Application of piperazine amide derivative in preparation of medicine for treating psoriasis

Abstract

The invention discloses an application of a piperazine amide derivative in preparation of a medicine for treating psoriasis, and belongs to the field of biological medicine, the piperazine amide derivative has the following structure: # imgabs0 # in a mouse psoriasis model induced by imiquimod cream, the scale, erythema and skin thickness can be obviously reduced by oral administration and local application of ID230301A-1, and the application of the ID230301A-1 in the mouse psoriasis model induced by imiquimod cream can be promoted. The hyperplasia of epithelial spine cells can be inhibited. Immunoblotting analysis is carried out on the skin tissue of an experimental mouse, and the result shows that the ID230301A-1 can regulate and control the expression of STAT3 signal related protein. Therefore, the medicine can be used as an active ingredient for regulating metabolism to prepare a medicine for treating psoriasis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of a piperazineamide derivative in the preparation of a drug for treating psoriasis. Background Art

[0002] Psoriasis is a chronic inflammatory disease of the skin that affects 2%-3% of the population worldwide. Psoriasis vulgaris is the most common clinical subtype, accounting for approximately 85% of cases, and is characterized by thick, erythematous plaques covered by silvery lamellar scales that are preferentially localized to the extensor surfaces, scalp, and trunk. Other less common clinical subtypes include pustular, maculopapular, palmoplantar, erythrodermic, and inverse psoriasis. Psoriasis can present as a relatively mild form with a limited number of lesions to a more severe form in which more than 10% of the skin surface is affected. Approximately one-third of patients will develop psoriatic arthritis. In addition, comorbidities of psoriasis include cardiometabolic disorders, stroke, metabolic syndrome, chronic kidney disease, mood disorders, and lymphoma, leading to a decrease in the patient's overall life expectancy. Therefore, psoriasis is not only a skin disease but also a systemic inflammatory disease.

[0003] Psoriasis is considered a T cell-mediated autoimmune disease influenced by genetic susceptibility and environmental stimuli such as trauma, infection, and drugs. Genome-wide association studies (GWAS) of psoriasis-related genes have shown that susceptibility to the disease involves both the adaptive and innate immune systems. The pathogenesis of psoriasis is associated with activation of dendritic cells residing in the dermis by epidermal antigens released by skin trauma or infection. Activated dendritic cells feedback upregulate the production of IFN-α and -β, TNF-α, IL-6, and IL-23. IL-6 plays a fundamental role in the differentiation of T lymphocytes into Th17 cells, which are generally considered to play a central role in the pathogenesis of psoriasis. The effects of IL-6 on naive T cells include the induction of STAT3 (Signal Transducer and Activator of Transcription 3)-dependent IL-23 receptors, which in turn is essential for conferring IL-23 responsiveness and full effector function to Th17 cells. In addition, resident γδ T cells, a population that expands in the dermis of patients with psoriasis, have also been shown to produce IL-17 and participate in the pathogenesis of the disease. Other effector cytokines include GM-CSF, TNF, IL-21, IL-22, IL-26, and IL-29. Together, these cytokines stimulate the formation and maintenance of psoriatic plaques, which are characterized by excessive proliferation and abnormal differentiation of keratinocytes, with keratinocyte nuclei retained in the uppermost differentiated epidermal layer (parakeratosis), including the expression of the specific psoriasis autoantigen Krt17 (K17) in epidermal keratinocytes, and promote the recruitment of leukocytes to the damaged skin.

[0004] Psoriasis cannot be cured and is prone to recurrence. The purpose of treatment is to control the disease, slow down or inhibit the progression of the disease, reduce clinical manifestations such as skin lesions, control related complications and improve the quality of life of patients. Lifelong treatment is required and personalized treatment plans are advocated. According to my country's 2022 Primary Diagnosis and Treatment Guidelines for Psoriasis, mild psoriasis is mainly treated with skin care products and topical medications such as emollients and glucocorticoids. Moderate patients are mainly treated with systemic treatment, including retinoids, immunosuppressants, biological agents and phototherapy, and are assisted by topical medications and skin care products. Severe patients are mainly treated with systemic treatment, including retinoids, immunosuppressants, biological agents and phototherapy, and are assisted by topical medications and skin care products. Acitretin capsules are a type of drug that belongs to the class of retinoids and are derivatives of vitamin A. However, the use of acitretin to treat psoriasis has side effects such as teratogenicity, increased blood lipids, and adverse reactions to the skin and mucous membranes, and it also has a certain impact on bones. The side effect of the immunosuppressant methotrexate (MTX) is that it can cause abnormal liver and kidney function. Clinically, for psoriasis vulgaris, including plaque psoriasis, acitretin capsules can be selected, while for severe psoriasis, methotrexate can be selected. Drugs can improve the condition faster or improve the efficacy, but adverse reactions such as skin infections should be closely observed. Combined ultraviolet light therapy can improve the efficacy, but combined ultraviolet light increases the risk of skin cancer.

[0005] Tofacitinib is an oral JAK1 and 3 inhibitor that has been approved for rheumatoid arthritis (RA) and is currently being evaluated for the treatment of plaque psoriasis and psoriatic arthritis. However, RA patients treated with Tofacitinib have an increased risk of pulmonary embolism and overall mortality. Ruxolitinib is a JAK1 and JAK1-2 inhibitor. In a Phase II clinical trial in patients with localized psoriasis (less than 20% of the body surface affected), Ruxolitinib was shown to be safe, well tolerated, and showed significant clinical activity in the topical treatment of psoriasis. However, the study was of relatively short duration and had a small sample size.

[0006] ID230301A-1 is a STAT3 protein inhibitor. A large amount of research data shows that STAT3 plays an important role in the pathogenesis of tumors and autoimmune diseases. Although there are currently no marketed drugs for STAT3 protein inhibitors, their research and development is in a boom stage. There are many clinical trial data of STAT3 protein inhibitors showing that they have great therapeutic potential in the treatment of tumors and autoimmune diseases such as psoriasis and ulcerative colitis. This application found in preclinical studies that the efficacy of ID230301A-1 for the treatment of psoriasis is better than methotrexate, and the toxic side effects of long-term use are small, showing good development and application prospects. Summary of the invention

[0007] In view of the problems of poor efficacy, serious side effects and easy recurrence of psoriasis treatment drugs in the prior art, the present invention provides a piperazinamide derivative (ID230301A-1) with known pharmacokinetic and toxicological effects, few side effects, low price and good development and application prospects for use in the preparation of psoriasis treatment drugs.

[0008] In order to achieve the above object, the present invention specifically adopts the following technical solutions:

[0009] Use of a piperazinamide derivative or a biologically acceptable salt thereof in the preparation of a drug for treating psoriasis, wherein the structure of the piperazinamide derivative is as follows:

[0010]

[0011] Furthermore, the drug is a mixture of one or more piperazinamide derivatives and biologically acceptable salts thereof in any proportion prepared into an injection, tablet, powder injection, granule, capsule, oral solution, ointment or cream.

[0012] The oral liquid is prepared by dissolving a piperazinamide derivative in polyethylene glycol to form a solution with a concentration of 1 to 20 mg / mL, and the molecular weight of the polyethylene glycol is 200 to 1000.

[0013] The ointment is prepared by dissolving a piperazinamide derivative in anhydrous ethanol, adding the mixture to melted white vaseline, stirring evenly at 50-70°C, and placing the mixture at 0-10°C until it becomes a semi-solid ointment, wherein the piperazinamide derivative accounts for 0.5-2% of the total weight of the piperazinamide derivative and white vaseline.

[0014] Furthermore, the piperazinamide derivative or a biologically acceptable salt thereof is administered alone or in combination with other pharmaceutically acceptable drugs.

[0015] Use of a piperazinamide derivative or a biologically acceptable salt thereof in the preparation of a STAT3 protein inhibitor, wherein the structure of the piperazinamide derivative is as follows:

[0016]

[0017] Furthermore, the above-mentioned biologically acceptable salt refers to a biologically acceptable salt formed by ID230301A-1 and at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, diphosphoric acid, hydrobromic acid, nitric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, succinic acid, ascorbic acid, boric acid, lactic acid, p-toluenesulfonic acid, salicylic acid and ethylenediaminetetraacetic acid.

[0018] The effects of ID230301A-1 were observed in an animal model of psoriasis, and it was found that ID230301A-1 effectively inhibited the clinical symptoms and histopathological changes of psoriasis.

[0019] The classic animal model of psoriasis is to apply 5% Imiquimod (IMQ) cream to the back skin of adult Balb / c mice (10 weeks old) for 6-8 consecutive days to induce skin lesions. This application uses this model to observe the therapeutic effect of ID230301A-1 on psoriasis. The results show that ID230301A-1 effectively inhibits the skin symptoms and pathological changes induced by Imiquimod.

[0020] Immunoblotting analysis was performed on the skin tissue of mice in the animal model to explore the molecular and signaling mechanisms of ID230301A-1 in inhibiting psoriasis. The results showed that ID230301A-1 could significantly downregulate the expression of STAT3 / IL-17 signaling-related molecules in the skin tissue of mice in the animal model.

[0021] Based on the above studies, it was found that ID230301A-1 has a good therapeutic activity against psoriasis, which is closely related to the inhibition of STAT3 signaling pathway activation by ID230301A-1. Therefore, it can be used as an active ingredient to prepare drugs for the treatment of psoriasis. In order to further improve the efficacy, ID230301A-1 can also be compounded with other active ingredients to prepare drugs for the treatment of psoriasis. When preparing these drugs, pharmaceutically acceptable carriers can also be added. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a gross morphological observation of the therapeutic effect of ID230301A-1 on the psoriasis mouse model;

[0023] Figure 2 Skin histological observation of the psoriasis mouse model treated with ID230301A-1;

[0024] Figure 3 PASI score (scaly) and statistical analysis of skin lesions in the psoriasis mouse model treated with ID230301A-1;

[0025] Figure 4 PASI score (erythema) and statistical analysis of skin lesions in the psoriasis mouse model treated with ID230301A-1;

[0026] Figure 5 PASI score (thickness) and statistical analysis of skin lesions in the psoriasis mouse model treated with ID230301A-1;

[0027] Figure 6PASI score (comprehensive score) and statistical analysis of skin lesions in the psoriasis mouse model treated with ID230301A-1;

[0028] Figure 7 These are the results of immunoblotting of skin tissues of mice in each group in the psoriasis model. DETAILED DESCRIPTION

[0029] In order to make the technical purpose, technical scheme and beneficial effects of the present invention clearer, the technical scheme of the present invention is further explained below in conjunction with specific embodiments. However, the implementation is intended to explain the present invention and cannot be understood as limiting the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in the field or the product manual shall be followed.

[0030] The preparation of ID230301A-1 is detailed in patent application CN116715657A.

[0031] Example 1. Preparation process of methotrexate solution, ID230301A-1 solution and 1% ID230301A-1 smear ointment

[0032] Preparation of 0.2mg / mL methotrexate sodium salt solution

[0033]

[0034]

[0035] Preparation process: Weigh the above-prescribed amount of raw materials into a 10mL EP tube, add the prescribed amount of purified water, and ultrasonicate for 30 minutes to obtain a clear and transparent liquid.

[0036] Preparation of 8 mg / mL ID230301A-1 solution

[0037] Components 8mg / mLID230301A-1 dosage (3mL) PEG-400 3mL ID230301A-1 API 24.48mg

[0038] Preparation process: Put the prescribed amount of reagent in a 5mL EP tube, add the prescribed amount of raw material, and ultrasonicate at 60℃ for 1h to obtain a clear, transparent light yellow liquid.

[0039] 1% ID230301A-1 paste formulation

[0040] Components Dosage (1wt%) ID230301A-1 87.4mg White Vaseline 8.73250g Anhydrous ethanol 0.437ml

[0041] Preparation process: Place the prescribed amount of white vaseline in a 10mL beaker, and melt the white vaseline in a 60℃ water bath to a nearly transparent liquid. Place the prescribed amount of ID230301A-1 and anhydrous ethanol in a 1.5mL EP tube, and ultrasonicate until clarified. Then, add them dropwise into the beaker at a speed of 1000r / min. Continue stirring for 30 minutes, and place in a 4-degree refrigerator. After half an hour, a semisolid ointment can be obtained.

[0042] Example 2: Gross morphological observation of the therapeutic effect of ID230301A-1 on a mouse model of psoriasis

[0043] The experimental animals were 10-week-old female Balb / c mice purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and raised in an SPF animal laboratory. After the mice were adapted for 1 week, the backs were shaved 2-3 days in advance and randomly divided into five groups. The normal mouse blank control group (n=5): normal saline was gavaged once a day, 0.4 mL each time; white vaseline preparation was applied to the back lesion area once a day. The model control group (IMQ group, n=5)): PBS was gavaged once a day, 0.4 mL each time; 62.5 mg, 5% IMQ (5% imiquimod cream) was applied to the back of the mice for 8 consecutive days, once a day. The positive control drug group (IMQ+MTX1mg / kg group, n=5): methotrexate solution was gavaged once a day according to methotrexate 1mg / kg, and 62.5 mg, 5% imiquimod cream was applied to the back lesion area once a day. Experimental group: Intragastric administration group (IMQ+ID230301A-1 40 mg / kg group): ID230301A-1 solution was administered intragastricly at 40 mg / kg (n=5) of ID230301A-1 compound once a day, and 62.5 mg, 5% imiquimod cream was applied to the back lesions once a day. Topical administration group (IMQ+ID230301A-1 1% group): 62.5 mg, 5% imiquimod cream was applied to the back lesions once a day, and 62.5 mg of 1% (n=5) ID230301A-1 ointment was applied to the same part after an interval of 8 hours. The above groups were treated continuously for 8 days. After the mice were killed, the back skin and subcutaneous tissue were collected, half of which was placed in a pre-prepared fixative (10% paraformaldehyde) for 24 h for HE staining and immunohistochemistry, and the other half of the back skin was placed in a cryovial and frozen in a liquid nitrogen tank for later experiments such as transcriptome sequencing and protein immunoblotting.

[0044] The results are as follows Figure 1As shown, compared with the normal group, the model control group IMQ-induced skin lesions showed typical psoriasis features including erythema, scaling, thickening and scaly skin lesions. The positive control methotrexate solution and 40mg / kg and 1% ID230301A-1 treatment groups significantly inhibited these skin lesions, showing only slight callous skin and a small amount of scales.

[0045] Example 3: ID230301A-1 inhibits IMQ-induced mouse epithelial cell proliferation

[0046] The fixed mouse skin tissue was dehydrated, waxed, paraffin-embedded, sliced, and attached to a slide, and baked at 60°C for 3h. The slices were then placed in xylene I for 10min, xylene II for 10min, anhydrous ethanol I for 5min, anhydrous ethanol II for 5min, 95% alcohol for 5min, 90% alcohol for 5min, 80% alcohol for 5min, 70% alcohol for 5min, and washed with distilled water for 5min for dewaxing and hydration. The slices were stained with Harris hematoxylin for 3min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water. The slices were stained with eosin stain for 3min. The slices were placed in 95% alcohol I for 5min, 95% alcohol II for 5min, anhydrous ethanol I for 5min, anhydrous ethanol II for 5min, xylene I for 5min, and xylene II for 5min to dehydrate and make them transparent. The slices were taken out of xylene to dry slightly and sealed with neutral gum. Observation and image acquisition and analysis were performed under an Olympus pathology microscope.

[0047] Figure 2 The results showed that compared with the epidermis of the normal (blank control) group of mice, under the continuous stimulation of IMQ, the stratum corneum of the epithelium of the model control (MQ) group of mice was significantly thickened and broken, and the prickle cells (keratinocytes) proliferated and formed large papillae extending into the subcutaneous tissue. The epithelial layer of mice in the methotrexate treatment group and the ID230301A-1 treatment group was significantly thinned, approaching the epithelial thickness of the normal group of mice. This result shows that ID230301A-1 can inhibit IMQ-induced mouse epithelial cell proliferation.

[0048] Example 4: ID230301A-1 significantly reduces the PASI score of psoriasis mice

[0049] The damaged areas of the back skin of each group of mice were scored using the scoring website http: / / pasi.corti.li / . The psoriasis score was calculated based on the proportion of psoriasis lesion area, redness (0-4), thickening (0-4), and severity of scaling symptoms (0-4) in the lesion area. For details, see Figures 3 to 6 .

[0050] Figures 3 to 6The results showed that IMQ could induce severe scaling, erythema and thickening of mouse skin, and the PASI scores and comprehensive scores were very high. Methotrexate could alleviate various symptoms, and ID230301A-1 was better than methotrexate in alleviating various symptoms and PASI scores.

[0051] Example 5: Analysis of immunoblotting results of psoriasis mouse skin tissue

[0052] The frozen skin tissues of mice in each group were obtained, fully ground and homogenized, and then lysed with RIPA lysis buffer. The proteins were collected, quantified and analyzed by Western blot.

[0053] 1. Steps for determining protein concentration (BCA method):

[0054] A. Dilute protein standards (Table 2) in 1× phosphate buffered saline (PBS):

[0055] Table 2 Preparation system of protein standards.

[0056]

[0057] B. Preparation of BCA working solution: Calculate the total amount of mixed working solution of A and B required according to the number of standards and samples to be tested. Prepare the working solution at a volume ratio of 50:1 between BCA reagent A and B, vortex and mix well for later use.

[0058] C. Take 25 μL of each of the protein standard solution and the sample supernatant diluted with phosphate buffered saline (PBS) (10-fold dilution) and add them to a new 96-well plate. Then add 200 μL of the pre-prepared BCA working solution and mix thoroughly. Be sure not to blow and create bubbles, cover the 96-well plate tightly, and react in a 37°C incubator for 30 minutes.

[0059] D. Take out the 96-well plate and restore it to room temperature for 3-5 minutes. Measure the absorbance value at a wavelength of 562nm on an enzyme reader, and make a standard curve to calculate the protein content of 1μL of each sample in preparation for protein loading.

[0060] 2. Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE)

[0061] (1) Fix the gel plate and prepare 10% SDS-PAGE separation gel (Table 3).

[0062] Prepare separation gel according to Table 3 below: 10 mL

[0063] Table 3 Separation gel formula information.

[0064]

[0065]

[0066] (2) Add the mixed separation gel to two gel plates, 1.0 cm from the top, fill the gel plates with anhydrous ethanol, and let stand for 30-45 minutes.

[0067] (3) After separation and gelation, pour out the remaining anhydrous ethanol and absorb it with filter paper.

[0068] (4) Prepare 5 mL of 5% concentrated gel according to Table 4

[0069] Table 4. Stacking gel recipe information.

[0070]

[0071] (5) Slowly add the prepared concentrated gel to the gel plate to avoid generating bubbles, insert the sample comb, and let it stand for 30-45 minutes.

[0072] (6) Take out the protein sample, heat it in a 100°C water bath for 5 min, and centrifuge it at 10,000 rpm for 10 min.

[0073] (7) Fix the gel plate into the electrophoresis tank, add SDS-PAGE electrophoresis buffer, remove the sample comb, and add the processed protein samples into the sample tank in order.

[0074] (8) Electrophoresis at 80 V for 40 min.

[0075] (9) Change the voltage to 120 V and perform electrophoresis for about 1.5 h until bromophenol blue runs out of the colloid;

[0076] 3. Western-blot transfer

[0077] (1) Rinse the SDS-PAGE gel after electrophoresis in TBST buffer once, and soak the protein gel in transfer buffer.

[0078] (2) Soak a layer of sponge pad in membrane transfer buffer and clamp it onto the membrane transfer apparatus with tweezers. Place the sponge pad, three layers of filter paper, protein glue, polyvinylidene fluoride (PVDF) membrane, three layers of filter paper, and sponge pad in order, align them, clamp them and place them onto the membrane transfer apparatus. During operation, both the filter paper and sponge pad should be soaked in transfer buffer. If there are bubbles between each layer, use a glass test tube to gently roll them out.

[0079] (3) Turn on the membrane transfer apparatus and transfer the membrane at 300 mA for 75 minutes.

[0080] (4) Remove the membrane, place it in TBST buffer, and rinse it three times on a 60 rpm horizontal shaker, each time for 8 min.

[0081] (5) Block with 20 mL of 5% bovine serum albumin (BSA) blocking solution on a 60 rpm horizontal shaker at room temperature for 2 h.

[0082] (6) Incubate the cells overnight in 3 mL of antibody incubation solution with 3 μL of primary antibody (1:1000) at 4°C on a horizontal shaker at 60 rpm.

[0083] (7) Wash the PVDF membrane three times with 10 mL of TBST on a horizontal shaker at 60 rpm at room temperature, each time for 10 min.

[0084] (8) Incubate the PVDF membrane with 20 mL of antibody incubation solution containing 2 μL of secondary antibody at room temperature on a horizontal shaker at 60 rpm for 2 h.

[0085] (9) Wash the PVDF membrane three times with 10 mL of TBST on a horizontal shaker at 60 rpm at room temperature, for 10 min each time.

[0086] (10) Take 1 mL each of chemiluminescent substrate reagent solution A and solution B, mix well, and apply them on the filter membrane.

[0087] (11) Use filter paper to absorb the liquid on the membrane and develop the color using a developer.

[0088] like Figure 7 The results of western blotting showed that the protein expression level of p-STAT3 (Y705) in the skin proteins of mice in the model group was increased. After treatment with ID230301A-1, the expression level of p-STAT3 (Y705) protein was reduced. This result suggests that ID230301A-1 may inhibit the proliferation, apoptosis resistance, lipid metabolism disorder and the formation and maintenance of inflammatory signaling networks of keratinocytes induced by IMQ by inhibiting STAT3 signaling, thereby reversing psoriasis-like skin lesions.

[0089] The above results show that ID230301A-1 has a good effect in inhibiting imiquimod-induced keratinocyte proliferation and lipid metabolism disorder, and shows good application prospects in the treatment of psoriasis.

Claims

1. Use of a piperazinamide derivative or a biologically acceptable salt thereof in the preparation of a drug for treating psoriasis, characterized in that: The structure of the piperazineamide derivative is as follows: 。 2. The use according to claim 1, characterized in that: The medicine is a medicine prepared from one or a mixture of two or more piperazinamide derivatives and biologically acceptable salts thereof in any proportion into injection, tablets, powder injection, granules, capsules, oral liquid, ointment or cream.

3. The use according to claim 2, characterized in that: The oral liquid is prepared by dissolving a piperazinamide derivative in polyethylene glycol to form a solution with a concentration of 1-20 mg / mL, and the molecular weight of the polyethylene glycol is 200-1000.

4. The use according to claim 2, characterized in that: The ointment is prepared by dissolving a piperazinamide derivative in anhydrous ethanol, adding the mixture to melted white vaseline, stirring evenly at 50-70°C, and leaving the mixture at 0-10°C until it becomes a semi-solid ointment, wherein the piperazinamide derivative accounts for 0.5-2% of the total weight of the piperazinamide derivative and the white vaseline.

5. The use according to claim 1, characterized in that: The piperazinamide derivative or its biologically acceptable salt is administered alone or in combination with other pharmaceutically acceptable drugs.

6. Use of a piperazinamide derivative or a biologically acceptable salt thereof in the preparation of a STAT3 protein inhibitor, characterized in that: The structure of the piperazineamide derivative is as follows: 。 7. The use according to claim 1, 2, 5 or 6, characterized in that: The biologically acceptable salt refers to a biologically acceptable salt formed by a piperazinamide derivative and at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, diphosphoric acid, hydrobromic acid, nitric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, succinic acid, ascorbic acid, boric acid, lactic acid, p-toluenesulfonic acid, salicylic acid and ethylenediaminetetraacetic acid.

Images