Use of a small molecule polypeptide and pharmaceutical composition thereof for treating psoriasis

Abstract

The application discloses a kind of small molecule polypeptide for treating psoriasis and its pharmaceutical composition and application.The application obtains a kind of small molecule polypeptide S1 from peptide library, which can be stably combined with cell receptor in psoriasis skin, and the small molecule polypeptide is composed of CNAGQRSEC ring peptide, and the small molecule polypeptide can inhibit the proliferation of human keratinocytes.In the application, further add medical acceptable adjuvant (such as transdermal absorption agent, fat-soluble matrix, emulsifying agent, humectant, pH regulator and antioxidant, etc.) on the basis of small molecule polypeptide S1, it is made into pharmaceutical cream, can significantly improve the stability and transdermal capacity of drug, realize the effective treatment of psoriasis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to application of a small-molecule polypeptide for treating psoriasis and a pharmaceutical composition thereof. BACKGROUND

[0002] Psoriasis, commonly known as psoriasis, is a common chronic inflammatory skin disease that affects 2% to 5% of the world's population. The World Health Organization defines it as a chronic, inflammatory, recurrent and incurable non-communicable disease, which is listed as one of the most serious non-communicable diseases due to the occurrence of complications and the impact on multiple organ systems during the course of the disease. Psoriasis is a heterogeneous disease, and genetic predisposing sites and environmental factors such as skin trauma, infection and stress are important factors in inducing or affecting the development of psoriasis. The clinical types of psoriasis can be divided into ordinary type, arthritis type, pustule type, palmoplantar pustulosis and erythroderma type, and the ordinary type of psoriasis accounts for 85% to 90% of the total, which is manifested as skin redness with silvery raised scales. The specific manifestations of pathological tissues are excessive proliferation of epidermal keratinocytes, incomplete keratinization, thickening of epidermal spire layer, expansion of superficial dermal microvessels, and infiltration of inflammatory cells in the dermal layer. Studies have shown that the quality of life and social and psychological health of psoriasis patients are usually greatly negatively affected. Compared with ordinary people, most psoriasis patients are more likely to have a tendency of self-abasement and even suffer from depression.

[0003] In view of the increasing trend of the incidence of psoriasis, which has become one of the diseases that are currently focused on in the field of dermatology, a variety of treatment options have been developed. For moderate to severe psoriasis that cannot be controlled by topical drugs, oral drugs such as methotrexate, cyclosporine and vitamin A are usually used for treatment, but these oral drugs also exhibit side effects such as hepatotoxicity and gastrointestinal discomfort. Although the biological agents developed for targeted immunotherapy of psoriasis have good efficacy and safety, the increase in the risk of infection and the high cost of medication mentioned in related reports still limit the application of such drugs. As the best local treatment for patient medication compliance, the first-line drug vitamin D derivative has a definite effect, but long-term use can easily cause skin dryness, redness, elevated blood calcium and other side effects. Similarly, the widely used glucocorticoids can easily cause local skin atrophy, thinning of the keratin layer, pigmentation and other adverse reactions, and are prone to relapse or rebound after drug withdrawal. Therefore, it is important to seek a topical drug for treating psoriasis with small toxic and side effects and good efficacy. SUMMARY

[0004] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a small-molecule polypeptide for treating psoriasis.

[0005] Another purpose of the present application is to provide a pharmaceutical composition for treating psoriasis.

[0006] Still another object of the present application is to provide a method for preparing the pharmaceutical composition for treating psoriasis.

[0007] Still another object of the present application is to provide the use of the small molecule polypeptide for treating psoriasis and the pharmaceutical composition for treating psoriasis.

[0008] The object of the present application is achieved by the following technical solutions:

[0009] A small molecule polypeptide for treating psoriasis, named S1, is a cyclic peptide composed of CNAGQRSEC.

[0010] The use of the small molecule polypeptide for treating psoriasis in the preparation of a medicine for treating psoriasis.

[0011] The medicine can be prepared into various dosage forms as needed, such as ointments (cream preparations), etc.

[0012] A pharmaceutical composition for treating psoriasis, comprising the above small molecule polypeptide (effective ingredient) and a medically acceptable excipient.

[0013] The small molecule polypeptide preferably accounts for 0.005% to 0.01% of the total mass of the pharmaceutical composition.

[0014] The excipient comprises at least one of a transdermal absorption agent, a liposoluble base, an emulsifier, a humectant, a pH regulator and an antioxidant.

[0015] The transdermal absorption agent preferably comprises one or more combinations of laurocapram, urea, turpentine oil and polyethylene glycol; and further preferably laurocapram.

[0016] The liposoluble base preferably comprises one or more combinations of stearic acid, glycerol monostearate, hydrogenated oil, vegetable oil, beeswax, paraffin, liquid paraffin and white petrolatum; and further preferably glycerol monostearate and white petrolatum.

[0017] The emulsifier preferably comprises one or more combinations of acrylamide dimethyl ammonium propane sulfonate / VP copolymer, cetyltrimethylammonium bromide, sodium laurate, sodium dodecyl sulfate, palmityl alcohol, lecithin and cholesterol; and further preferably acrylamide dimethyl ammonium propane sulfonate / VP copolymer, lecithin and cholesterol.

[0018] The humectant preferably comprises one or more combinations of glycerol, propylene glycol, betaine, 1,2-hexanediol and p-hydroxyacetophenone; and further preferably glycerol, 1,2-hexanediol and p-hydroxyacetophenone.

[0019] The pH regulator preferably comprises one or more combinations of triethanolamine, citric acid, isobutyl alcohol amine and potassium hydroxide; and further preferably triethanolamine.

[0020] The antioxidant is preferably vitamin E.

[0021] The pharmaceutical composition for treating psoriasis is preferably composed of the following components: 0.005-0.01% of small molecular polypeptide, 0-6% (preferably 0.4-6%) of transdermal absorption agent, 1-18% of liposoluble base, 0.15-13% of emulsifier, 3-15% of humectant, 0.1-0.2% of pH regulator, 0-0.4% of antioxidant, and water as the rest; and the pH of the final ointment is controlled to be 6.9-7.1.

[0022] The pharmaceutical composition for treating psoriasis is further preferably composed of the following components: 0.005-0.01% of small molecular polypeptide, 0-1% of transdermal absorption agent, 12% of liposoluble base, 0.15-0.57% of emulsifier, 4.1% of humectant, 0.15% of pH regulator, 0-0.4% of antioxidant, and water as the rest; and the pH of the final ointment is controlled to be 6.9-7.1.

[0023] The pharmaceutical composition for treating psoriasis is further preferably any one of the following compositions:

[0024] (1) The pharmaceutical composition of pharmaceutical ointment A

[0025] 0.01% of small molecular polypeptide, 1% of laurocapram, 2% of glycerin monostearate, 10% of white petrolatum, 0.15% of acrylamide dimethyl ammonium tosylate / VP copolymer, 3% of glycerin, 0.6% of 1,2-hexanediol, 0.5% of p-hydroxyacetophenone, 0.15% of triethanolamine, and 82.59% of water;

[0026] (2) The pharmaceutical composition of pharmaceutical ointment B

[0027] 0.005% of small molecular polypeptide, 0.32% of lecithin, 0.1% of cholesterol, 2% of glycerin monostearate, 10% of white petrolatum, 0.15% of acrylamide dimethyl ammonium tosylate / VP copolymer, 3% of glycerin, 0.6% of 1,2-hexanediol, 0.5% of p-hydroxyacetophenone, 0.15% of triethanolamine, 0.4% of vitamin E, and 82.775% of water.

[0028] The water is preferably purified water (deionized water).

[0029] A pharmaceutical ointment for treating psoriasis is prepared from the raw materials in the above pharmaceutical composition for treating psoriasis, and the specific steps are as follows:

[0030] S1, preparation of pharmaceutical ointment A

[0031] (a) Small molecule polypeptide pretreatment: a portion of water is used to dissolve a small molecule polypeptide at room temperature to obtain a small molecule polypeptide solution;

[0032] (b) Preparation of oil phase: white petrolatum and glycerol monostearate are heated and melted at 80±5°C, then cooled to 70±5°C and kept at this temperature, and the obtained mixture is used as the oil phase;

[0033] (c) Preparation of water phase: the remaining portion of water, triethanolamine, laurocapram, glycerol, 1,2-hexanediol, and p-hydroxyacetophenone, acrylamide dimethyl ammonium taurate / VP copolymer are heated to 85±5°C to melt, then cooled to 70±5°C and kept at this temperature, and the obtained mixture is used as the water phase;

[0034] (d) Emulsification: the water phase (solution) is slowly added to the oil phase under stirring at a temperature of 70±5°C, and then homogenized, stirred, cooled, and defoamed;

[0035] (e) Addition of small molecule polypeptide: when the emulsified system is cooled to 30±5°C, the small molecule polypeptide solution is added, and then stirred and mixed uniformly, and cooled to room temperature to obtain a drug cream A;

[0036] S2, preparation of a drug cream B:

[0037] (i) Preparation of small molecule polypeptide liposome: small molecule polypeptide, lecithin, and cholesterol are dissolved in chloroform, then most of the organic solvent is removed by rotary evaporation at 35±5°C to form a uniform transparent film, then vacuum dried at room temperature to remove residual organic solvent, then PBS buffer solution is added to the product, and then hydrated at 40±5°C, followed by ultrasonic treatment, centrifugation, filtration, resuspension in a liposome dispersion, and then the small molecule polypeptide liposome is obtained;

[0038] (ii) Preparation of oil phase: white petrolatum and glycerol monostearate are heated and melted at 80±5°C, then cooled to 70±5°C and kept at this temperature, and the obtained mixture is used as the oil phase;

[0039] (iii) Preparation of water phase: water, triethanolamine, glycerol, 1,2-hexanediol, p-hydroxyacetophenone, acrylamide dimethyl ammonium taurate / VP copolymer, and vitamin E are heated to 85±5°C to melt, then cooled to 70±5°C and kept at this temperature, and the obtained mixture is used as the water phase;

[0040] (iv) Emulsification: the water phase (solution) is slowly added to the oil phase under stirring at a temperature of 70±5°C, and then homogenized, stirred, cooled, and defoamed;

[0041] (v) Addition of small molecule polypeptide liposome

[0042] When the system to be emulsified is cooled to 30±5℃, the small-molecule polypeptide liposome is added, and after homogenization and stirring treatment, it is cooled to room temperature to obtain the drug cream B.

[0043] The stirring speed in steps (d) and (iv) is 300-700 r / min.

[0044] The homogenization speed in steps (d), (iv) and (v) is 15000-25000 r / min; preferably 20000 r / min.

[0045] The homogenization time in steps (d), (iv) and (v) is 6-10 min.

[0046] The continued stirring time in steps (e) and (v) is 5-15 min.

[0047] The pH value of the PBS buffer solution in step (i) is 6.3-6.7; preferably 6.5.

[0048] The constant temperature hydration time in step (i) is 1.5-2.5 hours; preferably 2 hours.

[0049] The pore size of the filter membrane in step (i) is 0.8 μm.

[0050] The present application has the following advantages and effects relative to the prior art:

[0051] (1) Since the pH value of the psoriasis-affected skin is acidic, in order to ensure the stability and effectiveness of the polypeptide in the microenvironment of the affected skin, the elution condition in the peptide library screening is reduced to 5.2, and the polypeptide S1 capable of stably binding to the cell receptor in the psoriasis-affected skin is screened. So far, it has not been reported that the polypeptide can be used for the treatment of psoriasis.

[0052] (2) In the present application, the small-molecule polypeptide is used as the drug active substance in the drug composition, and its screening condition is more suitable for the microenvironment of the affected skin. Moreover, it is screened from the natural amino acid peptide library, is natural in origin, has small toxic and side effects, and has the potential to prepare excellent psoriasis treatment drugs.

[0053] (3) The small molecule polypeptide in the present application can inhibit the proliferation of human keratinocytes, but the transdermal effect of the medicated ointment prepared by simply adding the small molecule polypeptide into the ointment base is relatively poor. Therefore, the present application uses the transdermal absorption agent laurocapram to improve the transdermal ability of the ointment, and the transdermal absorption amount of the drug is greatly increased. In addition, the small molecule polypeptide is added into the ointment base in the form of liposomes, so that the stability of the drug composition is obviously improved, the transdermal absorption amount of the drug almost linearly increases with time, and a relatively significant effect of treating psoriasis can be achieved at a low drug concentration (0.005%).

[0054] (4) The present application provides a medicament composition for treating psoriasis with small toxic side effects and good curative effect, which is composed of an effective component and a medically acceptable auxiliary material. The composition is scientific, the preparation process is simple, the medicament composition can inhibit the excessive proliferation of keratinocytes, significantly alleviate the phenotype of mouse psoriasis, increase the number of Tregs cell groups in the diseased skin lesions, has small toxicity and side effects, is suitable for a wide range of people, has good transdermal property, can continuously act on the treatment site, and the small molecule polypeptide can achieve effective treatment of psoriasis. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 is a CCK-8 experimental result graph of the inhibition of the proliferation of human keratinocytes by the small molecule polypeptide S1 (“****” represents P less than 0.0001).

[0056] Figure 2 is a CCK-8 detection result graph of the growth inhibition of the medicated ointment prepared in examples 2 and 3 on human keratinocytes (the comparison of the blank ointment base group with the example 2 group and the example 3 group is indicated by “*”, and “***” represents P less than 0.001).

[0057] Figure 3 is an Edu experimental result graph of the inhibition of DNA synthesis of human keratinocytes by the medicated ointment prepared in examples 2 and 3.

[0058] Figure 4 is an Edu positive rate statistical result graph of the inhibition of DNA synthesis of human keratinocytes by the medicated ointment prepared in examples 2 and 3.

[0059] Figure 5 is a colony formation experimental result graph of the inhibition of the growth of human keratinocytes (HaCaT) under the stimulation of inflammatory factors by the medicated ointment prepared in examples 2 and 3.

[0060] Figure 6is a statistical result graph of the relative percentage of each group of colonies in a colony formation experiment of human keratinocytes (HaCaT) under the stimulation of inflammatory factors for the drug cream prepared in Examples 2 and 3 (comparison of the blank cream base group with the model control group is indicated by "#", comparison of the blank cream base group with the Example 2 group and the Example 3 group is indicated by "*"; "##" indicates P less than 0.01, "***" indicates P less than 0.001).

[0061] Figure 7 is a symptom graph of psoriasis-like skin lesions of each group of mice after administration.

[0062] Figure 8 is a PASI score result graph of each group of mice after administration (comparison of the model control group with the Example 2 group and the Example 3 group is indicated by "*", "***" indicates P less than 0.001).

[0063] Figure 9 is a spleen index statistical result graph of each group of mice after administration (comparison of the blank control group with the model control group is indicated by "#", comparison of the model control group with the Example 2 group and the Example 3 group is indicated by "*"; "###" indicates P less than 0.001, "**" indicates P less than 0.01, "*" indicates P less than 0.05).

[0064] Figure 10 is a HE staining result graph of the back skin of each group of mice after administration.

[0065] Figure 11 is a relative thickness statistical result graph of the back skin of each group of mice after administration (comparison of the blank control group with the model control group is indicated by "#", comparison of the model control group with the Example 2 group and the Example 3 group is indicated by "*"; "####" indicates P less than 0.0001, "****" indicates P less than 0.0001, "***" indicates P less than 0.001).

[0066] Figure 12 is an immunohistochemical staining and quantification result graph of the back skin of each group of mice after administration (comparison of the blank control group with the model control group is indicated by "#", comparison of the model control group with the Example 2 group and the Example 3 group is indicated by "*"; "###" indicates P less than 0.001, "**" indicates P less than 0.01, "*" indicates P less than 0.05).

[0067] Figure 13 is a relative percentage statistical result graph of Treg cells in the back skin of each group of mice after administration (comparison of the blank control group with the model control group is indicated by "#", comparison of the model control group with the Example 2 group and the Example 3 group is indicated by "*";

[0068] "##" means P is less than 0.0001, and "***" means P is less than 0.001. DETAILED DESCRIPTION

[0069] The present application will be further described in conjunction with the following examples, but the embodiments of the present application are not limited thereto. Unless otherwise specified, the reagents, methods and apparatus used in the present application are the conventional reagents, methods and apparatus in the art. The test methods in the following examples, unless otherwise specified, are generally carried out according to the conventional experimental conditions or according to the experimental conditions suggested by the manufacturers. Unless otherwise specified, the reagents and raw materials used in the present application are commercially available.

[0070] Example 1

[0071] 1. Screening of small molecule polypeptide

[0072] The polypeptide sequence in the present application is screened by phage display peptide library (cyclic peptide-7 peptide library) screening technology. The extracellular segment of fibroblast growth factor receptor 2 (FGFR2) is used as a target, and the polypeptide sequence is identified by multiple rounds of screening. The specific steps are as follows:

[0073] First, add an appropriate amount of FGFR2 protein (Yiqiao God, F05-11G) to cover the bottom of the microwell plate, and incubate at 4°C with slight shaking overnight. After the protein solution is aspirated, fill the wells with blocking solution (formula: 0.1M NaHCO3(pH8.6), 5mg / ml BSA, 0.02% NaN3) and block for 2h. Phage peptide library (Ph.D. TM -C7C phage display peptide library kit, Catalog#:E8120) is incubated with the fixed protein at room temperature for 1h with gentle shaking. After the incubation, wash the unbound phage with TBST buffer (pH5.2) for 10 times. Finally, use elution buffer (TBST buffer) to dissociate the bound phage and infect E. coli ER2738 (BNCC359223, Shangcheng Beinaunai Liangshengye Technology Co., Ltd.) for amplification. After 5 rounds of screening, the selected phage is cloned and sequenced to determine the sequence of the peptide displayed by the phage. The final selected peptide is verified to determine its function. Sequencing shows that the small molecule polypeptide consists of 9 amino acid residues, which is a cyclic peptide derived from the sequence CNAGQRSEC, and is named S1.

[0074] 2. Inhibitory effect of small molecule polypeptide S1 on the growth of human keratinocytes (HaCaT)

[0075] Cell experiment: CCK-8 method was used to detect the growth inhibition effect of small molecule polypeptide solution on human keratinocytes (HaCaT), wherein the human keratinocytes HaCaT were purchased from Sibcro (Shanghai) Biotechnology Co., Ltd.

[0076] HaCaT cells in logarithmic growth phase were taken, and 96-well plates were plated at a density of 3000 cells per well. After adhering, they were starved for 24 hours. An appropriate amount of filtered and sterilized small molecule polypeptide S1 (small molecule polypeptide was dissolved in advance with sterile distilled water) and DMEM medium (gibco, C11995500BT) containing pentafactor (M5, containing 10 μg / L of IL-1α, IL-17, IL-22, TNF-α, Oncostatin M) were mixed to make the final concentration 0, 0.00256, 0.0128, 0.064, 0.32, 1.6, 8, 40, 200 μM. The cells were incubated for 48 hours, and then CCK-8 reagent was added. After placing in a 37°C incubator for 1 hour, the OD value of each well was detected by an enzyme-labeled instrument. Cell survival rate (%) = experimental group absorbance / control group absorbance x 100%. The experiment was set up in triplicate. The results are shown in Figure 1 Small molecule polypeptide S1 can significantly inhibit the proliferation of human keratinocytes.

[0077] Example 2

[0078] Preparation of a medicated cream for treating psoriasis (containing small molecule polypeptide S1):

[0079] By mass percentage, small molecule polypeptide S1 0.01% (synthesized by Suzhou Qiangyao Biotechnology Co., Ltd.), laurocapram 1% (Shanghai Jizhisheng Biotechnology Co., Ltd., 59227-89-3), glycerol monostearate 2% (Aladdin Biochemical Technology Co., Ltd., G196240), white vaseline 10% (Aladdin Biochemical Technology Co., Ltd., V105023), acrylamide dimethyl ammonium tosylate / VP copolymer 0.15% (Nanjing Aipulas Chemical Co., Ltd., APF3651C), glycerol 3% (Aladdin Biochemical Technology Co., Ltd., G358402), 1,2-hexanediol 0.6% (Aladdin Biochemical Technology Co., Ltd., H108067), p-hydroxyacetophenone 0.5% (American CATO Company, CCHM700219), triethanolamine 0.15% (Aladdin Biochemical Technology Co., Ltd., T108154), purified water 82.59%.

[0080] The specific preparation process is as follows:

[0081] (1) Small molecule polypeptide pretreatment: dissolve part of the purified water at room temperature to dissolve the small molecule polypeptide;

[0082] (2) Preparation of oil phase: white petrolatum, glycerol monostearate were heated to melt at 80°C, then cooled to 70°C and kept the temperature, the mixture was used as oil phase;

[0083] (3) Preparation of water phase: the rest of purified water, triethanolamine, laurocapram, glycerol, 1,2-hexanediol, p-hydroxyacetophenone, acrylamide dimethyl ammonium tosylate / VP copolymer were heated to melt at 85°C, then cooled to 70°C and kept the temperature, the mixture was used as water phase;

[0084] (4) Emulsification: under the condition of temperature 70°C and stirring speed 300-700 r / min, the water phase solution was slowly added into the oil phase, and homogenization was started for 6-10 min (speed 20000 r / min), then the system was kept for 5 min and stirred to reduce temperature and defoam;

[0085] (5) When the emulsified system was reduced to 30°C, the pretreated small molecule polypeptide was added, and stirring was continued for 5-15 min, then the system was cooled to room temperature.

[0086] Example 3

[0087] Preparation of a medicated cream for treating psoriasis (containing small molecule polypeptide S1): Different from Example 2, in this example, the small molecule polypeptide was prepared into a liposome solution first, and then mixed with the cream base. The specific process is as follows:

[0088] By mass percentage, small molecule polypeptide S1 0.005% (synthesized by Suzhou Qiangyao Biotechnology Co., Ltd.), lecithin 0.32% (Aidlab Biotech Co., Ltd., L105732), cholesterol 0.1% (Shanghai Macklin Biotechnology Co., Ltd., C804519), glycerol monostearate 2% (Aidlab Biotech Co., Ltd., G196240), white petrolatum 10% (Aidlab Biotech Co., Ltd., V105023), acrylamide dimethyl ammonium tosylate / VP copolymer 0.15% (Nanjing Aipulas Chemical Co., Ltd., APF3651C), glycerol 3% (Aidlab Biotech Co., Ltd., G358402), 1,2-hexanediol 0.6% (Aidlab Biotech Co., Ltd., H108067), p-hydroxyacetophenone 0.5% (Cato Corporation, CCHM700219), triethanolamine 0.15% (Aidlab Biotech Co., Ltd., T108154), vitamin E 0.4% (Huazhong Haiwei Gene Technology Co., Ltd., LS1680), purified water 82.775%.

[0089] The specific preparation process is as follows:

[0090] (1) Preparation of small molecule polypeptide liposome: dissolve small molecule polypeptide, lecithin and cholesterol in an appropriate amount of chloroform solution, then place the mixture in a rotary evaporator, remove most of the organic solvent at 35°C under reduced pressure, form a uniform transparent film, and finally dry the residual organic solvent at room temperature overnight. The next day, add PBS buffer solution (pH 6.5) to the product, and hydrate at 40°C for 2 hours. After ultrasonic treatment, centrifuge, and finally filter with a 0.8 μm pore size filter, resuspend in liposome dispersion (Guangzhou Chuang Sai Biomedical Materials Co., Ltd., CS-202407), and obtain.

[0091] (2) Preparation of oil phase: melt white vaseline and glycerol monostearate at 80°C, cool to 70°C and maintain the temperature, and the mixture is used as the oil phase;

[0092] (3) Preparation of water phase: melt purified water, triethanolamine, acrylamide dimethyl ammonium tosylate / VP copolymer, glycerol, 1,2-hexanediol, p-hydroxyacetophenone, and vitamin E at 85°C, cool to 70°C and maintain the temperature, and the mixture is used as the water phase;

[0093] (4) Emulsification: slowly add the water phase solution to the oil phase at a temperature of 70°C and a stirring speed of 300-700 r / min, turn on the homogenizer for 6-10 min (speed 20000 r / min), and after 5 min of incubation, reduce the stirring speed to defoam;

[0094] (5) When the emulsified system is cooled to 30°C, add the prepared small molecule polypeptide liposome, re-homogenize for 6-10 min (speed 10000 r / min), then stir at 300 r / min for 5-15 min, and cool to room temperature.

[0095] Example 4

[0096] 1. Cell experiment

[0097] 1.1 CCK-8 method was used to detect the growth inhibition effect of the drug cream prepared in Example 2 and Example 3 on human keratinocytes (HaCaT), wherein the human keratinocytes HaCaT were purchased from Sibcro (Shanghai) Biotechnology Co., Ltd.

[0098] Logarithmic growth of HaCaT cells, 3000 cells per hole density in 96-well plates, after adhering, starve for 24 hours, take the right amount of filtered sterile cream and DMEM medium (gibco, C11995500BT) containing pentuple factor (M5, containing 10 μg / L of IL-1α, IL-17, IL-22, TNF-α, Oncostatin M) mixed, so that the final concentration of cream is 50 μg / ml. Incubate the cells for 48 hours, then add CCK-8 reagent, place in 37℃ incubator for 1 hour, then detect the OD value of each well with enzyme marker. Cell survival rate (%) = experimental group absorbance / control group absorbance x 100%. The experiment is set up three times. The blank cream matrix is not added with small molecule polypeptide and small molecule polypeptide liposome, and the rest of the cream system is prepared as in Example 3, which is used as a blank control group for comparison.

[0099] As shown in Figure 2 The cream in Example 2 and Example 3 groups can significantly inhibit the proliferation of human keratinocytes.

[0100] 1.2 Edu experiment to determine the growth inhibition of the drug cream prepared in Example 2 and Example 3 on human keratinocytes (HaCaT).

[0101] DNA synthesis is a marker of cell proliferation. Edu is a thymidine analogue, which is taken into the nucleus during cell proliferation to form Edu-DNA complex. Based on the specific reaction of EdU and Apollo fluorescent dye to detect DNA replication activity, EdU labeling can accurately reflect the proliferation of cells. Refer to step 1.1 above, add the same concentration of drug medium mixture as in the CCK8 experiment to incubate the cells for 48 hours, then fix and stain, and observe under a fluorescence microscope. The experiment is set up three times.

[0102] As shown in Figure 3 and Figure 4 The cream in Example 2 and Example 3 groups can significantly inhibit the DNA synthesis of human keratinocytes.

[0103] 1.3 Cloning experiment to determine the growth inhibition of the drug cream prepared in Example 2 and Example 3 on human keratinocytes (HaCaT).

[0104] Logarithmic growth of HaCaT cells was digested with trypsin and blown into single cells, diluted to a certain multiple and inoculated in 6-well plates (1.5 x 10 3HaCaT cells were seeded in 6-well plates at a density of 1 x 105 cells / well and incubated overnight. The next day, the cells were stimulated with DMEM medium containing the pentafactor (M5, containing 10 μg / L of IL-1α, IL-17, IL-22, TNF-α, Oncostatin M) while an appropriate amount of the filtered and sterilized cream was added to the wells to make the final concentration 50 μg / ml. The cells were incubated for 2 to 3 weeks, the culture medium was discarded, and the cells were fixed and stained with crystal violet. Photographs were taken and the number of cell colonies was counted. The experiment was set in triplicate.

[0105] The results are shown in Tables 2 and 3: Figure 5 and Figure 6 The results show that both the drug creams can significantly inhibit the growth of human keratinocytes (HaCaT) stimulated by inflammatory factors. Notably, in this long-term cell experiment, the drug composition in Example 3 group has half the amount of drug polypeptides than that in Example 2 group, but Example 3 group shows a better inhibitory effect, proving that the polypeptide drugs wrapped in the liposome cream are more active and have a more lasting effect.

[0106] 2. Animal Experiment

[0107] 2.1 Experimental grouping and administration

[0108] Twenty-four male BalB / c mice weighing about 18-22 g (Guangdong Yaoke Biotechnology Co., Ltd.) were randomly divided into four groups, i.e. a blank control group, a model control group, an Example 2 group, and an Example 3 group, with 6 mice in each group. Except for the blank control group, the other groups were smeared with 5% imiquimod cream every morning: the model control group was smeared with blank cream base (without adding small molecule polypeptides and small molecule polypeptide liposomes, as described in 1.1 above) every afternoon, and the Example 2 and 3 groups were smeared with the drug creams prepared in Examples 2 and 3, respectively, each time 0.1 g.

[0109] 2.2 Establishment of psoriasis mouse model and administration

[0110] The day before the experiment, the back hair of the mice was shaved to form a skin exposure area of about 2 x 3 cm. The mice were smeared with 5% imiquimod cream (62.5 mg) on the back skin every morning, once a day, for 6 consecutive days, while the other administration groups were smeared with the corresponding cream (30 mg) every afternoon, once a day, for 6 consecutive days. On the 7th day, the mice were sacrificed by cervical dislocation, and part of the back skin lesions were taken, fixed, and paraffin-embedded for later use.

[0111] 2.3 Scoring of psoriasis-like skin lesion symptoms and disease conditions in mice

[0112] The skin lesions of psoriatic mice were photographed and recorded regularly. The severity of infiltration (I), erythema (E), and epidermal desquamation / scaling (D) of the lesions was scored according to the Psoriasis Area and Severity Index (PASI) criteria. The scores of these three factors were summed to obtain the total score. The PASI scoring criteria were: asymptomatic: 0; mild: 1; moderate: 2; severe: 3; very severe: 4. After collecting tissue samples from the mice, the spleen index was calculated for each group using the formula: Spleen Index = [mouse spleen weight (mg) / mouse body weight (g)] × 1000.

[0113] The results are as follows Figure 7 and Figure 8 As shown, the model control group exhibited significantly more erythema, thickening, and scaling on the back lesions compared to the blank control group, demonstrating that the application of imiquimod cream successfully induced the psoriasis model. Furthermore, compared to the model control group, groups 2 and 3 showed significant reductions in symptoms such as erythema, scaling, and epidermal thickening. Figure 9 The statistical results of the spleen index in mice showed that the drugs in Examples 2 and 3 could effectively inhibit imiquimod-induced splenomegaly, indicating that the drug compositions in Examples 2 and 3 could effectively treat psoriasis.

[0114] 2.4 Histopathology of mouse skin lesions

[0115] The paraffin-embedded tissue was cut into 5μm sections and baked in a 65℃ oven for 1 hour. After dewaxing, hematoxylin was stained for 10 min, rinsed with tap water, and then blued with a blue solution for 3 minutes. After rinsing again with tap water, eosin was stained for 2 min, dehydrated and cleared, mounted with neutral resin, observed and photographed under a microscope, and the thickness of the skin epidermis was calculated.

[0116] In the immunohistochemical staining experiment, paraffin sections were first dewaxed and then treated with 3% hydrogen peroxide at room temperature for 10 min. Microwave repair was then performed, followed by heating the sections to boiling point in 0.01M citrate buffer at maximum power, repeated twice. After the sections cooled to room temperature, they were washed three times with PBS and blocked with 5% (v / v) bovine serum albumin (BSA) for 2 hours, then incubated overnight with primary antibody. After washing three times with PBS, secondary antibody was added, followed by three more washes with PBS. A streptavidin-biotin-peroxidase (SABC) complex was added, and the sections were incubated at room temperature for 2 hours. After washing three times with PBS, 3,3'-diaminobenzidine (DAB) chromogenic solution was added, and the reaction was carried out for approximately 5 min. The sections were then rinsed thoroughly with tap water, counterstained with hematoxylin, dehydrated and cleared, mounted with neutral resin, and observed and photographed under a microscope.

[0117] The results are as follows Figure 10 and Figure 11As shown in the HE-stained micrographs of the skin lesions on the backs of mice in each group, compared with the blank control group, the epidermis of the model control group was significantly thickened, with scaling and significant infiltration of inflammatory cells. In contrast, the psoriasis phenotype and pathological phenotype in Example 2 and Example 3 groups were significantly reduced, along with decreased inflammatory cell infiltration and scaling. Further measurement of the epidermal thickness on HE-stained sections revealed a statistically significant reduction in epidermal thickness in both examples. Additionally, Figure 12 Immunohistochemical results showed that Ki67 expression in the basal layer cells of the epidermis was effectively inhibited in both Example 2 and Example 3 groups. This indicates that the cream formulations prepared in Example 2 and Example 3 can effectively treat the psoriasis mouse model.

[0118] 2.5 Determination of the proportion of Treg cells in mouse spleen

[0119] After euthanizing mice in each experimental group, spleens were removed, ground, and enzymatically digested. Cell suspensions were prepared by sieving through a sieve, centrifugation, and removal of the supernatant. After lysing the red blood cells, the precipitate was washed twice with cold PBS. Cells were counted, and 10-1 cells were collected. 6 The cells were resuspended in 110 μl PBS, and then anti-CD4-FITC antibody (Absin Biotech (Shanghai) Co., Ltd., abs180002) was added according to the instructions and incubated at room temperature in the dark for 30 min. The permeabilized cells were then fixed, and anti-Foxp3-APC antibody (Absin Biotech (Shanghai) Co., Ltd., FL2FP303S) was added and incubated at room temperature in the dark for 30 min. After washing once with PBS, the cells were resuspended in 320 μl PBS, and the proportion of Treg cells in the sample was analyzed by flow cytometry.

[0120] The results are as follows Figure 13 As shown, according to the flow cytometry results of the skin cells on the back of mice in each experimental group, the number of Tregs cells in the model control group was significantly reduced, while the number of Tregs cells induced by imiquimod in the Example 2 and Example 3 groups was significantly inhibited. This proves that the cream preparations prepared in Example 2 and Example 3 can reverse the trend of decreased immunosuppressive function in mice, thereby inhibiting the activation of inflammatory response in mice and finally achieving the purpose of alleviating psoriasis symptoms in mice.

[0121] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A small molecule polypeptide for treating psoriasis, characterized in that: A cyclic peptide consisting of CNAGQRSEC.

2. Use of the small molecular polypeptide for treating psoriasis in the preparation of a drug for treating psoriasis according to claim 1.

3. A pharmaceutical composition for treating psoriasis, characterized by: The small molecular polypeptide according to claim 1 and a pharmaceutically acceptable excipient.

4. The pharmaceutical composition for treating psoriasis according to claim 3, characterized by: The small molecular polypeptide accounts for 0.005% to 0.01% of the total mass of the pharmaceutical composition.

5. The pharmaceutical composition for treating psoriasis according to claim 3, wherein: The excipient comprises at least one of a transdermal absorption agent, a liposoluble base, an emulsifier, a humectant, a pH regulator and an antioxidant; The transdermal absorption agent is one or more combinations of laurocapram, urea, turpentine oil and polyethylene glycol; The liposoluble base is one or more combinations of stearic acid, glycerol monostearate, hydrogenated oil, vegetable oil, beeswax, paraffin, liquid paraffin and white petrolatum; The emulsifier is one or more combinations of acrylamide dimethyl ammonium propane sulfonate / VP copolymer, cetyltrimethylammonium bromide, sodium laurate, sodium dodecyl sulfate, palmitol, lecithin and cholesterol; The humectant is one or more combinations of glycerol, propylene glycol, betaine, 1,2-hexanediol and p-hydroxyacetophenone; The pH regulator is one or more combinations of triethanolamine, citric acid, isobutyl alcohol amine and potassium hydroxide; The antioxidant is vitamin E.

6. The pharmaceutical composition for treating psoriasis according to claim 3, characterized by: The small molecular polypeptide accounts for 0.005% to 0.01%, the transdermal absorption agent accounts for 0% to 6%, the liposoluble base accounts for 1% to 18%, the emulsifier accounts for 0.15% to 13%, the humectant accounts for 3% to 15%, the pH regulator accounts for 0.1% to 0.2%, the antioxidant accounts for 0 to 0.4%, and the rest is water.

7. The pharmaceutical composition for treating psoriasis according to claim 6, characterized by: The small molecular polypeptide accounts for 0.005% to 0.01%, the transdermal absorption agent accounts for 0 to 1%, the liposoluble base accounts for 12%, the emulsifier accounts for 0.15% to 0.57%, the humectant accounts for 4.1%, the pH regulator accounts for 0.15%, the antioxidant accounts for 0 to 0.4%, and the rest is water.

8. The pharmaceutical composition for treating psoriasis according to claim 7, characterized by, The pharmaceutical composition is any one of the following compositions: (1) The pharmaceutical composition of drug cream A The small molecular polypeptide accounts for 0.01%, laurocapram accounts for 1%, glycerol monostearate accounts for 2%, white petrolatum accounts for 10%, acrylamide dimethyl ammonium propane sulfonate / VP copolymer accounts for 0.15%, glycerol accounts for 3%, 1,2-hexanediol accounts for 0.6%, p-hydroxyacetophenone accounts for 0.5%, triethanolamine accounts for 0.15%, and water accounts for 82.59%; (2) The pharmaceutical composition of drug cream B The small molecular polypeptide accounts for 0.005%, lecithin accounts for 0.32%, cholesterol accounts for 0.1%, glycerol monostearate accounts for 2%, white petrolatum accounts for 10%, acrylamide dimethyl ammonium propane sulfonate / VP copolymer accounts for 0.15%, glycerol accounts for 3%, 1,2-hexanediol accounts for 0.6%, p-hydroxyacetophenone accounts for 0.5%, triethanolamine accounts for 0.15%, vitamin E accounts for 0.4%, and water accounts for 82.775%.

9. A medicated cream for the treatment of psoriasis, characterized in that: The raw material for the pharmaceutical composition for treating psoriasis according to any one of claims 6 to 8 is prepared according to the following steps: S1, preparation of drug cream A (a) small molecule polypeptide pretreatment: part of water is used to dissolve small molecule polypeptide at room temperature to obtain a small molecule polypeptide solution; (b) preparation of oil phase: white vaseline and glycerol monostearate are heated and melted at 80±5℃, then cooled to 70±5℃ and kept at this temperature, and the obtained mixture is used as oil phase; (c) preparation of water phase: the remaining part of water, triethanolamine, laurocapram, glycerol, 1,2-hexanediol, and p-hydroxyacetophenone, acrylamide dimethyl ammonium tosylate / VP copolymer are heated to 85±5℃ to melt, then cooled to 70±5℃ and kept at this temperature, and the obtained mixture is used as water phase; (d) emulsification: under the condition of temperature 70±5℃ and stirring, the water phase is slowly added to the oil phase, and after homogenization treatment, stirring, cooling and defoaming are performed; (e) addition of small molecule polypeptide: when the emulsified system is cooled to 30±5℃, the small molecule polypeptide solution is added, and stirring is continued until uniformity is achieved, and then the system is cooled to room temperature to obtain the drug cream A; S2, preparation of drug cream B: (i) preparation of small molecule polypeptide liposome: small molecule polypeptide, lecithin and cholesterol are dissolved in chloroform, then most of the organic solvent is removed by rotary evaporation at 35±5℃ to form a uniform transparent film, then the residual organic solvent is removed by vacuum drying at room temperature, then PBS buffer solution is added to the product, and the product is hydrated at 40±5℃, then subjected to ultrasonic treatment, centrifugation, filtration, resuspension in liposome dispersion to obtain small molecule polypeptide liposome; (ii) preparation of oil phase: white vaseline and glycerol monostearate are heated and melted at 80±5℃, then cooled to 70±5℃ and kept at this temperature, and the obtained mixture is used as oil phase; (iii) preparation of water phase: water, triethanolamine, glycerol, 1,2-hexanediol, p-hydroxyacetophenone, acrylamide dimethyl ammonium tosylate / VP copolymer and vitamin E are heated to 85±5℃ to melt, then cooled to 70±5℃ and kept at this temperature, and the obtained mixture is used as water phase; (iv) emulsification: under the condition of temperature 70±5℃ and stirring, the water phase is slowly added to the oil phase, and after homogenization treatment, stirring, cooling and defoaming are performed; (v) addition of small molecule polypeptide liposome When the emulsified system is cooled to 30±5℃, the small molecule polypeptide liposome is added, and after homogenization and stirring treatment, the system is cooled to room temperature to obtain the drug cream B.

10. The drug cream for treating psoriasis according to claim 9, characterized in that: the stirring speed in steps (d) and (iv) is 300-700 r / min; the homogenization speed in steps (d), (iv) and (v) is 15000-25000 r / min; the homogenization time in steps (d), (iv) and (v) is 6-10 min; the stirring time in steps (e) and (v) is 5-15 min; the pH value of the PBS buffer solution in step (i) is 6.3-6.7; the time for constant temperature hydration in step (i) is 1.5-2.5 hours; the pore size of the filter membrane in step (i) is 0.8 μm.

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