Pharmaceutical composition for treating psoriasis as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a pharmaceutical composition for treating psoriasis as well as a preparation method and application of the pharmaceutical composition. The invention provides a strategy of combining the AhR activator and the TOP2A inhibitor, the psoriasis can be effectively treated, the definite synergistic treatment and relapse prevention effects of the psoriasis are shown in multiple aspects of clinical manifestations, histopathology, gene transcription to specific pathways and biological processes and the like on an animal model, the effect is superior to that of a single therapy, and the application has the advantages of being simple in preparation method, low in cost and good in application prospect. Good application prospects are realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a pharmaceutical composition for treating psoriasis and a preparation method and application thereof. BACKGROUND

[0002] Psoriasis is a chronic, recurrent, inflammatory skin disease with the core pathological features of excessive proliferation of skin keratinocytes and uncontrolled inflammatory response. Its global incidence is about 2%-3%, and has shown a gradually increasing trend in recent years. The disease not only causes appearance damage such as redness, scaling and thickening of the skin of patients, but also often accompanies discomfort symptoms such as itching and pain, seriously affecting the quality of life of patients. Some patients may also have comorbidities such as joint disease (psoriatic arthritis) and cardiovascular disease, which brings significant challenges to personal health and social medical burden.

[0003] In recent years, with the in-depth study of the pathogenesis of psoriasis, the molecular network of “inflammation-proliferation-differentiation imbalance” has been gradually clarified. Among them, the aryl hydrocarbon receptor (AhR) signaling pathway, the cell proliferation regulation mediated by topoisomerase ⅡA (TOP2A), the cytokine-cytokine receptor interaction pathway (such as IL-6, IL-1β pathway) and keratinization abnormalities have become research hotspots in the field. Based on these mechanisms, new targeted drugs have emerged, such as the AhR activator benvitimod (approved in China in 2019 for the topical treatment of psoriasis, reference: A double-blind, randomized, placebo- and positive-controlled phase III trial of 1% benvitimod cream in mild-to-moderate plaque psoriasis, Chinese Medical Journal (English Version) 2020, Vol. 133, No. 24), TOP2A inhibitors (such as amonafide, originally used for tumor treatment, and found in recent years to have inhibitory effect on overproliferating keratinocytes, reference: Research Progress of Topoisomerase Ⅱ Inhibitors in Skin Proliferative Diseases, Journal of Dermatovenereology 2021, Vol. 28, No. 4), which provide new molecular targets and drug options for the treatment of psoriasis.

[0004] Currently, the existing technologies related to AhR activators and TOP2A inhibitors for psoriasis mainly fall into two categories: AhR activator monotherapy and TOP2A inhibitor monotherapy. The AhR activator monotherapy is represented by bexarotene, which activates the local AhR pathway in the skin, regulates the balance of downstream anti-inflammatory factors (such as IL-10) and pro-inflammatory factors (such as IL-17), and promotes the normal differentiation of keratinocytes, thereby improving psoriatic lesions. This technology has entered the clinical application stage, and phase III clinical trials have shown that after 8 weeks of treatment with bexarotene cream, the PASI 75 (more than 75% improvement in skin lesions) response rate was about 50%-60%, which is better than traditional vitamin D3 derivatives (reference: same as above, Chinese Medical Journal (English Edition) 2020, Vol. 133, No. 24). In addition, related basic research (such as “Therapeutic Effects of AhR Activator Bexarotene on Imiquimod-induced Psoriasis-like Mouse Model”, Chinese Pharmacological Bulletin 2018, Vol. 34, No. 8) has confirmed that bexarotene can reduce the epidermal thickening and inflammatory infiltration of psoriasis-like skin lesions in mice, but has limited preventive effect on skin lesion recurrence. TOP2A is a key enzyme that regulates DNA replication and division in cells. In psoriatic lesions, the expression of TOP2A in keratinocytes is significantly increased, leading to excessive cell proliferation. TOP2A inhibitors (such as amonafide) inhibit TOP2A activity and block cell cycle progression, thereby inhibiting the excessive proliferation of keratinocytes. Currently, this technology is mainly in the basic research stage, and studies have shown that amonafide can inhibit the proliferation of primary keratinocytes from psoriasis patients in vitro (reference: “Effects and Mechanisms of TOP2A Inhibitor Amonafide on Psoriasis Keratinocyte Proliferation”, Chinese Journal of Dermatovenereology 2022, Vol. 36, No. 6), and in the imiquimod (IMQ)-induced psoriasis-like mouse model, the use of amonafide alone can slightly reduce the epidermal thickness, but has weak regulatory effects on inflammatory factors (such as IL-6, IL-1β), and cannot improve the splenomegaly and other systemic inflammatory responses in mice.

[0005] The prior art has the problem of limited single-drug treatment effect, and it is difficult to achieve the synergistic effect of "anti-inflammatory-anti-proliferation-prevention of recurrence". For example, when an AhR activator (such as bexarotene) is used alone, it can effectively regulate local inflammation and keratinocyte differentiation, but has weak inhibitory effect on the over-proliferation of keratinocytes, and cannot prevent psoriasis recurrence. When a TOP2A inhibitor (such as amonafide) is used alone, it can only inhibit keratinocyte proliferation, and has insufficient regulation ability on inflammatory factor pathways (such as cytokine-cytokine receptor interaction pathways), and cannot effectively reduce inflammation-related skin lesions such as skin redness and scales, and has no improvement effect on systemic inflammation such as splenomegaly in a mouse psoriasis model. Because the pathogenesis of psoriasis is the result of the synergistic effect of "uncontrolled inflammatory response", "abnormal keratinocyte proliferation", and "epidermal differentiation disorder", single-target drugs can only play a role in one pathological link - AhR activators mainly act on "inflammation regulation and differentiation regulation", and TOP2A inhibitors mainly act on "cell proliferation inhibition", and neither of them can cover multiple key links of psoriasis pathogenesis at the same time, resulting in that single-drug treatment is difficult to achieve comprehensive pathological improvement. Therefore, developing a synergistic treatment scheme that can cover multiple pathological links such as "inflammation regulation, proliferation inhibition, and differentiation regulation" at the same time has become the key direction to solve the defects of the prior art.

[0006] Therefore, the present application is proposed. SUMMARY

[0007] The purpose of the present application is to overcome the deficiencies of the prior art, and to propose a combination strategy of AhR activators and TOP2A inhibitors, which can effectively treat psoriasis and achieve synergistic technical effects.

[0008] To achieve the above-mentioned purpose, the technical solutions of the present application are as follows:

[0009] One aspect of the present application relates to a pharmaceutical composition for treating psoriasis, comprising the following active ingredients:

[0010] (a) bexarotene as an AhR activator;

[0011] (b) amonafide as a TOP2A inhibitor;

[0012] Wherein, the active ingredients (a) and (b) have synergistic effect on treating psoriasis.

[0013] In some embodiments of the present application, the pharmaceutical composition, wherein the concentration of bexarotene is 1%, and the concentration of amonafide is 0.1%.

[0014] In some embodiments of the present application, the pharmaceutical composition further comprises pharmaceutically acceptable adjuvants, including glycerin, stearic acid, triethanolamine, liquid paraffin, paraffin and propyl gallate.

[0015] In some embodiments of the present application, the pharmaceutical composition is in the form of an external preparation, preferably a cream.

[0016] Another aspect of the present application relates to a method for preparing the pharmaceutical composition, comprising the following steps:

[0017] Preparation of cream base: mix glycerin, stearic acid, triethanolamine, liquid paraffin, paraffin, propyl gallate and water, heat to dissolve, stir until condensation;

[0018] Preparation of the pharmaceutical cream: uniformly mix the binimetinobe powder and the amonafide powder with the cream base to prepare the pharmaceutical composition cream.

[0019] Still another aspect of the present application relates to the use of the pharmaceutical composition in the preparation of a medicament for treating psoriasis.

[0020] The beneficial effects of the present application are as follows:

[0021] 1. Synergistic effect in disease treatment. The combination of AhR activator and TOP2A inhibitor can more significantly alleviate the psoriasis-like skin lesions induced by imiquimod in mice (confirmed by skin lesion photos, PASI score and epidermal thickness, etc.), and more effectively improve the systemic symptoms such as splenomegaly, compared with single drug treatment.

[0022] 2. Synergistic advantage in preventing disease recurrence. The combination treatment shows better preventive effect than single drug treatment in the relapse model induced by low-dose imiquimod again, and can more effectively inhibit the recurrence of skin lesions and related abnormal proliferation of epidermal structure, and prevent the recurrence of splenomegaly.

[0023] 3. Synergistic regulatory effect at the level of gene expression regulation. Transcriptomic analysis (PCA, cluster heat map) shows that the combination of drugs can synergistically reverse the gene expression profile related to psoriasis, making it closer to the healthy state, and the regulated genes and protein interaction networks are much more than single drug treatment, indicating that the effect is not simply additive, but a new synergistic regulatory mode is generated.

[0024] 4. Synergistic effect on key psoriasis-related pathways. KEGG enrichment analysis shows that the combination of drugs can significantly synergistically regulate multiple signaling pathways, especially in the "cytokine-cytokine receptor interaction" key pathway of psoriasis, the combination of drugs can more effectively regulate the abnormal expression of IL-6, IL-1β and other pathogenic cytokines.

[0025] 5. Synergistically regulate the key biological process of keratinization. GO enrichment analysis showed that the combination of drugs can significantly synergistically regulate the "keratinization" process, and more effectively reverse the abnormal expression of keratin (KRT6b, KRT10, KRT14, KRT16), thereby playing a synergistic role in improving epidermal differentiation disorders.

[0026] In summary, the combined strategy of AhR activation and TOP2A inhibition showed a clear synergistic effect on treating psoriasis and preventing its recurrence in animal models at multiple levels from clinical manifestations, histopathology, gene transcription to specific pathways and biological processes, and the effect was better than that of single therapy. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Representative pictures of the back skin of mice in the primary psoriasis experiment;

[0028] Figure 2 PASI score graph of psoriasis-like skin lesions in mice treated with different drugs in the primary psoriasis experiment;

[0029] Figure 3 Spleen images of mice treated with different drugs in the primary psoriasis experiment;

[0030] Figure 4 Spleen index graph of mice treated with different drugs in the primary psoriasis experiment;

[0031] Figure 5 HE staining and Ki67 immunohistochemical images of the back skin of mice in each group in the primary psoriasis experiment;

[0032] Figure 6 Epidermal protrusion thickness graph of mice in each group in the primary psoriasis experiment;

[0033] Figure 7 Ki67 expression amount statistical graph of mice in each group in the primary psoriasis experiment;

[0034] Figure 8 Representative pictures of the back skin of mice in each group in the secondary psoriasis experiment;

[0035] Figure 9 PASI score graph of psoriasis-like skin lesions in mice treated with different drugs in the secondary psoriasis experiment;

[0036] Figure 10 Spleen images of mice treated with different drugs in the secondary psoriasis experiment;

[0037] Figure 11 Spleen index graph of mice treated with different drugs in the secondary psoriasis experiment;

[0038] Figure 12 Figure for epidermis thickness, epidermal ridge number and epidermal ridge length of mice treated with different drugs in the experiment of recurrent psoriasis;

[0039] Figure 13 Figure for HE staining and Ki67 immunohistochemistry of the back skin of mice in each group in the experiment of recurrent psoriasis;

[0040] Figure 14 Figure for the expression amount of Ki67 of mice in each group in the experiment of recurrent psoriasis. DETAILED DESCRIPTION

[0041] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not noted in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not noted for the manufacturer are all conventional products that can be obtained by purchase in the market.

[0042] Example

[0043] (I) Preparation of drug cream

[0044] Preparation of cream base: 80 g of glycerol, 120 g of stearic acid, 30 g of triethanolamine, 180 g of liquid paraffin, 80 g of paraffin, 1.5 g of nipagin ethyl ester and ddH2O (up to 1000 g) are heated to 85-90℃ for sufficient dissolution, and stirred until condensation.

[0045] Preparation of drug cream: according to the target drug concentration (bivertimod 1%, amnafide 0.1%), the fine drug powder is precisely weighed and uniformly mixed with the cream base, and then stored at 4℃.

[0046] (II) Pharmacological experiment

[0047] The present application prepares a psoriasis animal model by using imiquimod, and when inflammation occurs, the prepared drug composition cream is given for intervention, and the effect of the prepared cream is observed and verified by observing the relief of inflammation. The specific method is as follows:

[0048] 1. Establishment of psoriasis and recurrent psoriasis mouse model

[0049] Male BALB / c mice (7 weeks old, body weight 20 ± 2 g) were purchased from Beijing Charles River Laboratories. All animal experiments were performed in accordance with the “Helsinki Declaration” and “Ethical Standards” guidelines and were approved by the Shanxi University Research Ethics Committee (License No: SXULL2022081). The feeding conditions were room temperature (22 ± 2) ~ ℃, humidity 45 ~ 55%, natural day-night rhythm (12 h day, 12 h night), free access to standard laboratory feed and tap water.

[0050] For the primary psoriasis mouse model, the mice were adaptively fed for 1 week before the experiment. During this period, the hair on the back of the mice was removed 3 × 3 cm 2 After an additional 2 days of back exposure adaptation, 62.5 mg of imiquimod (IMQ) cream was applied to the skin on the back of the mice to induce the occurrence of psoriasis-like lesions in mice. 8 h later, 62.5 mg of drug cream was applied to the back of the mice for treatment of IMQ-induced psoriasis lesions. Modeling and treatment were performed continuously for 6 days.

[0051] For the recurrent psoriasis mouse model, the mice were first modeled and treated in the same way as the primary psoriasis, and after 6 days, modeling and treatment were stopped to allow the psoriasis lesions in the mice to recover for 14 days, and the body weight and back skin condition of the mice were recorded daily. Then, a low dose (20.8 mg) of IMQ cream was applied to the back of all mice for 6 consecutive days to induce the recurrence of psoriasis.

[0052] 2. PASI score and spleen index analysis

[0053] The psoriasis area and severity index (PASI) score was based on the severity of erythema, scaling, and thickening. After the experiment, the skin and spleen were taken. The ratio of spleen to body weight was the spleen index. Then the skin and spleen were stored at -80℃ for further analysis.

[0054] 3. HE staining experiment

[0055] The skin tissue on the back of the mice was fixed with formalin solution, paraffin-embedded, sectioned, deparaffinated, rehydrated, stained with hematoxylin for the nucleus, and stained with eosin for the cytoplasm, dehydrated with ethanol, transparentized with xylene, and mounted for preservation. Microscopy was used for observation, and image analysis was performed. Image J software was used to quantify the HE-stained sections, including changes in epidermal thickness, number and length of epidermal ridges.

[0056] 4. Immunohistochemical staining experiment

[0057] The tissues were fixed in formalin solution, paraffin-embedded, and then sectioned. After deparaffinization, the sections were rehydrated and subjected to antigen retrieval. Washed with double distilled water and incubated with permeabilization blocking solution (50 g / L BSA) for 60 min. Remove the permeabilization blocking solution and wash with TBST for 3 times. Incubate the sections with primary antibody working solution at 4°C overnight. After removing the primary antibody working solution, wash with TBST for 3 times, then add secondary antibody working solution and incubate at room temperature for 2 hours. Remove the secondary antibody working solution and wash with TBST for 3 times. Add appropriate amount of DAB solution, stain for 2-5 min, and rinse quickly with double distilled water. After dehydration with ethanol and clearing with xylene, the sections are mounted and stored. Finally, observe and take photos under a microscope for analysis.

[0058] Experimental results:

[0059] (1) AhR activation and TOP2A inhibition in combination can synergistically reduce psoriasiform skin lesions in mice

[0060] Skin photographs and PASI scores showed that the combination of benveztide and aminonafalt reduced the psoriatic skin lesions induced by imiquimod in mice, and the therapeutic effect was better than that of single use ( Figure 1 and Figure 2 ). The combination of benveztide and aminonafalt can reduce the splenomegaly induced by imiquimod in mice, while the single use has no significant effect on the spleen of mice ( Figure 3 and Figure 4 ). The results of skin HE staining showed that the combination of benveztide and aminonafalt improved the epidermal structure changes induced by imiquimod by reducing the epidermal thickness, reducing the number and length of epidermal ridges ( Figure 5 and Figure 6 ). For the reduction of epidermal thickness and the number of epidermal ridges, the combination of benveztide and aminonafalt was better than single treatment ( Figure 6 ). IHC staining of Ki67 showed that the combination of benveztide and aminonafalt reduced the up-regulation of Ki67 in the skin induced by imiquimod, and there was no significant difference between the combination and single drug treatment ( Figure 5 and Figure 7 ). Based on these results, it can be proved that the synergistic effect of AhR activation and TOP2A inhibition reduces psoriasiform lesions in mice.

[0061] (2) AhR activation and TOP2A inhibition in combination can synergistically improve the recurrence of psoriasiform skin lesions in mice Figure 8

[0062] Skin photographs and PASI scores showed that the combination of benveztide and aminonafalt significantly reduced the skin lesions induced by low-dose (20.8 mg) imiquimod in mice, and the effect was better than that of benveztide and aminonafalt alone ( Figure 9 , Figure 10). Low-dose imiquimod-induced psoriasis in mice did not cause splenomegaly after binimetinib and amonafide combination treatment, while both monotherapy groups or the no-treatment group did cause splenomegaly Figure 11 、 Figure 12 ) Low-dose imiquimod caused alterations in epidermal structure, including increased epidermal thickness, and increased number and length of epidermal ridges, which were ameliorated by binimetinib, amonafide or their combination treatment Figure 12 ) Combination treatment with binimetinib and amonafide was superior to monotherapy in terms of reduction in epidermal thickness and number of epidermal ridges Figure 13 ) IHC staining for Ki67 showed that low-dose imiquimod induced upregulation of Ki67 in the skin, which was ameliorated by binimetinib and amonafide combination treatment, but not by their monotherapy Figure 14 、 ​ ) In summary, combination treatment with AhR activation and TOP2A inhibition synergistically ameliorated the recurrence of psoriasis-like lesions in mice.

Claims

1. A pharmaceutical composition for treating psoriasis, characterized in that, Comprising the following active ingredients: (a) Belinostat as an AhR activator; (b) Amnafide as a TOP2A inhibitor; Wherein the active ingredients (a) and (b) have synergistic efficacy in treating psoriasis.

2. The pharmaceutical composition of claim 1, wherein The concentration of the belinostat is 1%, and the concentration of the amnafide is 0.1%.

3. The pharmaceutical composition of claim 1, wherein Further comprising pharmaceutically acceptable adjuvants, which include glycerin, stearic acid, triethanolamine, liquid paraffin, paraffin, and nipagin ethyl ester.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that, It is an external dosage form.

5. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that, It is a cream.

6. A process for the preparation of a pharmaceutical composition according to any one of claims 1 to 5, characterized in that, Including the following steps: Preparation of cream base: mix glycerin, stearic acid, triethanolamine, liquid paraffin, paraffin, nipagin ethyl ester and water, heat to dissolve, stir to condense; Preparation of medicinal cream: uniformly mix belinostat powder, amnafide powder and cream base to prepare medicinal composition cream.

7. Use of the pharmaceutical composition of any one of claims 1-5 in the preparation of a medicament for treating psoriasis.

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