Application of cobra neurotoxin or preparation thereof in preparation of medicine for treating psoriasis

Abstract

The invention relates to the technical field of biological medicines, in particular to application of cobra neurotoxin or a preparation thereof to preparation of a medicine for treating psoriasis, and the cobra neurotoxin or the preparation thereof has a beautifying effect when used for treating skin diseases. The snake venom neurotoxin is cobra neurotoxin which is selected from Chinese cobra neurotoxin (cobratoxin) (CTX) and / or Thailand cobra neurotoxin (cobratoxin). Through an imiquimod-induced psoriasis model, the snake venom neurotoxin disclosed by the invention has the effects of relieving and treating psoriasis and has a beautifying effect, and can effectively improve skin lesion of mouse psoriasis and repair skin to achieve the beautifying effect; the treatment effect of certain anti-cytokine monoclonal antibodies can also be enhanced. The invention provides a new application and a new thought of the snake venom neurotoxin, brings a new hope for patients with psoriasis, and can also provide a new skin beautifying medicinal preparation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the use of cobra neurotoxin or its preparation in the preparation of a drug for treating skin immune inflammatory diseases and having cosmetic effects. BACKGROUND

[0002] Psoriasis is a complex and common clinical condition of the skin, the pathological features of which mainly manifest as a vicious cycle of skin barrier dysfunction, accompanied by skin damage, hyperplasia, roughness, dryness and mossification, which seriously affects the quality of life of patients. The characteristics of psoriasis are well-defined red plaques covered with silvery white scales, and its pathogenesis involves genetic variations, environmental triggers (such as streptococcal infection) and abnormal activation of the IL-23 / IL-17 axis in the epidermal microenvironment (EIME). Keratinocytes and Langerhans cells in the epidermis form a dynamic network with DCs and macrophages in the dermis, and in psoriasis lesions, keratinocytes recruit T cells and neutrophils, which damage the skin barrier function.

[0003] The current treatment system mainly includes five types of intervention strategies, but all have clinical limitations: local treatment: long-term use of glucocorticoids can easily cause iatrogenic damage such as skin atrophy and telangiectasia, and once the drug is stopped, the disease is likely to rebound; systemic treatment: although antihistamines can relieve pruritus, the regulation is limited, and may cause side effects such as drowsiness; physical therapy: although phototherapy can avoid drug metabolism problems, the cumulative radiation dose is positively correlated with the risk of skin tumors; biological targeted therapy: although monoclonal antibodies can precisely block key mediators, the high cost, potential infection risk and drug resistance in some patients limit their widespread use; traditional Chinese medicine intervention: traditional prescriptions have the advantage of multi-target regulation, but there are practical bottlenecks such as slow onset and lack of standardization of preparations. SUMMARY

[0004] Based on the limitations of existing therapies, it is of great clinical significance to find new and safer and more effective drugs for treating psoriasis. Therefore, the technical problem to be solved by the present application is to provide the use of cobra neurotoxin or its preparation in the preparation of a drug for treating psoriasis and having cosmetic effects.

[0005] To this end, the present application provides the following technical solutions: The use of cobra neurotoxin or its preparation in the preparation of a drug for treating psoriasis; or the use of cobra neurotoxin or its preparation in the preparation of a cosmetic drug.

[0006] The use of cobra neurotoxin or its preparation in combination with monoclonal antibodies in the preparation of a drug for treating psoriasis; or the use of cobra neurotoxin or its preparation in combination with monoclonal antibodies in the preparation of a cosmetic drug.

[0007] A medicine for treating psoriasis and / or cosmetically, comprising a cobra neurotoxin or a preparation thereof. Preferably, it further comprises an additional therapeutic agent. Further, the additional therapeutic agent comprises a monoclonal antibody, a synthetic small molecule drug, a genetically engineered drug, a biological preparation or a gene therapy drug.

[0008] In the present application, the cobra neurotoxin is cobrotoxin of Chinese cobra and / or cobratoxin of Thai cobra.

[0009] Optionally, the cobra neurotoxin comprises at least one of cobroxin, Nyloxin or cobraxin, or a clinically acceptable injection or non-injection dosage form prepared from the above.

[0010] In the present application, the monoclonal antibody comprises at least one of etanercept, infliximab, efalizumab, recombinant human interleukin or recombinant human tumor necrosis factor receptor-antibody fusion protein, or a clinically acceptable injection or non-injection dosage form prepared from the above.

[0011] In the present application, the medicine comprises a therapeutically effective amount of the cobra neurotoxin; preferably, the content of the cobra neurotoxin is 0.0001-1% by mass percentage.

[0012] Optionally, the medicine further comprises a pharmaceutically acceptable carrier or salt.

[0013] Optionally, the pharmaceutically acceptable carrier or salt is selected from at least one of a pharmaceutically acceptable solvent, a solubilizer, a cosolvent, an emulsifier, a coloring agent, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a thickening agent, a complexing agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculating agent, a filter aid, a release retardant, a high molecular skeleton material and a film-forming material. Optionally, the pharmaceutically acceptable carrier or salt is selected from physiological saline, water or a buffer.

[0014] Optionally, the dosage form of the medicine is an injection, a lotion, a liniment, a tincture, an oil, a paste, a paste, a patch, a film, a gel or a patch, a nasal (drop) spray; preferably, it is an injection.

[0015] The technical solution of the present application has the following advantages: The application provides use of cobra neurotoxin or a preparation thereof in preparation of a medicine for treating psoriasis and having a skin cosmetic effect, the cobra neurotoxin being Chinese cobra neurotoxin cobrotoxin and / or Thai cobra neurotoxin cobratoxin; currently, there is no relevant research report on use of the cobra neurotoxin or the preparation thereof in treatment of psoriasis and having a cosmetic effect, and the application discloses that the cobra neurotoxin or the preparation thereof has the effect of treating psoriasis, can effectively alleviate infiltration of skin tissues, improve skin lesion symptoms and cosmetic effect, has a similar effect intensity to dexamethasone, is stronger than a single antibody drug, and does not have any side effect, and thus can be used for preparation of a medicine for treating psoriasis, so that the cobra neurotoxin or the preparation thereof can be used for preparation of a medicine for treating psoriasis and having a cosmetic effect.

[0016] The application provides use of cobra neurotoxin or a preparation thereof in combination with a single antibody in preparation of a medicine for treating psoriasis and having a cosmetic effect, the single antibody including at least one of etanercept, infliximab, efalizumab, recombinant human interleukin or recombinant human type II tumor necrosis factor receptor-antibody fusion protein, or a clinically acceptable injection or non-injection dosage form prepared from the above; currently, there is no research report on use of the cobra neurotoxin or the preparation thereof in combination with the single antibody in treatment of psoriasis, and the application discloses that the cobra neurotoxin or the preparation thereof has the effect of treating psoriasis in combination with the single antibody, so that the cobra neurotoxin or the preparation thereof in combination with the single antibody can be used for preparation of a medicine for treating psoriasis, to achieve better treatment effect. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative effort.

[0018] Figure 1 The back photos of mice in a normal control group (Control), a psoriasis model group (Model), a positive drug group dexamethasone (PC (Dex)), and three cobra a-neurotoxin cobrotoxin (CTX) drug groups (CTX (3 μg / kg / d), CTX (6 μg / kg / d), and CTX (12 μg / kg / d)) in Example 1 of the application.

[0019] Figure 2The PASI score statistical chart of the back skin lesion of the mice of the normal control group (Control), the psoriasis model group (Model), the positive drug group (PC (Dex)), and the three groups of CTX drug groups (CTX (3 μg / kg / d), CTX (6 μg / kg / d), and CTX (12 μg / kg / d)) provided for the embodiment 1 of the present application.

[0020] Figure 3 The statistical data of the change of the body weight of the mice of the normal control group (Control), the psoriasis model group (Model), the positive drug group (PC (Dex)), and the three groups of CTX drug groups (CTX (3 μg / kg / d), CTX (6 μg / kg / d), and CTX (12 μg / kg / d)) on the 7th day of drug treatment and the initial body weight, the picture of the size of the spleen, and the statistical data of the spleen index provided for the embodiment 1 of the present application; A is the statistical data of the change of the body weight of the mice of each group; B is the picture of the size of the spleen of each group; and C is the statistical data of the spleen index of each group.

[0021] Figure 4 The HE staining microscopic chart of the back skin of the mice of the normal control group (Control), the psoriasis model group (Model), the positive drug group (PC (Dex)), and the three groups of CTX drug groups (CTX (3 μg / kg / d), CTX (6 μg / kg / d), and CTX (12 μg / kg / d)) provided for the embodiment 1 of the present application; A is the HE staining microscopic chart under a 20-fold objective lens; and B is the HE staining microscopic chart under a 40-fold objective lens.

[0022] Figure 5 The back photo of the mice of the normal control group (Control), the psoriasis model group (Model), the IXE drug group (20 mg / kg), the CTX drug group (CTX (12 μg / kg)), and the combined treatment group (IXE+CTX) in the animal experiment of the embodiment 2 of the present application.

[0023] Figure 6 The PASI score statistical chart of the back skin lesion of the mice of the normal control group (Control), the psoriasis model group (Model), the IXE drug group (20 mg / kg), the CTX drug group (CTX (12 μg / kg)), and the combined treatment group (IXE+CTX) provided for the embodiment 2 of the present application.

[0024] Figure 7Micrograph of HE staining of the back skin of the normal control group (Control), the psoriasis model group (Model), the IXE drug group (20 mg / kg), the CTX drug group (CTX (12 μg / kg)), and the combined treatment group (IXE+CTX) provided for Embodiment 2 of the present application; A is the micrograph of HE staining under a 20x objective lens; B is the micrograph of HE staining under a 40x objective lens.

[0025] Figure 8 Inhibition of CTX on the proliferation of the Hacat cell psoriasis model cells induced by M5 provided for Embodiment 3 of the present application. The experiment takes Hacat cells as the research object, and is divided into a blank control group (CTL), a psoriasis model group (M5), a CTX low-concentration treatment group (CTX 50 ng / mL), and a CTX high-concentration treatment group (CTX 250 ng / mL). The CCK-8 method is used to detect the cell proliferation. DETAILED DESCRIPTION

[0026] As a natural bioactive substance, the neurotoxin component of snake venom has been increasingly concerned by the academic community due to its unique pharmacological mechanism. Studies have shown that this type of protein polypeptide composed of 50-80 amino acids has the function of neurotransmitter regulation. From the perspective of structural biology, although neurotoxins from different snake families show conformational diversity, they all have the two characteristics of high targeting and ligand binding specificity. From the mechanism level, specific neurotoxins can regulate the release of neurotransmitters, thereby affecting the function of the nervous system. For example, the alpha-neurotoxin of cobra venom (cobrotoxin injection) has been approved by the state as a clinical analgesic drug. This type of bioactive molecule with nerve regulation shows unique advantages, and has higher targeting and safety compared to traditional preparations. However, the application of snake venom neurotoxin as a substance with special biological activity in the treatment of psoriasis has not been reported.

[0027] The potential of snake venom neurotoxin as a substance with special biological activity in the treatment of psoriasis has not been fully explored and utilized. Whether snake venom a-neurotoxin can improve the skin symptoms of psoriasis patients by regulating key cytokines in the skin and thus become a new, effective and safe solution for treating psoriasis has not been reported.

[0028] Therefore, the present embodiment provides the following technical solutions: Use of cobra neurotoxin or its preparation in the preparation of a drug for treating skin immune / inflammatory diseases and having a cosmetic effect, characterized in that the cobra neurotoxin is Chinese cobra neurotoxin cobrotoxin and / or Thai cobra neurotoxin cobratoxin.

[0029] The following examples are provided to better enable those skilled in the art to which the application pertains to further understand the application and are not intended to limit the scope of the application, nor are they intended to limit the scope of equivalents of the application. Any product that is the same as or similar to the present application that is derived from the present application or from the combination of the present application and other prior art features falls within the scope of the present application.

[0030] When the specific experimental steps or conditions are not mentioned in the examples, the operation or conditions can be carried out according to the conventional experimental steps described in the literature in the art. When the reagents or instruments are not mentioned by the manufacturer, they are conventional reagent products that can be obtained by purchase.

[0031] In some embodiments, the cobra neurotoxin includes at least one of cobratope, Nyloxin, or cobraxin, or a clinically acceptable injection or non-injection dosage form prepared from the above. In some embodiments, the amino acid sequence of the cobra neurotoxin is shown in SEQ ID NO. 1.

[0032] In some embodiments, the use of the cobra neurotoxin or preparation thereof in combination with a monoclonal antibody in the preparation of a drug for treating psoriasis is also included. In some embodiments, the monoclonal antibody includes at least one of etanercept, infliximab, efalizumab, recombinant human interleukin, or recombinant human type II tumor necrosis factor receptor-antibody fusion protein, or a clinically acceptable injection or non-injection dosage form prepared from the above.

[0033] Further, experimental and clinical studies have shown that the pathological mechanism of drug-induced psoriasis is similar to that of psoriasis itself, and imiquimod can induce the formation of psoriasis lesions in the skin. The psoriasis model selected in the present application is an imiquimod (IMQ)-induced psoriasis model, and the effect of cobra venom a-neurotoxin cobrotoxin on the IMQ-induced psoriasis model is investigated. The results show that cobra venom a-neurotoxin cobrotoxin can reduce the skin symptoms such as erythema, skin thickening, and scaling of the epidermis caused by IMQ and the spread of the disease. The PASI score results and the lesion HE staining results also demonstrate that cobra venom a-neurotoxin cobrotoxin has a significant improvement effect on the pathology of psoriasis. The data of mouse body weight and spleen size and index show that the cobra venom neurotoxin (cobrotoxin) group can alleviate the psoriasis-like skin damage and immune dysfunction caused by 5% imiquimod cream.

[0034] In some embodiments, the content of the cobra neurotoxin is 0.01-1% by mass percentage.

[0035] In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier or salt.

[0036] In some embodiments, the pharmaceutically acceptable carrier or salt is selected from at least one of a pharmaceutically acceptable solvent, a solubilizer, a cosolvent, an emulsifier, a coloring agent, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure adjusting agent, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH adjusting agent, a buffer, a plasticizer, a surfactant, a thickening agent, an inclusion agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculating agent, a filtration aid, a release retardant, a high molecular backbone material, and a film-forming material. In some embodiments, the pharmaceutically acceptable carrier or salt is selected from physiological saline, water, or a buffer.

[0037] In some embodiments, the medicament is in the form of an injection, a lotion, a liniment, a tincture, an oil, a paste, a paste, a patch, a film, a gel, or a patch, a nasal (drop) spray; preferably, an injection.

[0038] In some embodiments, the medicament further comprises another additional therapeutic agent for treating skin immune / inflammatory diseases, and the additional therapeutic agent is selected from a synthetic small molecule drug, a genetically engineered drug, a biological agent, or a gene therapy drug.

[0039] The present application adopts the administration mode of ixekizumab (IXE) alone, IXE combined with cobrotoxin, and cobrotoxin alone, and discloses the similarities and differences between cobra a-neurotoxin cobrotoxin and monoclonal antibody drugs for treating psoriasis. The experimental results show that IXE alone and cobrotoxin alone have the effects of inhibiting psoriasis and reducing the symptoms of skin lesions, and the drug efficacy of the combination of IXE and cobrotoxin is stronger than that of cobrotoxin alone and IXE alone, which indicates that there is a significant synergistic effect between IXE and cobrotoxin.

[0040] In conclusion, the application discloses an effect of cobra venom a-neurotoxin on psoriasis treatment, can effectively alleviate the infiltration of skin tissues, improves the lesion symptoms (has certain cosmetic effect), the effect intensity is similar to that of dexamethasone, and is stronger than that of monoclonal antibody drugs. And cobrotoxin does not have any side effects, and can be used for preparing a treatment drug for psoriasis and having a cosmetic effect. Meanwhile, the application also discloses that the drug effect of ixekizumab (IXE) combined with cobrotoxin is stronger than that of cobrotoxin alone and IXE alone, which indicates that there is a significant synergistic effect between IXE and cobrotoxin. The application provides a new use of cobra venom a-cobra venom a-neurotoxin and combination of the cobra venom a-neurotoxin with ixekizumab, expands the application range of the cobra venom a-neurotoxin, and has important economic benefits and social benefits.

[0041] Psoriasis lesion severity score of the mouse. Referring to the Psoriasis Area and Severity Index (PASI) content commonly used in clinical evaluation of psoriasis, the severity of the redness, scales and thickening of the stratum corneum of the lesion area of the mouse model is selected as the measure of the inflammatory symptom evaluation. Only the score of the fixed area treated by the above grouping scheme is evaluated, and other affected skin areas are not included. The redness, scales and thickening of the stratum corneum are independently calculated by 0-4 points, and the higher the score, the more serious the lesion of the lesion site. The lesion severity score is obtained by adding the severity scores of the redness, scales and thickening of the stratum corneum.

[0042] Table 1 Score scale

[0043] Body weight, spleen index and spleen size. The body weight change of the mouse before and after the experiment is measured, the body weight change (g)=the body weight after the experiment (g)-the body weight before the experiment (g), and after the mouse is dissected, the mouse spleen is taken, photographed and weighed, and the formula is used to calculate the spleen index (Spleen Index)=the weight of the mouse spleen (g) / the weight of the mouse (g).

[0044] Hematoxylin and Eosin (HE) staining is used to observe the histopathological changes of the psoriasis lesion tissue of the mouse. First, the mouse is killed, the back bare skin is cut, the frozen section is fixed with paraformaldehyde for 10 min, the fixed section is immersed in hematoxylin staining solution for about 5 min, the section is washed with water for 2 min, then differentiation solution is differentiated for 3-5 s, the section is washed with water for 2 min, then eosin staining is performed for 3 min, the section is washed with water for 3-5 s, gradient alcohol is used for dehydration, neutral balsam is used for sealing, and observation and photography are performed under a microscope.

[0045] Experimental Example 1 Experiment of cobrotoxin (CTX) of Naja naja a- neurotoxin for treating psoriasis Cobrotoxin solution (CTX, the amino acid sequence of which is shown as SEQ ID NO. 1, and the concentration of which is 150 μg / mL) was provided by Suzhou Renben Pharmaceutical Co., Ltd. 5% Imiquimod cream and compound dexamethasone acetate cream (999 Bioderm Cream) were commercially available. SPF 6-8 week old BALB / C male mice were purchased from the Model Animal Research Center of Nanjing University, and the animal experiments met the ethical requirements. Data processing: statistical analysis was performed by GraphPad Prism 6.0 software. Two-way ANOVA was used for comparison between groups.

[0046] SEQ ID NO. 1: LECHNQQSSQTPTTTGCSGGETNCYKKRWRDHRGYRTERGCGCPSVKNGIEINCCTTDRCNN Experimental grouping. Grouping: normal control group (Control), psoriasis model group (Model), positive drug group (PC (Dex), dexamethasone cream), and three CTX drug groups (CTX (3 μg / kg / d), CTX (6 μg / kg / d), and CTX (12 μg / kg / d), subcutaneous injection), a total of 6 groups, 6 mice in each group.

[0047] Psoriasis model of mice. First, the mice were anesthetized, and the smooth skin tissue was exposed after the back skin was depilated as the experimental drug application area. The skin of the normal control group of mice was only smeared with an appropriate amount of vaseline, and the back skin of the rest of the mice was smeared with 5% imiquimod (IMQ) cream, 62.5 mg each time (the drug application area was 2×3 cm), once a day, to prepare an IMQ-induced psoriasis-like mouse model; from the 4th day, the positive drug group and the CTX drug group were smeared or injected with the corresponding drugs 4 hours after IMQ smearing, the positive drug group was smeared with dexamethasone cream, 20 mg per mouse (the concentration of dexamethasone was 0.75 mg / g), and the CTX drug group was subcutaneously injected, and the three groups were administered at doses of 3 μg / kg / d, 6 μg / kg / d, and 12 μg / kg / d (after dilution of the 150 μg / mL stock solution to 0.3, 0.6, and 1.2 μg / mL with normal saline, the treatment was continuously performed for 7 days.

[0048] Experimental results.

[0049] 1. Effect of cobrotoxin (CTX) on psoriasis model mice skin lesions As Figure 1As shown, the Control group mice had pinkish skin on the back, smooth, without scales, erosion and erythema on day 7. The Model group mice had erythema and thick layer of silver scales on the back. Compared with the Model group mice, the positive drug group and the CTX administration group mice had reduced erythema and scales on the back, among which the medium dose CTX (6 μg / kg) and high dose CTX (12 μg / kg) groups were significantly better than the positive drug PC (Dex) group.

[0050] 2. Results of the severity score of the psoriasis-like skin lesions in mice Compared with the Model group, the positive drug PC (Dex) group and the CTX administration group mice had reduced scores of erythema, scales and thickening of the stratum corneum on the back (***P<0.001 or ****P<0.0001). The scores of erythema, scales and thickening of the stratum corneum decreased with the increase of the CTX concentration, showing a certain dose-dependent manner. The skin of the CTX administration group was smooth without wrinkles on day 7, showing a certain cosmetic effect. The low dose CTX (3 μg / kg) group was slightly better than the positive drug PC (Dex) group, but the difference was not statistically significant. The medium dose CTX (6 μg / kg) group and the high dose CTX (12 μg / kg) group were significantly improved compared with the positive drug PC (Dex) group (##P<0.01), as shown in Figure 2 .

[0051] 3. Effects of Cobrotoxin (CTX) on the body weight and spleen of psoriasis model mice Compared with the Control group, the Model group and the PC (Dex) positive drug group mice showed significant decrease in body weight (##P<0.01 or ####P<0.0001) induced by IMQ. Compared with the Model group, the CTX administration group mice had significantly increased body weight ( *P<0.05 or **P<0.01), as shown in Figure 3 A. As shown in Figure 3 B, the size of the spleen of each group, Figure 3 C, the spleen index of each group. As shown in the figure, compared with the Model group, the spleen index of the CTX administration group was significantly reduced ( *P<0.05 or **P<0.01).

[0052] 4. Effects of Cobrotoxin (CTX) on the histopathological changes of the skin tissues of each group of mice Figure 4HE staining results of mouse skin, where Fig. A is a 20x objective lens collected picture, Fig. B is a 40x objective lens collected picture, and the histopathological changes of the skin lesions of the model mice after administration of each group are observed. It can be seen that the stratum corneum of the back skin of the control group (Control) mice is thinner, and the epidermis does not show obvious hyperplasia, and the dermal blood vessels do not show obvious dilation. The epidermis of the model group (Model) mice was HE stained, and under the microscope, cell proliferation, epidermal thickening, and subcutaneous inflammatory cell infiltration were observed. Compared with the model group (Model), the positive administration group PC (Dex) and each CTX administration group showed different degrees of pathological improvement in the skin of the mice, the thickness of the stratum corneum and stratum spinosum was reduced, and the inflammatory cell infiltration was reduced, and the skin became smoother. Comparison between different administration concentrations, the skin pathology of mice gradually recovered, showing a certain dose-dependent relationship.

[0053] Experimental Example 2 Treatment of Psoriasis by Ophiophagus hannah a-neurotoxin CTX alone and its combination with ixekizumab IXE Cobrotoxin solution (CTX, concentration 1.2 μg / mL) of Ophiophagus hannah neurotoxin was provided by Suzhou Renben Pharmaceutical Co., Ltd. 5% imiquimod cream was commercially available, and ixekizumab (ixekizumab, IXE 2 mg / mL) was produced by Eli Lilly and Company. SPF level 8-10 week old BALB / C female mice, weighing 20-22 g, were purchased from Changzhou Cavens Experimental Animal Co., Ltd., and the animal experiments met the ethical requirements. Data processing: statistical analysis was performed using GraphPad Prism 6.0 software. Two-way ANOVA was used for comparison between groups.

[0054] Experimental grouping. Grouping: normal control group (Control), psoriasis model group (Model), IXE drug group (IXE (20 mg / kg), subcutaneous injection), CTX drug group (CTX (12 μg / kg), subcutaneous injection), combined treatment group (IXE+CTX, subcutaneous injection) a total of 5 groups, 8 mice in each group.

[0055] Mouse psoriasis model. First, the mice were anesthetized, and the skin on the back was shaved to expose the smooth skin tissue as the experimental drug application area. The normal control group of mice was only smeared with a proper amount of vaseline, and the rest of the mice were smeared with 5% imiquimod (IMQ) cream on the back skin, 25 mg each time (the drug application area was 2x2 cm, once a day to prepare the IMQ-induced psoriasis-like mouse model; from the 4th day, the drug groups and the combination drug groups were injected with the corresponding drugs 4 hours after IMQ was applied. The IXE drug group was subcutaneously injected with 20 mg / kg / d of ixekizumab, and the CTX drug group was subcutaneously injected with 12 μg / kg / d (physiological saline was diluted to 1.2 μg / mL after the mother liquor of 150 μg / mL was injected) of Chinese cobra neurotoxin solution. The combination drug group was subcutaneously injected with 20 mg / kg / d of IXE and 12 μg / kg / d of CTX at the same time, for 7 consecutive days.

[0056] Experimental results.

[0057] 1. Effect of cobrotoxin (CTX) and combination of ixekizumab (IXE) on psoriasis mouse skin lesions As Figure 5 shown, the skin on the back of the control group (Control) mice was light red on the 7th day, smooth, without scales, erosion, and erythema. The skin on the back of the model group (Model) mice was red, covered with scales, and had obvious thickening of the stratum corneum. Compared with the model group (Model) mice, the redness and scale appearance of the skin on the back of the mice in the IXE drug group, the CTX drug group, and the combination drug group (IXE+CTX) were reduced, and the skin improvement of the mice in the CTX drug group and the IXE+CTX combination drug group was significantly better than that in the IXE drug group.

[0058] 2. Psoriasis-like mouse skin lesion severity score results As Figure 6As shown, compared with the control group (Control), the PASI score of the model group (Model) mice was always significant (P<0.0001), indicating that the psoriasis modeling was successful. Compared with the model group (Model), the IXE drug group began to show a decrease in the PASI score of the mouse back skin on the 4th day of administration, but there was no significant difference on the 4th to 6th day, and the PASI score was significantly reduced on the 7th day (**P<0.01); the PASI scores of the combination administration group (IXE+CTX) and the CTX drug group mice were not significantly different, but showed a downward trend on the 3rd day after administration, and the PASI scores were significantly reduced on the 4th to 7th day (*P<0.05, **P<0.01, ***P<0.001 or ****P<0.0001). Compared with the IXE drug group, the PASI scores of the CTX drug group and the IXE+CTX combination administration group began to decrease on the 3rd day of administration and continued to the 7th day, and the PASI scores were significantly reduced on the 3rd to 6th day (*P<0.05 or **P<0.01). Further, the PASI scores of the CTX drug group and the IXE+CTX combination administration group were better than those of the single IXE drug group, and the administration of 4-7 days had a role in improving the psoriasis condition.

[0059] 3、Cobrotoxin (CTX) and combined eculizumab (IXE) on the histopathological changes of the skin tissues of each group of mice Figure 7 The results of HE staining of the mouse skin are shown in Figure A, which is a picture taken under a 20x objective lens, and Figure B is a picture taken under a 40x objective lens. Observation of the histopathological changes in the skin lesions of the model mice after administration showed that the skin of the control group (Control) mice had a thin stratum corneum, and the epidermis showed no obvious hyperplasia, and the dermal blood vessels showed no obvious dilation. HE staining of the epidermis of the model group (Model) mice showed that the cells were hyperplastic, the epidermis was thickened, and there was inflammatory cell infiltration under the skin. Compared with the model group mice, the erythema and scaling of the back skin of the IXE drug group, the CTX drug group and the IXE+CTX combination administration group mice began to decrease within 2 to 3 days after administration, and the improvement of the CTX group and the IXE+CTX combination administration group was significantly better than that of the IXE drug group.

[0060] Based on the results of Examples 1-2 above, Cobrotoxin (CTX) has a significant improvement effect on psoriasis lesion models and has a certain cosmetic effect. Compared with the commonly used dexamethasone and eculizumab for psoriasis, the treatment effect of CTX is better, and the side effects are less.

[0061] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

[0062] Experimental Example 3: Cobra α-neurotoxin CTX inhibits skin cell proliferation Human immortalized keratinocytes (Hacat) were purchased from Hastar Biotechnology. DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was purchased from Hastar Biotechnology. M5 cytokines (IL-1α, IL-17A, IL-22, Oncostatin M, TNF-α) were purchased from Merck. CCK-8 kit was purchased from Beyotime Biotechnology Co., Ltd.

[0063] Preparation and seeding of Hacat cells. Hacat cells in the logarithmic growth phase were harvested, the old culture medium was discarded, and the cells were gently rinsed twice with sterile PBS buffer to remove residual culture medium and metabolic waste. 2 mL of 0.25% trypsin-EDTA digestion solution was added to the culture flask, and the flask was incubated at 37°C for 2-3 minutes. Observation was performed under an inverted phase-contrast microscope. When the intercellular spaces increased and the cells became round, 4 mL of DMEM medium containing 10% FBS was immediately added to terminate the digestion. Use a pipette to gently agitate the culture flask walls to detach the cells and form a single-cell suspension. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant and add 5 mL of fresh DMEM medium containing 10% FBS to the centrifuge tube. Gently agitate the cells to resuspend them and count them using a cell counting chamber.

[0064] Based on the cell count results, the cell concentration was adjusted to 5 × 10⁻⁶ cells using fresh culture medium. 4 Cells / mL were then seeded into 96-well cell culture plates at a volume of 100 μL per well, with approximately 5 × 10⁶ cells per well. 3 Place the 96-well plate inoculated with cells in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere fully. Grouping and Model Construction. Cells in 96-well plates after 24 hours of culture were divided into four groups, with six replicates per group to minimize experimental error: Blank control group (CTL): Only 100 μL of DMEM medium was added, without any inducers or inhibitors; Psoriasis model group (M5): Discard the original culture medium in the hole, add 100 μL of DMEM culture medium containing M5 cytokine mixture (M5 final concentration is 100 ng / mL of each cytokine), construct Hacat cell psoriasis model; CTX low concentration treatment group (CTX 50 ng / mL): Discard the original culture medium in the hole, first add 50 μL of DMEM culture medium containing M5 cytokine mixture (M5 final concentration is 200 ng / mL of each cytokine), then add 50 μL of DMEM culture medium containing CTX (CTX final concentration is 50 ng / mL), so that the total volume of the culture medium in the hole is 100 μL, and the M5 final concentration is consistent with the model group; CTX high concentration treatment group (CTX 250 ng / mL): The operation is the same as the CTX 50 ng / mL treatment group, only the final concentration of CTX is adjusted to 250 ng / mL. After the grouping treatment is completed, the 96-well plate is continued to be placed in a 37°C, 5% CO2 incubator for 60h, so that the cells can fully respond to various treatment factors. CCK-8 method for detecting cell proliferation. After 60h of culture, the 96-well plate was taken out, 100 μL of supernatant was discarded from each well (to avoid damaging the adherent cells), and then 10 μL of CCK-8 reagent was added to each well. The 96-well plate was gently shaken to mix the reagent and the culture medium thoroughly. The 96-well plate was re-placed in a 37°C, 5% CO2 incubator for 2h to allow the CCK-8 reagent to fully react with the dehydrogenase in the cells to generate a colored product. After incubation, the 96-well plate was taken out and placed in a microplate reader, and the absorbance value (OD value) of each well was measured at 450nm as the detection wavelength and 630nm as the reference wavelength. The OD value is positively correlated with the proliferation activity of the cells, and the higher the OD value, the more vigorous the cell proliferation. Data statistics and analysis. Record the OD values of the 6 replicate wells in each group, calculate the average OD value of each group (± standard deviation, x±s), as the representative value of the cell proliferation activity of the group. Taking the OD value of the blank control group as the reference, calculate the cell proliferation rate of the M5 model group, the CTX 50 ng / mL treatment group and the CTX 250 ng / mL treatment group relative to the blank control group, the calculation formula is: cell proliferation rate (%) = (experimental group OD value / blank control group OD value) x 100%. The experimental results are as follows Figure 8As shown, the present embodiment successfully constructed a psoriasis model of Hacat cells by M5 cytokines, and the cells in the model showed obvious characteristics of active proliferation (##P<0.01); while CTX at concentrations of 50ng / mL and 250ng / mL could effectively inhibit the abnormal proliferation of Hacat cells caused by M5 stimulation, and the inhibition effect was enhanced with the increase of CTX concentration, and the inhibition effect of 250ng / mL CTX was more significant (*P<0.05 or **P<0.01). The experimental results of this experiment provide experimental basis at the in vitro cell level for the application of CTX in the treatment of psoriasis, and lay a foundation for subsequent related drug research and clinical research.

Claims

1. The use of cobra neurotoxin or its preparations in the preparation of medicaments for treating psoriasis; or the use of cobra neurotoxin or its preparations in the preparation of cosmetic medicaments.

2. The use of cobra neurotoxin or its preparations in the preparation of drugs that enhance the efficacy of monoclonal antibody drugs for the treatment of psoriasis.

3. The use of cobra neurotoxin or its preparations in combination with monoclonal antibodies in the preparation of drugs for the treatment of psoriasis and / or cosmetic purposes.

4. The application according to claim 1, 2, or 3, characterized in that, The formulation includes at least one of the clinically acceptable injectable or non-injectable dosage forms; the drug also includes a pharmaceutically acceptable carrier or salt; the dosage form of the drug is one or more of the following: injection, lotion, liniment, tincture, oil, ointment, paste, patch, film, gel or tablet, nasal spray, nasal drop.

5. The application according to claim 1, 2, or 3, characterized in that, The content of cobra neurotoxin in the drug is 0.0001% to 1% by mass.

6. The application according to claim 1, 2, or 3, characterized in that, The cobra neurotoxin is Chinese cobra neurotoxin and / or Thai cobra neurotoxin.

7. The application according to claim 6, characterized in that, The cobra neurotoxins include cobraxin, nyloxin, or cobraxin.

8. The application according to claim 2 or 3, characterized in that, Monoclonal antibodies include ixekizumab, infliximab, efalizimab, recombinant human interleukin, or recombinant human type II tumor necrosis factor receptor-antibody fusion protein.

9. A medicament for treating psoriasis and / or for cosmetic purposes, characterized in that, It contains cobra neurotoxin or its preparations.

10. The medicament according to claim 9, characterized in that, The medication also contains additional therapeutic agents.

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