Application of paeonoside b4 derivative in preparation of a drug for treating atopic dermatitis

Abstract

This invention belongs to the field of biomedical technology and relates to a drug for treating dermatitis, specifically the application of Pulsatilla chinensis saponin B4 derivatives in the preparation of drugs for treating atopic dermatitis. This invention discloses for the first time the use of this Pulsatilla chinensis saponin B4 derivative A3-6 in the treatment of atopic dermatitis, achieving significant technological progress compared to other structurally similar Pulsatilla chinensis saponin B4 derivatives, demonstrating inventiveness. The Pulsatilla chinensis saponin derivative group has an immunomodulatory effect and does not produce the severe immunosuppression seen with dexamethasone; therefore, it can alleviate the symptoms of atopic dermatitis through immunomodulation. Crucially, compared to the model group, the Pulsatilla chinensis saponin B4 derivative not only did not reduce body weight but also showed a trend of weight recovery from day 4, indicating that the Pulsatilla chinensis saponin B4 derivative has a certain restorative effect on weight loss caused by atopic dermatitis, effectively solving the problem of rapid weight loss in mice caused by existing dexamethasone treatments.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a drug for treating dermatitis, specifically the application of Pulsatilla saponin B4 derivative in the preparation of a drug for treating atopic dermatitis. Background Technology

[0002] Atopic dermatitis (AD) is a common chronic, inflammatory, systemic skin disease clinically characterized by dry skin, persistent itching, and chronic eczematous lesions. It is often accompanied by other atopic diseases such as allergic rhinitis and asthma, severely impacting patients' quality of life. There are many pathogenic factors for atopic dermatitis, primarily genetic and environmental factors. Genetic factors are the strongest risk factor for AD; numerous studies show that children of parents with AD have a higher incidence rate. Related pathogenic genes mainly fall into three categories: genes related to the skin barrier, innate immune response, and specific immune response. Mutations in the filaggrin gene, which involve loss of function, were initially considered a major risk factor. Other gene mutations, such as those involving the fusion of proteins from the S-100 family (interkeratin filaggrin-2, keratin capsule precursor SPRR3), transmembrane protein 79 (TMEM79), and serine protease inhibitors (SPINK5), also play important roles in the pathogenesis of AD. Gene mutations can lead to the loss of proteins that make up the epidermal structure, causing epidermal barrier defects. Environmental factors, including air pollution, passive smoking, and environmental allergens such as house dust mites and pollen, all increase the prevalence of Alzheimer's disease (AD). Besides impairing skin barrier function, environmental risk factors also act as pruritogens and inflammatory agents, triggering or exacerbating dermatitis in susceptible individuals. Environmental antigens stimulate antigen-presenting cells through the damaged skin barrier, causing Th2 cells in local lymph nodes to interact with T cells / B cells, leading to excessive IgE conversion. Simultaneously, memory T cells redistribute into the bloodstream, worsening AD through T cell infiltration of the skin and spreading beyond the skin, causing other atopic diseases.

[0003] Currently, medications for treating atopic dermatitis are mainly divided into topical and oral medications. Oral medications primarily include antihistamines, immunosuppressants, modulators, and biologics. However, oral medications have obvious adverse effects on the digestive system, and their side effects are significantly greater than those of topical medications. Topical medications mainly employ topical corticosteroids, calcineurin inhibitors (CPIs), and moisturizers. CPIs (tacrolimus and pimecrolimus) can repair the skin barrier, while moisturizers reduce moisture evaporation and better protect the skin barrier; however, both of these drugs have a slow onset of action. Corticosteroids can relieve itching and reduce local inflammation and are currently the most effective clinical drugs for treating atopic dermatitis, but they have many side effects. In addition, many biologics have been marketed in recent years, but they are expensive, their efficacy is unclear, their safety is uncertain, and their application prospects remain unclear. Therefore, it is essential to develop new drugs for atopic dermatitis. Summary of the Invention

[0004] There are many existing drugs for atopic dermatitis, with topical corticosteroids being the most effective. These topical medications possess anti-inflammatory, anti-endotoxin, immunosuppressive, anti-shock, and stress-response-enhancing pharmacological effects. However, they also have side effects, easily leading to weakened constitution and poor disease resistance. This invention discloses a Pulsatilla saponin B4 derivative for the treatment of atopic dermatitis, exhibiting better technical efficacy than existing positive control drugs. In particular, it solves the problem of weight loss in mice caused by existing drugs.

[0005] The present invention adopts the following technical solution: This invention discloses the application of a Pulsatilla saponin B4 derivative in the preparation of a drug for treating atopic dermatitis.

[0006] This invention discloses a pharmaceutical composition for treating atopic dermatitis, using the above-mentioned Pulsatilla saponin B4 derivative as the active ingredient, and further comprising a pharmaceutically acceptable carrier; the active ingredient and the pharmaceutical composition are used to prepare a drug for treating atopic dermatitis.

[0007] The present invention also discloses the application of the above-mentioned Pulsatilla saponin B4 derivative or pharmaceutical composition in the preparation of immunomodulatory drugs.

[0008] This invention also discloses the application of the above-mentioned Pulsatilla saponin B4 derivative or pharmaceutical composition in the preparation of a medicament for treating atopic dermatitis and maintaining body weight. Maintaining body weight, as described in this invention, means that during the treatment of atopic dermatitis, there is no statistically significant difference in body weight between the affected individual and a healthy individual (non-affected individual).

[0009] This invention also discloses the application of the above-mentioned Pulsatilla chinensis saponin B4 derivative or pharmaceutical composition in the preparation of synergistic drugs for the treatment of atopic dermatitis. As is common knowledge, synergistic treatment refers to the combined use of Pulsatilla chinensis saponin B4 derivative or pharmaceutical composition with other drugs capable of treating atopic dermatitis to enhance the therapeutic effect of the other drugs.

[0010] This invention also discloses the application of the above-mentioned Pulsatilla saponin B4 derivative or pharmaceutical composition in the preparation of a medicament for treating atopic dermatitis and regulating spleen weight. Regulating spleen weight as described in this invention refers to reducing the spleen weight of a diseased individual to a level not lower than that of a healthy individual (non-disease-prone individual).

[0011] The present invention also discloses the application of the above-mentioned Pulsatilla saponin B4 derivative or pharmaceutical composition in the preparation of drugs for reducing ear thickness and ear weight caused by atopic dermatitis.

[0012] The present invention also discloses the application of the above-mentioned Pulsatilla saponin B4 derivative or pharmaceutical composition in the preparation of cell membrane markers that inhibit the expression of dendritic cell (DC) activation.

[0013] In this invention, the chemical structural formula of the Pulsatilla saponin B4 derivative is as follows: .

[0014] In this invention, the drug includes topical, oral, rectal, or parenteral medications. The drug is formulated into pharmaceutically permissible dosage forms, such as pills, tablets, powders, capsules, granules (powders), ointments, solutions, gels, or suppositories. Solutions include drop pills, drops, sprays, injections, and suspensions. Preferably, the drug of this invention is a topical medication, with the above-mentioned Pulsatilla chinensis saponin B4 derivative as the active ingredient. For example, a drug for treating atopic dermatitis is a solution, with the above-mentioned Pulsatilla chinensis saponin B4 derivative as the active ingredient, and water and / or an organic solvent as the solvent; preferably, the solvent is water containing ethanol and glycerin.

[0015] In this invention, a pharmaceutically acceptable carrier refers to one or more compatible solid or liquid fillers or gel substances that are pharmaceutically usable, have sufficient purity and low toxicity, and can be mixed with other components in the pharmaceutical composition and with the active ingredient of this invention without reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), cyclodextrins (such as hydroxypropyl β-cyclodextrin), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0016] The Pulsatilla chinensis saponin B4 derivative used in this invention did not show significant cytotoxicity against THP-1 macrophages, demonstrating biocompatibility. Skin sensitizers can induce the expression of cell membrane markers (CD54 and CD86) associated with dendritic cell (DC) activation; after administration of the Pulsatilla chinensis saponin derivative, the expression of both decreased, indicating a certain therapeutic effect on atopic dermatitis. The Eczema Area and Severity Index (EASI) score is one of the important indicators in atopic dermatitis; the Pulsatilla chinensis saponin derivative group (6.6 mg / kg) showed better therapeutic effects than the unmodified AB4, and even better than dexamethasone. In atopic dermatitis, ear thickness and ear weight are important indicators reflecting disease progression; the Pulsatilla chinensis saponin derivative group (6.6 mg / kg) showed better therapeutic effects than Pulsatilla chinensis saponin B4 (6.6 mg / kg) and dexamethasone. The Pulsatilla chinensis saponin derivative group (6.6 mg / kg) exhibited immunomodulatory effects without producing the severe immunosuppression seen with dexamethasone. Therefore, the Pulsatilla chinensis saponin derivative can alleviate the symptoms of atopic dermatitis through immunomodulation. Crucially, compared to the model group, the Pulsatilla chinensis saponin B4 derivative not only did not reduce body weight but also showed a tendency to increase weight from day 4 onwards. This indicates that the Pulsatilla chinensis saponin B4 derivative (6.6 mg / kg) has a certain restorative effect on the weight loss caused by atopic dermatitis, effectively addressing the problem of rapid weight loss in mice caused by existing dexamethasone treatments. Attached Figure Description

[0017] Figure 1 Effects of Pulsatilla saponin B4 derivative A3-6 on the skin of mice with atopic dermatitis (appearance image).

[0018] Figure 2 The effect of Pulsatilla saponin B4 derivative A3-6 on eczema area and severity index (EASI) score in mice with atopic dermatitis.

[0019] Figure 3 The effect of Pulsatilla saponin B4 derivative A3-6 on body weight in mice with atopic dermatitis.

[0020] Figure 4 The effect of Pulsatilla saponin B4 derivative A3-6 on ear thickness and ear weight in mice with atopic dermatitis.

[0021] Figure 5 The effects of Pulsatilla saponin B4 derivative A3-6 on organs of mice with atopic dermatitis.

[0022] Figure 6 The effect of Pulsatilla saponin B4 derivative A3-6 on the expression of dendritic cell surface markers CD54 and CD86. Detailed Implementation

[0023] The specific embodiments of the present invention will be further described in detail below with reference to examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. The Pulsatilla chinensis saponin B4 derivative used in this invention is an existing substance, referred to as Pulsatilla chinensis saponin B4 derivative A3-6. No research has been found on its use in atopic dermatitis. This invention is the first to disclose the use of this Pulsatilla chinensis saponin B4 derivative A3-6 in the treatment of atopic dermatitis, achieving significant technical progress compared to other structurally similar Pulsatilla chinensis saponin B4 derivatives, and is therefore inventive.

[0024] The modeling and experimental methods of this invention utilize conventional techniques, employing DNCB to induce atopic dermatitis; data analysis utilizes conventional statistical methods. The chemical structural formula of the Pulsatilla saponin B4 derivative A3-6 is as follows: .

[0025] Establishment of atopic dermatitis: Balb / c mice weighing approximately 20 g were acclimatized for one week and then divided into 5 groups according to their weight. The day before the experiment, the hair on a 2cm × 2cm area on the back of each mouse was removed. Except for the normal control group, the other groups were sensitized by 50 μL of 5% DNCB solution on the back on the first and second days of the experiment. The standard for successful modeling was that repeated stimulation of the back skin with DNCB solution resulted in different degrees of redness, papules, desquamation, crusting, and other symptoms. Starting from the third day, the right ear of the mice was challenged with 50 μL of 1% DNCB solution for 3 consecutive days. The left ear was given an equal volume of matrix. Increased ear thickness indicated successful modeling.

[0026] Body weight effect test: Mice were divided into 5 groups: normal control group, model group, Pulsatilla saponin B4 group (6.6 mg / kg), dexamethasone group, and A3-6 group (6.6 mg / kg). From day 1 of the experiment, the A3-6 group received A3-6 solution applied to the back and ears 4 hours before and 2 hours after modeling, twice a day for 7 consecutive days. Simultaneously, the model group received an equal volume of solvent (water containing 70% ethanol and 2% glycerol, volume percentage). The dexamethasone group received dexamethasone applied to the back and ears once a day for 7 consecutive days, 2 hours after modeling. Mice were weighed daily during the experiment, photographed on days 1, 3, 5, and 7 after modeling, and scored. On the last day of the experiment, ear thickness was measured with calipers and photographed; mice were then euthanized for tissue collection. Example 1

[0027] Effects of Pulsatilla saponin B4 derivative A3-6 on eczema area and severity index (EASI) score in atopic dermatitis mice

[0028] The Eczema Area and Severity Index (EASI) score is one of the important indicators of atopic dermatitis. On days 1, 3, 5, and 7 after modeling, the backs of mice were photographed using a digital camera. Figure 1 On the last day, two people rated the photos, and the average score was taken. The rating criteria included four aspects: erythema, papules, desquamation, and crusting. Each aspect was further divided into three degrees, expressed as a rating scale: 0 points = none, 1 point = mild, 2 points = moderate, and 3 points = severe. The defining criteria were: 0 points = none, meaning no obvious skin lesions were visible to the naked eye; 1 point = mild, meaning the skin lesions were only visible upon close inspection; 2 points = moderate, meaning the skin lesions were clearly visible; and 3 points = severe, meaning the skin lesions were severe and very obvious. Figure 2 On days 1, 3, 5, and 7 after modeling, the EASI scores of the control group increased significantly compared with the normal control group (P<0.0001). On days 3 and 5 after modeling, the EASI scores of the A3-6 group (6.6 mg / kg) of Pulsatilla saponin B4 derivative decreased significantly compared with the model group (P<0.05). However, the EASI scores of the control group, the Pulsatilla saponin B4 group (6.6 mg / kg) and the dexamethasone group, did not decrease significantly and were not significantly different from the model group. This indicates that the therapeutic effect of the A3-6 group (6.6 mg / kg) of Pulsatilla saponin derivative (compared with the normal group, ####<0.0001; compared with the model group, *p<0.05) is better than that of the AB4 group before structural modification, and the effect is also better than that of dexamethasone. Example 2

[0029] Effects of Pulsatilla saponin B4 derivative A3-6 on body weight in mice with atopic dermatitis

[0030] Experimental results show that, Figure 3After DNCB modeling, the mice showed a decreasing trend in body weight on day 2, especially after administration of dexamethasone, with a sharp drop in body weight. On day 7, there was a statistically significant difference compared with the model group (P<0.05). The groups treated with Pulsatilla saponin B4 derivative A3-6 (6.6 mg / kg) and Pulsatilla saponin B4 (6.6 mg / kg) did not show a significant decrease in body weight compared with the normal group (nor was there a statistically significant difference). In particular, there was a trend of body weight recovery from day 4. This indicates that Pulsatilla saponin B4 derivative A3-6 (6.6 mg / kg) has a certain restorative effect on the weight loss caused by atopic dermatitis. It also indicates that Pulsatilla saponin B4 derivative A3-6 (6.6 mg / kg) has a certain therapeutic effect on AD and effectively solves the problem of significant weight loss in mice caused by dexamethasone (*p<0.05 compared with the model group). Example 3

[0031] Effects of Pulsatilla saponin B4 derivative A3-6 on ear thickness and ear weight in mice with atopic dermatitis

[0032] In atopic dermatitis, ear thickness and ear weight are important indicators reflecting the progression of the disease. In this experiment, the difference between the two ears was used to reflect the degree of ear thickening. The ear thickness of the mice was measured and recorded using calipers on the last day of the experiment. The ear thickness difference was calculated as: right ear thickness - left ear thickness. Figure 4 After modeling, the ear thickness difference increased significantly, showing a statistically significant difference compared with the normal control group (P<0.0001). The ear thickness difference of Pulsatilla saponin B4 (6.6mg / kg) and dexamethasone also showed a statistically significant difference compared with the model group (P<0.0001). The ear thickness difference of Pulsatilla saponin derivative A3-6 group (6.6mg / kg) also decreased significantly, showing a statistically significant difference compared with the model group (P<0.0001). After euthanizing the mice, both ears were taken, and a hole was punched in the middle of the ear with a diameter of 7mm. The weight was measured on a balance, and the ear weight difference was: right ear weight - left ear weight. The model group showed a statistically significant difference compared to the normal control group (P<0.0001), and the dexamethasone group showed a statistically significant difference compared to the model group (P<0.05). However, the Pulsatilla saponin B4 (6.6 mg / kg) group showed no statistically significant difference compared to the model group, while the Pulsatilla saponin derivative A3-6 group (6.6 mg / kg) showed a significant statistically significant difference compared to the model group (P<0.0001). In conclusion, the Pulsatilla saponin derivative A3-6 group (6.6 mg / kg) showed a superior therapeutic effect on atopic dermatitis compared to the normal group (####<0.0001; compared to the model group (*p<0.05, ****P<0.0001)) compared to Pulsatilla saponin B4 (6.6 mg / kg) and dexamethasone. Example 4

[0033] Effects of Pulsatilla saponin B4 derivative A3-6 on organs of mice with atopic dermatitis

[0034] Atopic dermatitis is a type IV hypersensitivity reaction closely related to the immune system, and the spleen, to some extent, reflects the state of the immune system. On the last day, the mouse was weighed before sacrifice, and after sacrifice, the spleen was removed and weighed on a balance. The spleen index was calculated as spleen weight / mouse body weight * 100. Figure 5 After modeling, the spleens of mice were significantly enlarged, showing a statistically significant difference compared to the normal control group (P<0.0001). After administration, the levels of Pulsatilla saponin B4 (6.6 mg / kg) did not change significantly compared to the model group. However, the spleen weight in the Pulsatilla saponin derivative A3-6 group (6.6 mg / kg) was significantly lower than that in the model group (P<0.05). The spleen weight in the dexamethasone group was significantly reduced, even falling below that of the normal control group, showing a significant statistical difference compared to the model group (P<0.0001). In summary, the Pulsatilla saponin derivative A3-6 group (6.6 mg / kg) has an immunomodulatory effect without producing the severe immunosuppression seen with dexamethasone. Therefore, Pulsatilla saponin derivative A3-6 can alleviate the symptoms of atopic dermatitis through immunomodulation. (Compared to the normal group, ####<0.0001; compared to the model group, *p<0.05, ****P<0.0001). Example 5

[0035] Effects of Pulsatilla saponin B4 derivative A3-6 on the expression of dendritic cell surface markers CD54 and CD86

[0036] Dendritic cells (DCs) are the most important antigen-presenting cells (APCs), playing a crucial role in connecting innate and adaptive immunity. Dendritic cells are activated by microbial stimulation through their pattern recognition receptors, and are then programmed to direct different CD4+ receptors. + T cell fate was determined by skin sensitizers, which induce the expression of cell membrane markers (CD54 and CD86) associated with dendritic cell (DC) activation. These surface molecules are typical markers of DC activation and play a crucial role in the initiation of T cells and activation of the immune response by dendritic cells. THP-1 cells were stimulated using the hapten DNCB, with 8 × 10⁸ cells per well. 5 Cell suspension of / ml was seeded into 12-well plates. One hour prior to incubation, cells were injected with A3-6 (10µM), followed by stimulation with DNCB (20µM) for 24 hours. Cells harvested at 800rpm for 5min were added to each well with 100µl of PBS (CD54:CD86 = 100:1:4) and incubated at 4°C in the dark for 30min. After washing once with PBS, the expression of the DC surface markers CD54 and CD86 could be directly measured using flow cytometry. Figure 6 By calculating the relative fluorescence intensity, it was found that the expression of both markers increased after cells were stimulated with DNCB, and the expression of both decreased after administration of the Pulsatilla saponin derivative A3-6. This indicates that it has a certain therapeutic effect on allergic diseases such as atopic dermatitis (compared with the normal group, ###<0.0001; compared with the model group, *p<0.05).

[0037] There are many existing drugs for atopic dermatitis, with topical corticosteroids being the most effective, possessing pharmacological effects such as anti-inflammatory, anti-endotoxin, immunosuppressive, anti-shock, and stress-increasing properties. However, they also have some side effects, easily leading to physical weakness and poor disease resistance. This invention discloses a Pulsatilla saponin B4 derivative, which is used to treat atopic dermatitis and has better technical effects than existing positive control drugs. In particular, it solves the problem of weight loss in mice caused by existing drugs. Furthermore, among the Pulsatilla saponin B4 derivatives previously disclosed by the applicant, compounds with similar structures or properties to the Pulsatilla saponin B4 derivative A3-6 of this invention are less effective in treating atopic dermatitis than A3-6.

[0038] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a Pulsatilla saponin B4 derivative in the preparation of a drug for treating atopic dermatitis, characterized in that, The chemical structural formula of the Pulsatilla saponin B4 derivative is as follows: 。 2. The application according to claim 1, characterized in that, The medication can be used externally or orally.

3. The application according to claim 1, characterized in that, The drug in question is a rectal medication.

4. The application according to claim 1, characterized in that, The drug is a parenteral drug.

5. The application according to claim 1, characterized in that, The drug is a solution.

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