream, a cream, or an ointment.

[0026] Another aspect of the present invention provides an ointment for treating psoriasis, wherein the ointment comprises lefamulin or retapamulin,

[0027] The structural formula of lefamulin is as follows:

[0028] The structural formula of retapamulin is shown below:

[0029] In some embodiments, the ointment comprises the following:

[0030] In some embodiments, in the ointment of the present invention, the amount of lefamulin or retapamulin is 0.25g to 0.75g, specifically, the amount of lefamulin or retapamulin is 0.25g to 0.75g, 0.25g to 0.70g, 0.25g to 0.65g, 0.25g to 0.60g, 0.25g to 0.55g, 0.25g to 0.50g, 0.25g to 0.45g , 0.25g to 0.40g, 0.25g to 0.35g, 0.30g to 0.75g, 0.30g to 0.70g, 0.30g to 0.65g, 0.30g to 0.60g, 0.30g to 0.55g, 0.30g to 0.50g, 0.30g to 0.45g, 0.30g to 0.40g, 0.35g to 0.75g, 0.35g to 0.70g, 0.3 5g to 0.65g, 0.35g to 0.60g, 0.35g to 0.55g, 0.35g to 0.50g, 0.35g to 0.45g, 0.40g to 0.75g, 0.40g to 0.70g, 0.40g to 0.65g, 0.40g to 0.60g, 0.40g to 0.55g, 0.40g to 0.50g, 0.45g to 0.75g, 0.45g to 0 .70g, 0.45g to 0.65g, 0.45g to 0.60g, 0.45g to 0.55g, 0.50g to 0.75g, 0.50g to 0.70g, 0.50g to 0.65g, 0.50g to 0.60g, 0.50g to 0.75g, 0.55g to 0.70g, 0.55g to 0.65g, 0.60g to 0.75g, 0.60g to 0.70g.

[0031] In some embodiments, in the ointment of the present invention, the amount of the polyoxyethylene stearate is 2.0 to 3.0 g, specifically, the amount of the polyoxyethylene stearate is 2.0 to 3.0 g, 2.0 to 2.5 g, or 2.5 to 3.0 g.

[0032] In some embodiments, in the ointment of the present invention, the amount of propylene glycol is 2.0 to 3.0 g, specifically, the amount of propylene glycol is 2.0 to 3.0 g, 2.0 to 2.5 g, or 2.5 to 3.0 g.

[0033] In some embodiments, in the ointment of the present invention, the amount of the white petrolatum is 4.0 to 5.0 g, specifically, the amount of the white petrolatum is 4.0 to 5.0 g, 4.0 to 4.5 g, or 4.5 to 5.0 g.

[0034] The present invention also provides a use of a compound in the preparation of a drug for inhibiting the TNF-α pathway by interacting with heterogeneous nuclear ribonucleoprotein U (hnRNP-U), wherein the compound is lefamulin, retapamulin or a derivative thereof,

[0035] The structural formula of lefamulin is as follows:

[0036] The structural formula of retapamulin is shown below:

[0037] Unless otherwise indicated, the term "therapeutically effective amount" as used herein refers to the amount of a drug required to produce an effective effect. The term "therapeutically effective amount" is adjustable and variable and can be determined by the attending physician, taking into account factors such as the specific condition of the subject being treated, the duration of treatment, the severity of the disease, and sound medical judgment. The therapeutically effective amount of a drug administered to a human can be determined mathematically based on the results of animal studies. The therapeutically effective amount of a drug to be administered for treatment can be readily determined by those skilled in the art, and the optimal dose will vary depending on the specific compound used, the mode of administration, the formulation strength, and the progression of disease symptoms. Furthermore, the dose may need to be adjusted to an appropriate therapeutic level due to factors related to the specific subject being treated, including the subject's age, weight, diet, and time of administration.

[0038] The present invention has at least the following beneficial effects: (1) Retapamulin and Lefamulin can improve the symptoms of psoriasis vulgaris. They are non-hormonal compounds and can be administered externally. External administration can significantly reduce side effects and have less impact on the whole body, and can be better used in the treatment of psoriasis; (2) The present invention has discovered new medicinal value for the known small molecule compounds Retapamulin and Lefamulin, and used them to treat psoriasis, thereby opening up a new application field for the application of Retapamulin and Lefamulin; (3) The pharmacological effects of Retapamulin and Lefamulin of the present invention are strong. Cell death modeling is carried out in the L929 cell line using TNF-α, and TNF-α The induced L929 cell death effect was inhibited by lefamulin and retapamulin, and the inhibition efficiency was dose-dependent; (4) A photocrosslinking group was added to lefamulin, and chemical proteomics screening found that hnRNP-U could interact with lefamulin. In HaCaT cells, hnRNP-U expression in HaCaT cells was found to be decreased after co-incubation with lefamulin, indicating that lefamulin can inhibit the TNF-α pathway through hnRNP-U; (5) In psoriatic-like lesions of mice and lesions of psoriasis patients, it was found that the expression level of hnRNP-U was correlated with the severity of the disease, indicating that lefamulin can achieve the purpose of treating psoriasis by inhibiting hnRNP-U. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is an HE staining image of the changes and lesions on the back skin of mice in Example 2A and Example 2B, wherein A is an HE staining image of the changes and lesions on the back skin of mice in Example 2A, wherein the left side is an image of the changes on the back skin and the right side is an HE staining image of the lesions; B is an HE staining image of the changes and lesions on the back skin of mice in Example 2B, wherein the left side is an image of the changes on the back skin and the right side is an HE staining image of the lesions;

[0040] FIG2A is a statistical graph showing the inhibitory effect of lefamulin on TNF-α-induced L929 cell death in Example 3A;

[0041] FIG2B is a statistical graph showing the inhibitory effect of retapamulin on TNF-α-induced L929 cell death in Example 3B;

[0042] FIG3A is the molecular structure of lefamulin with added photocrosslinking groups (lef-probe) in Example 4;

[0043] FIG3B is a statistical graph showing the inhibitory effect of Lef-probe on TNF-α-induced L929 cell death in Example 4;

[0044] FIG3C is a schematic diagram of the grouping status of the proteomic screening of lefamulin protein substrates in Example 4;

[0045] FIG3D is a schematic diagram of three replicate experiments in Example 4 for screening potential protein substrates from L929 and HaCaT cells;

[0046] FIG3E is a graph showing the ratios of proteins interacting with lefamulin identified in L929 and HaCaT cells in Example 4;

[0047] FIG4A is an immunoblot image showing the inhibition of hnRNP-U expression in HaCaT cells using Lefamulin in Example 5;

[0048] FIG4B is a statistical diagram showing the attenuation of the function of lefamulin in inhibiting TNF-α-induced L929 cell death in Example 5;

[0049] FIG5A is an observation diagram of hnRNP-U immunohistochemical staining of mouse skin in Example 6;

[0050] FIG5B is a statistical diagram of hnRNP-U immunohistochemical staining of mouse skin under different treatment groups;

[0051] Figure 5C shows the immunohistochemical staining of hnRNP-U in skin lesions of patients with psoriasis. DETAILED DESCRIPTION

[0052] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0053] The beneficial effects of the drug of the present invention are further illustrated below through test examples, which include the efficacy test of the present invention.

[0054] Example 1 Preparation of Lefamulin Ointment and Retapamulin Ointment

[0055] Example 1A Preparation of Lefamulin Ointment

[0056] 1A) Prepare the ingredients according to the following formula:

[0057] 2A) Preparation of Lefamulin Ointment

[0058] Weigh lefamulin (0.5 g) and place it in a beaker. Add white vaseline and heat to promote dissolution. Then add polyoxyethylene stearate and propylene glycol in sequence. Heat and stir to melt. Stir until solidified to obtain lefamulin ointment.

[0059] Example 1B Preparation of Retapamulin Ointment

[0060] 1B) Prepare the ingredients according to the following recipe:

[0061] 2B) Preparation of Retapamulin Ointment

[0062] Weigh retapamulin (0.5 g) and place it in a beaker. Add white vaseline and heat to promote dissolution. Then add polyoxyethylene stearate and propylene glycol in sequence. Heat while stirring to melt. Stir until solidified to obtain retapamulin ointment.

[0063] Example 2 Effects of Topical Administration of Lefamulin or Retapamulin on IMQ-Induced Mouse Psoriasis Model Example 2A Effects of Topical Administration of Lefamulin on IMQ-Induced Mouse Psoriasis Model

[0064] 2A.1 Test materials

[0065] 2A.1.1 Experimental animals and reagents

[0066] The experimental animals used were 7-8 week old male BALB / c mice, provided by Jicui Yaokang Animal Biotechnology Experimental Center, certificate number: SCXK(Su)2018-0008.

[0067] The experimental reagents used were IMQ cream (purchased from Sichuan Mingxin Pharmaceutical Co., Ltd.) and the lefamulin ointment prepared in Example 1A.

[0068] 2A.1.2 Experimental Methods

[0069] Twenty-four male BALB / c mice aged 7-8 weeks were randomly divided into two groups, with 6 mice in each group. The backs of the mice were shaved (area: 2 cm × 3 cm) and the mice were raised normally. Three days later, different treatments were given to the shaved areas of the mice as follows:

[0070] Model group (IMQ): IMQ 62.5 mg / mouse / day was applied to the back skin of mice for 5 days;

[0071] Treatment group (IMQ+5% Lefamulin): IMQ 62.5 mg / mouse / day was applied to the back skin of mice for 5 days; 12 hours after applying IMQ every day, an equal dose of 5% Lefamulin ointment prepared in Example 1A was applied.

[0072] The mice in each group were fed and drank normal water. The skin manifestations of the mice were observed every day. The mice were killed on the 6th day and the skin lesions were obtained for (H&E) staining.

[0073] 2A.1.3 Experimental Results

[0074] The changes in the dorsal skin of mice in each group on the 6th day and the lesions were stained with HE (4X). The results are shown in Figure 1A.

[0075] As shown in Figure 1A, compared to the blank control group, the dorsal skin of mice in the modeling group (IMQ) showed significant erythema and scaling. HE staining revealed significant hyperkeratosis and parakeratosis in the epidermis, and numerous inflammatory cells were infiltrated into the superficial dermis. The treatment group (IMQ + 5% Lefamulin) showed a significant alleviation of erythema and scaling after topical drug application.

[0076] Example 2B Effect of Topical Administration of Retapamulin on IMQ-Induced Psoriasis Model in Mice

[0077] 2B.1 Test Materials

[0078] 2B.1.1 Experimental animals and reagents

[0079] The experimental animals used were 7-8 week old male BALB / c mice, provided by Jicui Yaokang Animal Biotechnology Experimental Center, certificate number: SCXK(Su)2018-0008.

[0080] The experimental reagents used were the retapamulin ointment prepared in Example 1B and IMQ cream (purchased from Sichuan Mingxin Pharmaceutical Co., Ltd.).

[0081] 2B.1.2 Experimental Methods

[0082] Twenty-four male BALB / c mice aged 7-8 weeks were randomly divided into two groups, with 6 mice in each group. The backs of the mice were shaved (area: 2 cm × 3 cm) and the mice were raised normally. Three days later, different treatments were given to the shaved areas of the mice as follows:

[0083] Model group (IMQ): IMQ 62.5 mg / mouse / day was applied to the back skin of mice for 5 days;

[0084] Treatment group (IMQ+5% Retapamulin): IMQ 62.5 mg / mouse / day was applied to the back skin of mice for 5 days; 12 hours after applying IMQ every day, an equal dose of 5% Retapamulin ointment prepared in Example 1B was applied.

[0085] The mice in each group were fed and drank normal water. The skin manifestations of the mice were observed every day. The mice were killed on the 6th day and the skin lesions were obtained for (H&E) staining.

[0086] 2B.1.3 Experimental Results

[0087] The changes in the dorsal skin of mice in each group on the 6th day and the skin lesions were stained with HE (4X). The results are shown in Figure 1B.

[0088] As shown in Figure 1B, compared to the blank control group, the mice in the modeling group (IMQ) showed significant erythema and scaling on the back skin. HE staining revealed significant hyperkeratosis and parakeratosis in the epidermis, and a large number of inflammatory cells infiltrated into the superficial dermis. The treatment group (IMQ + 5% Retapamulin) showed a significant alleviation of erythema and scaling after topical treatment.

[0089] The experiment in Example 2 showed that in the IMQ-induced psoriasis mouse model, topical administration of famolin or retapamulin can alleviate IMQ-induced psoriatic dermatitis, and topical drug administration is more efficient and convenient in treating skin diseases.

[0090] Example 3 Effects of Lefamulin and Retapamulin on TNF-α-induced L929 cell death model

[0091] Example 3A Effect of Lefamulin on TNF-α-induced L929 cell death model

[0092] 3A.1 Test Materials

[0093] Mouse fibroblast L929 cell line in the logarithmic growth phase was used for the experiment;

[0094] Human TNF-α was dissolved in purified water at a concentration of 10 ng / ml; lefamulin was dissolved in dimethyl sulfoxide to prepare solutions with concentrations of 10 μM, 30 μM, 50 μM, 70 μM, and 100 μM, respectively; the CCK8 cell death detection kit was purchased from MCE.

[0095] 3A.2 Experimental Methods

[0096] Divided into 3 groups, the specific operations are as follows:

[0097] Negative control group: complete DMEM medium 100 μl / well, 4 replicates per 96-well plate;

[0098] Cell death group: 10 ng / ml TNF-α and 1 μg / ml actinomycin D were added, with four replicates per 96-well plate;

[0099] Drug group: Lefamulin (dissolved in DMSO, DMSO concentration ≤ 0.1%), 10 ng / ml TNF-α, and 1 μg / ml actinomycin D were added at five different concentrations of 10 μM, 30 μM, 50 μM, 70 μM, and 100 μM, with four replicate wells for each concentration;

[0100] The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 7 hours. CCK-8 reagent (10 μl / well) was then added to the 96-well plate and incubated in a 37°C, 5% CO2 incubator for 2 hours. The optical density of each well was measured at a wavelength of 450 nm using a microplate reader. The background OD value (complete medium plus CCK8 detection solution, no cells) was subtracted from the OD value of each test well. The mean and standard deviation were calculated as (drug group / cell death group) / (negative control / cell death group).

[0101] 3A.3 Experimental Results

[0102] After treating L929 cells with 10 ng / ml of TNF-α and 1 μg / ml of actinomycin D for 7 hours, cell viability was assessed using the CCK-8 assay, revealing that approximately 90% of the L929 cells underwent cell death. In the drug groups, lefamulin at varying concentrations significantly inhibited TNF-α-induced L929 cell death. P values ​​were calculated using a two-tailed Student's t-test (Figure 2A).

[0103] Example 3B Effect of Retapamulin on TNF-α-induced L929 cell death model

[0104] 3B.1 Test Materials

[0105] Mouse fibroblast L929 cell line in the logarithmic growth phase was used for the experiment;

[0106] Human TNF-α was dissolved in purified water at a concentration of 10 ng / ml; retapamulin was dissolved in dimethyl sulfoxide to prepare solutions with concentrations of 10 μM, 30 μM, 50 μM, 70 μM, and 100 μM, respectively; the CCK8 cell death detection kit was purchased from MCE.

[0107] 3B.2 Experimental Methods

[0108] Divided into 3 groups, the specific operations are as follows:

[0109] Negative control group: complete DMEM medium 100 μl / well, 4 replicates per 96-well plate;

[0110] Cell death group: 10 ng / ml TNF-α and 1 μg / ml actinomycin D were added, with four replicates per 96-well plate;

[0111] Drug group: Retapamulin (dissolved in DMSO, DMSO concentration ≤ 0.1%), 10 ng / ml TNF-α, and 1 μg / ml actinomycin D were added at five different concentrations of 10 μM, 30 μM, 50 μM, 70 μM, and 100 μM, with four replicate wells for each concentration;

[0112] The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 7 hours. CCK-8 reagent (10 μl / well) was then added to the 96-well plate and incubated in a 37°C, 5% CO2 incubator for 2 hours. The optical density of each well was measured at a wavelength of 450 nm using a microplate reader. The background OD value (complete medium plus CCK8 detection solution, no cells) was subtracted from the OD value of each test well. The mean and standard deviation were calculated as (drug group / cell death group) / (negative control / cell death group).

[0113] 3B.3 Experimental Results

[0114] After treating L929 cells with 10 ng / ml of TNF-α and 1 μg / ml of actinomycin D for 7 hours, cell viability was assessed using the CCK-8 assay, revealing that approximately 90% of the L929 cells underwent cell death. In the drug groups, retapamulin at various concentrations significantly inhibited TNF-α-induced L929 cell death. P values ​​were calculated using a two-tailed Student's t-test (Figure 2B).

[0115] Thus, the results of Example 3 show that both lefamulin and retapamulin can directly inhibit TNF-α-induced L929 cell death in in vitro experiments. Therefore, lefamulin and retapamulin can inhibit TNF-α and ultimately achieve a therapeutic effect on psoriasis.

[0116] Example 4: Using chemical proteomics to discover the interaction between lefamulin and hnRNP-U

[0117] 4.1 Experimental Materials and Methods

[0118] The experiment was conducted using the logarithmic growth phase human epidermal immortalized cell line HaCaT and the mouse fibroblast L929 cell line. Lefamulin was added with a photocrosslinking group (Lef-probe). The molecular structure of the photocrosslinking group (lef-probe) of Lefamulin is shown in Figure 3A.

[0119] The effect of Lef-probe on the TNF-α-induced L929 cell death model was detected using the same detection method as in Example 3. As shown in FIG3C , the grouping and steps of the proteomic screening of lefamulin protein substrates are as follows:

[0120] 1) L929 or HaCaT cells were co-incubated with Lef-probe. To improve the specificity of mass spectrometry data, the experiment was divided into two pairs (as shown in Figure 3C): ① UV irradiation and non-UV irradiation groups (calculation of UV enrichment intensity), ② UV irradiation, lefamulin and non-lefamulin groups (calculation of lefamulin competition intensity);

[0121] 2) Using click chemistry to couple the probe-target protein complex to biotin;

[0122] 3) Enrichment using streptavidin coupling to obtain a probe-target protein-biotin complex;

[0123] 4) labeling target proteins using tandem mass spectrometry;

[0124] 5) Liquid chromatography tandem mass spectrometry analysis.

[0125] 4.2 Experimental Results

[0126] As shown in Figure 3B , Lef-probe inhibited TNF-α-induced cell death, indicating that the photocrosslinking group did not affect the biological function of lefamulin. In the chemical proteomics experiment, three independent experiments were repeated in two cell lines (as shown in FIG3D , potential protein substrates screened from L929 and HaCaT cells). 97 potential target proteins were obtained in the L929 cell line and 69 potential target proteins were obtained in the HaCaT cell line. The data of the two cell lines were cross-analyzed, as shown in FIG3E , which is a ratio diagram of proteins interacting with lefamulin identified in L929 and HaCaT cells. Among them, the black spot with the largest area is hnRNP-U. In FIG3E , the Y-axis is the UV enrichment ratio (Ruv = UV irradiation mass spectrum abundance value / non-UV irradiation mass spectrum abundance value, Ruv>1.1 is positive), and the X-axis is the lefamulin competition ratio (Rcomp = Lef-probe group mass spectrum abundance value / lefamulin mass spectrum abundance value, Rcomp>1.1 is positive). The applicant found that lefamulin is most likely to interact with heterogeneous nuclear ribonucleoprotein U (hnRNP-U). ribonucleoprotein-U, hnRNP-U) interact with each other, which shows that

[0127] The results of Example 4 indicate that lefamulin interacts with hnRNP-U in in vitro experiments.

[0128] Example 5 Lefamulin can affect the function of hnRNP-U protein and thus inhibit the TNF-α pathway

[0129] 5.1 Test Materials

[0130] The experiments were conducted using the human epidermal immortalized cell line HaCaT and the mouse fibroblast line L929 in the logarithmic growth phase.

[0131] Falfamulin was dissolved in dimethyl sulfoxide to prepare solutions with concentrations of 50 μM and 100 μM, respectively. The primary antibody for immunoblotting was purchased from Abcam: hnRNP-U (ab180952). The mouse hnRNP-U siRNA sequences were: GGAGCAAUAUAAAGAAGAATT and UUCUUCUUUUAUAUUGCUCCTT. siRNA transfection reagent (Lipofectamine TM 3000 kit) was purchased from ThermoFisher. The CCK8 cell death detection kit was purchased from MCE. Human TNF-α was dissolved in purified water at a concentration of 10 ng / ml.

[0132] 5.2 Experimental methods

[0133] 5.2.1 Immunoblotting

[0134] Divided into 3 groups, the specific operations are as follows:

[0135] Blank control group (Control): Protein extracted from HaCaT cell line, that is, protein extracted from HaCaT cells using RIPA lysis buffer was used as blank control;

[0136] 12-hour incubation lefamulin group (TNF-α + 50 μM Lefamulin): HaCaT cells were treated with 50 μM lefamulin for 12 hours; HaCaT cell proteins were then extracted using RIPA lysis buffer;

[0137] 24-hour incubation lefamulin group (TNF-α + 50 μM Lefamulin): HaCaT cells were treated with 50 μM lefamulin for 24 hours; then, HaCaT cell proteins were extracted using RIPA lysis buffer;

[0138] At room temperature, 100 μl of RIPA lysis buffer was added to the cells. After lysis for 2 minutes, the cells were collected into centrifuge tubes and centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was aspirated to obtain the protein sample. Equal amounts of protein samples were separated by electrophoresis on a 4-20% sodium dodecyl sulfate polyacrylamide gel (Bio-RAD) at 150 V constant voltage at room temperature. Proteins were transferred to a PVDF membrane (Bio-RAD) using a semi-dry transfer method. The PVDF membrane was blocked with 5% skim milk powder for 1 hour at room temperature. The PVDF membrane was incubated with the primary antibody (i.e., the first antibody for immunoblotting) overnight at 4°C and then incubated with a horseradish peroxidase secondary antibody (ABclonal) for 1 hour at room temperature. Chemiluminescent signals were detected and analyzed using a Gel-Doc XR Imaging Laboratory System (Bio-RAD).

[0139] 5.2.2 Cell death assay

[0140] Divided into 5 groups, the specific operations are as follows:

[0141] Negative control group: complete DMEM medium 100 μl / well, 4 replicates per 96-well plate;

[0142] Cell death group: 10 ng / ml TNF-α and 1 μg / ml actinomycin D were added, with four replicates per 96-well plate;

[0143] hnRNP-U knockdown cell death group: cells were transfected with siRNA for 48 hours to knock down hnRNP-U expression, and 10 ng / ml TNF-α and 1 μg / ml actinomycin D were added, with four replicates per 96-well plate;

[0144] Drug group: lefamulin (dissolved in DMSO, DMSO concentration ≤ 0.1%), 10 ng / ml TNF-α, and 1 μg / ml actinomycin D were added at a concentration of 50 μM, with 4 replicates for each concentration;

[0145] hnRNP-U knockdown drug group: cells were transfected with siRNA for 48 hours to knock down hnRNP-U expression, and lefamulin (dissolved in DMSO, DMSO concentration ≤ 0.1%), 10 ng / ml TNF-α, and 1 μg / ml actinomycin D were added at a concentration of 50 μM, with four replicate wells for each concentration.

[0146] The cell death detection method is the same as that in Example 3.

[0147] 5.3 Experimental Results

[0148] As shown in Figure 4A, in the treatment group, lefamulin significantly inhibited hnRNP-U expression in HaCaT cells at both 12 and 24 hours. As shown in Figure 4B, knockdown of hnRNP-U using specific siRNA in the L929 cell death model revealed that knockdown of hnRNP-U weakened the ability of lefamulin to inhibit the TNF-α pathway.

[0149] As shown in Figures 4A and 4B, the results of Example 5 indicate that lefamulin can directly inhibit the expression of hnRNP-U in vitro. Therefore, lefamulin may inhibit TNF-α through hnRNP-U, ultimately achieving a therapeutic effect on psoriasis.

[0150] Example 6hnRNP-U is associated with psoriasis disease activity

[0151] 6.1 Experimental Animals, Psoriasis Lesions, and Reagents

[0152] 7-8 week old male C57 mice were provided by Jicui Yaokang Animal Biotechnology Laboratory Center, with the certificate number: SCXK(Su)2018-0008.

[0153] The patient's skin lesions were provided by the Dermatology Hospital of the Chinese Academy of Medical Sciences, and the experiment was approved by the Ethics Committee of the Dermatology Hospital of the Chinese Academy of Medical Sciences.

[0154] IMQ cream (purchased from Sichuan Mingxin Pharmaceutical Co., Ltd.), hnRNP-U antibody (purchased from Abcam).

[0155] 6.2 Experimental methods

[0156] 6.2.1 Animal experiments

[0157] Six male C57 mice aged 7-8 weeks were randomly divided into two groups, with 6 mice in each group. The backs of the mice were shaved (area: 2 cm × 3 cm) and the mice were raised normally. Three days later, different treatments were given to the shaved areas of the mice. The specific procedures were as follows:

[0158] Blank control group (Control): 62.5 mg of Vaseline was applied to the back skin of mice per day for 5 days.

[0159] Model group (IMQ): IMQ 62.5 mg / mouse / day was applied to the back skin of mice for 5 days;

[0160] Mice in each group were fed and watered normally. Skin manifestations were observed daily. On the sixth day, mice were sacrificed, and skin lesions were paraffin-embedded and stained with hematoxylin and eosin. 5-μm-thick paraffin sections were then cut for hnRNP-U immunohistochemical staining using the primary antibody (Abcam, ab180952, 1:100 dilution). Images were taken at 4X magnification, and the number of hnRNP-U-positive cells was counted at 40X magnification using Image J software. The average number of hnRNP-U-positive cells in three random fields of view per mouse was calculated.

[0161] 6.2.2 Human Tissue Specimen Experiments

[0162] Human psoriatic lesions were surgically harvested, embedded in paraffin, and stained with hematoxylin and eosin. 5 μm-thick paraffin sections were then cut and stained with hnRNP-U immunohistochemistry using the primary antibody (Abcam, ab180952, 1:100 dilution).

[0163] 6.3 Experimental Results

[0164] As shown in Figures 5A to 5C, in the mouse experiments, hnRNP-U immunohistochemical staining was significantly enhanced in the skin modeling area compared with the blank control group. In the human tissue specimen experiments, hnRNP-U expression was significantly higher in the lesional area than in the non-lesional area.

[0165] Thus, the experiment in Example 6 shows that hnRNP-U is related to the disease activity of psoriasis. Therefore, lefamulin can inhibit TNF-α through hnRNP-U, ultimately achieving a therapeutic effect on psoriasis.

[0166] As can be seen, the present invention first used imiquimod to establish a psoriasis model in mice, resulting in psoriatic dermatitis on the dorsal skin of the mice. The psoriatic dermatitis was then treated with 5% lefamulin and retapamulin ointments, and it was found that both drugs could treat imiquimod-induced psoriatic dermatitis. Furthermore, the present invention used TNF-α to establish a cell death model in the L929 cell line. Lefamulin and retapamulin inhibited TNF-α-induced L929 cell death in a dose-dependent manner. Furthermore, the present invention added a photocrosslinking group to lefamulin, and chemical proteomics screening revealed that hnRNP-U interacts with lefamulin. Furthermore, in L929 cells, hnRNP-U was knocked down using hnRNP-U-specific siRNA. It was found that knockdown of hnRNP-U reduced lefamulin's ability to inhibit TNF-α-induced L929 cell death. In HaCaT cells, co-incubation with lefamulin revealed decreased hnRNP-U expression in HaCaT cells. These experiments demonstrate that lefamulin can inhibit the TNF-α pathway through hnRNP-U. No inhibitors or agonists targeting hnRNP-U have been reported in the prior art. Finally, hnRNP-U expression in psoriatic-like lesions in mice and in psoriasis patients was found to correlate with disease severity, suggesting that lefamulin can treat psoriasis by inhibiting hnRNP-U.

[0167] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of a compound in the preparation of a drug for treating psoriasis, characterized in that: The compound is lefamulin, retapamulin or a derivative thereof, Wherein, the structural formula of lefamulin is as follows: The structural formula of retapamulin is shown below:

2. The use according to claim 1, characterized in that The derivatives are in the form of salts and / or solvates.

3. The use according to claim 1, characterized in that The derivatives of lefamulin are in the form of lefamulin acetate and lefamulin hydrochloride.

4. The use according to claim 1, characterized in that The psoriasis is psoriasis vulgaris.

5. The use according to claim 1, characterized in that The medicine contains therapeutically effective amounts of lefamulin, retapamulin and a pharmaceutically acceptable carrier.

6. The use according to claim 5, characterized in that The therapeutically effective amount is 1% to 10%.

7. The use according to claim 5, characterized in that The pharmaceutically acceptable carrier includes one or more of a diluent, a solubilizer, a latent solvent, a disintegrant, a dispersant, a lubricant, a flavoring agent, an antioxidant, a binder, an absorbent, a wetting agent, a buffer and a cross-linking agent.

8. The use according to claim 1, characterized in that The drug is prepared into a pharmaceutically acceptable dosage form, wherein the dosage form includes pills, tablets, powders, capsules, granules, powders, pellets, drops, patches, tinctures, pastes, lotions, sprays, injections, suspensions, creams, frosts, ointments, gels or suppositories.

9. An ointment for treating psoriasis, wherein the ointment comprises lefamulin or retapamulin, in, The structural formula of lefamulin is shown below: The structural formula of retapamulin is shown below:

10. The ointment according to claim 9, wherein the composition of the ointment is as follows:

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