WO2025093003A1 - Use of compound in preparation of drug for treating psoriasis - Google Patents

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

Application of a compound in the preparation of a drug for treating psoriasis Technical Field

The present invention belongs to the field of medical technology and relates to the application of a compound in the preparation of a drug for treating chronic inflammatory skin diseases, and in particular to the application of a compound in the preparation of a drug for treating psoriasis.

Background Art

Psoriasis is a common clinical chronic inflammatory and proliferative skin disease with erythematous scaling and epidermal thickening as the main clinical manifestations. It can affect multiple tissues such as the patient's skin, mucous membranes and joints. Psoriasis has a long course, is difficult to cure, and is prone to recurrence. Most people suffer from it for life. The itching and pain it causes have a serious impact on the patient's physical and mental health, and also bring a huge economic burden to the patient's family and the entire society. The cause and pathogenesis of psoriasis are complex. Genetics, infection, immunity, metabolic disorders, mental, environmental and other factors are closely related to the disease. Various factors interact with each other to form a network, which also leads to the multi-center onset of psoriasis and the complexity of psoriasis prevention and treatment.

There is currently no effective cure for psoriasis. Commonly used clinical treatment measures can be divided into physical therapy, local drug therapy and systemic drug therapy. Physical therapy and local drug therapy are suitable for patients with milder symptoms, while moderate and severe patients require systemic drug therapy. Traditional local treatment for psoriasis mainly uses topical corticosteroids, but long-term use of topical corticosteroids may cause a series of adverse reactions such as skin atrophy and hirsutism.

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 many inherent defects, such as high price, inability to be taken orally or topically, and serious side effects caused by long-term use.

Therefore, the development of drugs for psoriasis is one of the major issues that need to be solved urgently in current experimental research and clinical practice. Studies in recent years have shown that small molecule drugs have many advantages such as low cost and flexible administration methods. Therefore, finding and developing small molecule drugs for psoriasis is a hot topic in current research.

Retapamulin has a molecular formula of C ₃₀ H ₄₆ NO ₄ S and a structural formula as shown in formula (I).

It is a local 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.

Lefamulin has a molecular formula of C ₂₈ H ₄₅ NO ₅ S and a structural formula as shown in formula (II).

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.

Summary of the invention

In view of 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 treating psoriasis. Such compounds are lefamulin or retapamulin or their derivatives. Such compounds are non-hormonal compounds and can be administered externally, which significantly reduces side effects and has less impact on the whole body, and can be better used in drugs for the treatment of psoriasis.

The present invention mainly found that lefamulin and retapamulin have a significant improvement effect on the mouse psoriasis-like model induced by imiquimod. The results of immunoblotting experiments indicate that lefamulin and retapamulin have the ability to antagonize TNF-α and its pathway effects, so it can be used in the treatment of psoriasis.

The present invention uses imiquimod (IMQ) to model psoriasis in mice, psoriasis-like dermatitis appears on the back skin of mice, and retapamulin and lefamulin topical medicines are prepared, and lefamulin and retapamulin topical medicines are used to treat psoriasis-like dermatitis, and it is found that both drugs can treat imiquimod-induced psoriasis-like dermatitis. In addition, the present invention uses TNF-α to model cell death in L929 cell line, and then conducts experiments. The experimental results show that the administration of retapamulin and lefamulin by the present invention 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 action 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. The results of immunoblotting suggested that lefamulin could inhibit the expression of hnRNP-U in HaCaT cells. In immunohistochemistry, hnRNP-U was found to be highly expressed in psoriatic-like lesions and psoriasis lesions in mice, indicating that hnRNP-U was associated with the activity of psoriasis. The above results showed that retapamulin and lefamulin could inhibit the TNF-α pathway through hnRNP-U and thus improve the symptoms of IMQ-induced psoriasis. Moreover, the small molecule can be administered externally, so the side effects are relatively small and the impact on the whole body is also small. Therefore, it can be used in the treatment of psoriasis.

To achieve the above objectives, the present invention provides the following technical solutions: On the one hand, the present invention provides an application of a compound in the preparation of a drug for treating psoriasis, wherein the compound is Retapamulin or Lefamulin or a derivative thereof.

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

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

In some embodiments, the psoriasis is psoriasis vulgaris.

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

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%~5%, 3%~5%, 4%~5%, 1%~4%, 2%~4%, 3%~4%, 1%~3%, 2%~3%.

In some embodiments, the pharmaceutically acceptable carrier includes one or more of a diluent, a solubilizer, a co-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.

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

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

In some embodiments, the drug is a topical drug.

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%.

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.

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

Wherein, the structural formula of lefamulin is as follows:

The structural formula of retapamulin is shown below:

In some embodiments, the ointment is composed of:

In some embodiments, in the ointment of the present invention, the amount of lefamulin or retapamulin is 0.25 g to 0.75 g, specifically, the amount of lefamulin or retapamulin is 0.25 g to 0.75 g, 0.25 g to 0.70 g, 0.25 g to 0.65 g, 0.25 g to 0.60 g, 0.25 g to 0.55 g, 0.25 g to 0.50 g, 0.25 g to 0.45 g , 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.

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.

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.

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

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,

Wherein, the structural formula of lefamulin is as follows:

The structural formula of retapamulin is shown below:

Unless otherwise indicated, the term "therapeutically effective amount" as used herein is the amount of a drug required to produce an effective effect; "therapeutically effective amount" is adjustable and variable, and can be determined by the attending physician, taking into account factors such as the specific circumstances of the subject being treated, the duration of treatment, the severity of the disease, and reasonable medical judgment, and can be determined mathematically based on the results of animal experiments to determine the therapeutically effective amount of the drug to be administered to humans. The therapeutically effective amount of the drug to be administered for treatment can be easily determined by those skilled in the art, and the optimal dose will vary with the specific compound used, the mode of administration, the formulation specifications, and the progression of disease symptoms. In addition, due to factors related to the specific subject to be treated, including the subject's age, weight, diet, and administration time, the dose needs to be adjusted to an appropriate treatment level.

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. They can be better used in the treatment of psoriasis; (2) The present invention The invention has discovered new medicinal value for the known small molecule compounds retapamulin and lefamulin, and used them to treat psoriasis, thus opening up a new application field for the application of retapamulin and lefamulin; (3) the pharmacological effects of retapamulin and lefamulin of the invention are strong, and cell death modeling is performed in L929 cell line using TNF-α. The L929 cell death effect induced by TNF-α is inhibited by lefamulin and retapamulin, and the inhibition efficiency is dose-dependent; (4) photo-crosslinking groups are added to lefamulin, Through chemical proteomics screening, it was found that hnRNP-U can interact with lefamulin. In HaCaT cells, the expression of hnRNP-U in HaCaT cells was decreased after co-incubation with lefamulin, indicating that lefamulin can inhibit the TNF-α pathway through hnRNP-U. (5) In psoriasis-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

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

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

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

FIG3A is a molecular structure of lefamulin with added photo-crosslinking groups (lef-probe) in Example 4;

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

FIG3C is a schematic diagram of the grouping state of the proteomic screening of lefamulin protein substrates in Example 4;

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

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

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

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

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

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

FIG5C is an observation image of hnRNP-U immunohistochemical staining of skin lesions of patients with psoriasis.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

The beneficial effects of the drug of the present invention are further described below through test examples, which include the efficacy test of the present invention.

Example 1 Preparation of Lefamulin Ointment and Retapamulin Ointment

Example 1A Preparation of Lefamulin Ointment

1A) Prepare the ingredients according to the following formula:

2A) Preparation of Lefamulin Ointment

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.

Example 1B Preparation of Retapamulin Ointment

1B) Prepare the ingredients according to the following formula:

2B) Preparation of Retapamulin Ointment

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 and stir to melt. Stir until solidified to obtain retapamulin ointment.

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

2A.1 Test materials

2A.1.1 Experimental animals and reagents

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.

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

2A.1.2 Experimental Methods

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 they were raised normally. Three days later, different treatments were given to the shaved areas of the mice. The specific operations were as follows:

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

Treatment group (IMQ+5% Lefamulin): 62.5 mg of IMQ/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.

The mice in the above groups were fed and drank normally, and the skin manifestations of the mice were observed every day. On the 6th day, the mice were killed and the skin lesions were taken for (H&E) staining.

2A.1.3 Experimental Results

The changes in the back skin of each group of mice on the 6th day and the HE staining of the skin lesions (4X) are shown in Figure 1A.

As shown in Figure 1A, compared with the blank control group, obvious erythema and scaling were observed on the back skin of mice in the modeling group (IMQ); HE staining showed obvious hyperkeratosis and parakeratosis of the epidermis, and a large number of inflammatory cells infiltrated in the superficial dermis. The treatment group (IMQ + 5% Lefamulin) showed that erythema and scaling were significantly alleviated after topical drug treatment.

Example 2B Effect of topical administration of retapamulin on IMQ-induced psoriasis model in mice

2B.1. Test materials

2B.1.1 Experimental animals and reagents

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.

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

2B.1.2 Experimental methods

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 they were raised normally. Three days later, different treatments were given to the shaved areas of the mice. The specific operations were as follows:

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

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.

The mice in the above groups were fed and drank normally, and the skin manifestations of the mice were observed every day. On the 6th day, the mice were killed and the skin lesions were taken for (H&E) staining.

2B.1.3 Experimental Results

The changes in the back skin and skin lesions of each group of mice on the 6th day were analyzed by HE staining (4X). The results are shown in FIG1B .

As shown in Figure 1B, compared with the blank control group, obvious erythema and scaling were observed on the back skin of mice in the modeling group (IMQ); HE staining showed obvious hyperkeratosis and parakeratosis of the epidermis, and a large number of inflammatory cells infiltrated in the superficial dermis. The treatment group (IMQ + 5% Retapamulin) showed that erythema and scaling were significantly alleviated after topical drug treatment.

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

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

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

3A.1 Test Materials

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

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; CCK8 cell death detection kit was purchased from MCE.

3A.2 Experimental methods

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

Negative control group: complete DMEM medium 100 μl/well, 4 replicate wells per 96-well plate;

Cell death group: 10 ng/ml TNF-α and 1 μg/ml actinomycin D were added, with 4 replicates per 96-well plate;

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 4 replicate wells for each concentration;

The 96-well plate was placed in a 37°C, 5% carbon dioxide incubator for 7 hours, and 10 μl/well of CCK-8 reagent was added to the 96-well plate and incubated in a 37°C, 5% carbon dioxide incubator for 2 hours. The optical density of each well was detected by a microplate reader at a wavelength of 450 nm, and the background OD value (complete culture medium plus CCK8 detection solution, no cells) was subtracted from the OD value of each test well to calculate the mean and standard deviation, and the calculated value = (drug group/cell death group)/(negative control/cell death group).

3A.3 Experimental Results

After L929 cells were treated with 10 ng/ml TNF-α and 1 μg/ml actinomycin D for 7 hours, the cell viability was detected by CCK-8 method, and about 90% of L929 cells showed cell death. In the drug group, different concentrations of lefamulin could significantly inhibit TNF-α-induced L929 cell death, and the P value was calculated by two-tailed Student's t test (as shown in Figure 2A).

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

3B.1. Test materials

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

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; CCK8 cell death detection kit was purchased from MCE.

3B.2 Experimental methods

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

Negative control group: complete DMEM medium 100 μl/well, 4 replicate wells per 96-well plate;

Cell death group: 10 ng/ml TNF-α and 1 μg/ml actinomycin D were added, with 4 replicates per 96-well plate;

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 4 replicate wells for each concentration;

The 96-well plate was placed in a 37°C, 5% carbon dioxide incubator for 7 hours, and 10 μl/well of CCK-8 reagent was added to the 96-well plate and incubated in a 37°C, 5% carbon dioxide incubator for 2 hours. The optical density of each well was detected by a microplate reader at a wavelength of 450 nm, and the background OD value (complete culture medium plus CCK8 detection solution, no cells) was subtracted from the OD value of each test well to calculate the mean and standard deviation, and the calculated value = (drug group/cell death group)/(negative control/cell death group).

3B.3 Experimental results

After L929 cells were treated with 10 ng/ml TNF-α and 1 μg/ml actinomycin D for 7 hours, the cell viability was detected by CCK-8 method, and about 90% of L929 cells showed cell death. In the drug group, different concentrations of retapamulin could significantly inhibit TNF-α-induced L929 cell death, and the P value was calculated by two-tailed Student's t test (as shown in Figure 2B).

It can be seen that the results of Example 3 show that in in vitro experiments, lefamulin or retapamulin can directly inhibit TNF-α-induced L929 cell death, so lefamulin or retapamulin can inhibit TNF-α and ultimately achieve a therapeutic effect on psoriasis.

Example 4 Using chemical proteomics to find that lefamulin interacts with hnRNP-U

4.1 Experimental materials and methods

The human epidermal immortalized cell HaCaT cell line and mouse fibroblast L929 cell line in the logarithmic growth phase were used for the experiment, and a photo-crosslinking group (Lef-probe) was added to lefamulin. The molecular structure of the photo-crosslinking group (lef-probe) added to lefamulin is shown in FIG3A .

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

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 of combinations (as shown in Figure 3C): ① UV irradiation and non-UV irradiation groups (calculation of UV enrichment intensity), ② UV irradiation, with lefamulin and without lefamulin groups (calculation of lefamulin competition intensity);

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

3) Use streptavidin coupling to enrich the probe-target protein-biotin complex;

4) Labeling of target proteins using tandem mass spectrometry;

5) Liquid chromatography tandem mass spectrometry analysis.

4.2 Experimental Results

As shown in Figure 3B, Lef-probe can inhibit TNF-α-induced cell death, indicating that the photocrosslinking group does not affect the biological function of lefamulin. In the chemical proteomics experiment, after three independent experiments were repeated in two cell lines (as shown in Figure 3D, 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 Figure 3E, which is a ratio diagram of proteins interacting with lefamulin identified in L929 and HaCaT cells, in which the black spot with the largest area is hnRNP-U. In Figure 3E, the Y axis is the UV enrichment ratio (Ruv = UV irradiation mass spectrum abundance value/no 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). Abundance value, Rcomp＞1.1 is positive), the applicant found that lefamulin is most likely to interact with heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), which shows that

The results of Example 4 show that lefamulin interacts with hnRNP-U in in vitro experiments.

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

5.1 Test materials

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

Famolin 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. The 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.

5.2 Experimental methods

5.2.1 Immunoblotting

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

Blank control group (Control): Protein extracted from HaCaT cell line, i.e., protein extracted from HaCaT cells using RIPA lysis buffer was used as blank control;

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

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

At room temperature, 100 μl of RIPA lysis buffer was added to the cells. After lysis for 2 minutes, the liquid was collected into a centrifuge tube and centrifuged at 10,000 rpm for 10 minutes in a high-speed centrifuge at 4°C. The supernatant was aspirated to obtain the protein sample. Equal amounts of protein samples were separated by electrophoresis using a constant voltage method of 150 V and 4-20% sodium dodecyl sulfate polyacrylamide gel (Bio-RAD) at room temperature; the protein was 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) at 4°C overnight, and then incubated with the horseradish peroxidase secondary antibody (ABclonal) for 1 hour at room temperature. The chemiluminescent signal was detected and analyzed using the Gel-Doc XR imaging laboratory system (Bio-RAD).

5.2.2 Cell death assay

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

Negative control group: complete DMEM medium 100 μl/well, 4 replicate wells per 96-well plate;

Cell death group: 10 ng/ml TNF-α and 1 μg/ml actinomycin D were added, with 4 replicates per 96-well plate;

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 4 replicates per 96-well plate;

Drug group: 50 μM lefamulin (dissolved in DMSO, DMSO concentration ≤ 0.1%), 10 ng/ml TNF-α and 1 μg/ml actinomycin D were added, with 4 replicate wells for each concentration;

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 4 replicates for each concentration;

The cell death detection method is the same as in Example 3.

5.3 Experimental Results

As shown in Figure 4A, in the drug-treated group, lefamulin could significantly inhibit the expression of hnRNP-U in HaCaT cells at both 12 hours and 24 hours. As shown in Figure 4B, using specific siRNA to knock down hnRNP-U, it was found that knocking down hnRNP-U led to a weakened function of lefamulin in inhibiting the TNF-α pathway in the L929 cell death model.

As shown in Figures 4A and 4B, the results of Example 5 show that lefamulin can directly inhibit the expression of hnRNP-U in in vitro experiments, so lefamulin may inhibit TNF-α through hnRNP-U and ultimately achieve a therapeutic effect on psoriasis.

Example 6hnRNP-U is associated with psoriasis disease activity

6.1 Experimental animals, psoriasis lesions, and reagents

C57 mice, male, 7-8 weeks old, were provided by Jicui Yaokang Animal Biotechnology Experimental Center, certificate number: SCXK(Su)2018-0008.

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

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

6.2 Experimental methods

6.2.1 Animal experiments

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: 2cm×3cm) and they were raised normally. Three days later, different treatments were given to the shaved areas of the mice. The specific operations were as follows:

Blank control group (Control): 62.5 mg of vaseline was applied to the back skin of mice per day for 5 days;

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

The mice in the above groups were fed and watered normally, and the skin manifestations of the mice were observed every day. The mice were killed on the 6th day and the skin lesions of the mice were embedded in paraffin and stained with HE. Paraffin sections with a thickness of 5 μm were cut for hnRNP-U immunohistochemical staining, and the primary antibody was used (Abcam, ab180952, 1:100 dilution). Pictures were taken under a 4X field of view, and the number of hnRNP-U positive cells was counted under a 40X field of view using the software Image J, and the average value of hnRNP-U positive cells in 3 random fields of view for each mouse was calculated.

6.2.2 Experiments with human tissue specimens

Human psoriasis lesions were obtained surgically, embedded in paraffin and stained with HE. 5 μm thick paraffin sections were cut for hnRNP-U immunohistochemical staining, and the primary antibody used was (Abcam, ab180952, 1:100 dilution).

6.3 Experimental Results

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

It can be seen that the experiment in Example 6 shows that hnRNP-U is related to the disease activity of psoriasis, so lefamulin can inhibit TNF-α through hnRNP-U and ultimately achieve a therapeutic effect on psoriasis.

It can be seen that the present invention firstly models psoriasis in mice by using imiquimod, and psoriatic dermatitis appears on the back skin of mice. Psoriatic dermatitis is treated by using 5% lefamulin and retapamulin ointment, and it can be found that both drugs can treat imiquimod-induced psoriatic dermatitis. In addition, the present invention uses TNF-α to model cell death in L929 cell line, and the cell death effect of L929 induced by TNF-α is inhibited by lefamulin and retapamulin, and the inhibition efficiency is dose-dependent. In addition, the present invention adds a photocrosslinking group to lefamulin, and through chemical proteomics screening, it is found that hnRNP-U can interact with lefamulin. On this basis, in L929 cells, hnRNP-U in cells is knocked down using hnRNP-U specific siRNA, and it is found that the function of lefamulin to inhibit TNF-α-induced L929 cell death is weakened after knocking down hnRNP-U. In HaCaT cells, lefamulin is used for co-incubation, and it is found that the expression of hnRNP-U in HaCaT cells decreases. The above experiments show that lefamulin can inhibit the TNF-α pathway through hnRNP-U, but there is no report on the prior art. Finally, in the psoriatic-like lesions of mice and the lesions of psoriasis patients, it was found that the expression of hnRNP-U was correlated with the severity of the disease, which suggests that lefamulin can achieve the purpose of treating psoriasis by inhibiting hnRNP-U.

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 embodiments of the present invention.

Claims (10)

A 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:
The use according to claim 1, characterized in that the derivative is in the form of a salt and/or a solvate. The use according to claim 1, characterized in that the derivative of lefamulin is in the form of lefamulin acetate or lefamulin hydrochloride. The use according to claim 1, characterized in that the psoriasis is psoriasis vulgaris. The use according to claim 1, characterized in that the drug contains a therapeutically effective amount of lefamulin, retapamulin and a pharmaceutically acceptable carrier. The use according to claim 5, characterized in that the therapeutically effective amount is 1% to 10%. The use according to claim 5, characterized in that the pharmaceutically acceptable carrier comprises 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. 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. An ointment for treating psoriasis, wherein the ointment comprises lefamulin or retapamulin, Wherein, the structural formula of lefamulin is as follows:
The structural formula of retapamulin is shown below:
The ointment according to claim 9, wherein the composition of the ointment is as follows: