Small molecule polypeptide for treating psoriasis and application of pharmaceutical composition of small molecule polypeptide

Abstract

The invention discloses a small molecule polypeptide for treating psoriasis and application of a pharmaceutical composition of the small molecule polypeptide. According to the present invention, the small molecule polypeptide S1 capable of stably binding to the cell receptor in the psoriasis skin is screened from the peptide library, the small molecule polypeptide S1 is the cyclic peptide composed of CNAGQRSEC, and the small molecule polypeptide can inhibit the proliferation of human keratinocytes. According to the invention, medically acceptable auxiliary materials (such as a transdermal absorbent, a fat-soluble matrix, an emulsifier, a humectant, a pH regulator and an antioxidant) are further added on the basis of the small molecule polypeptide S1 to prepare the medicine cream, so that the stability and transdermal capacity of the medicine can be remarkably improved, and effective treatment of psoriasis is realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of a small molecule polypeptide for treating psoriasis and its pharmaceutical composition. Background Art

[0002] Psoriasis, commonly known as psoriasis vulgaris, is a common chronic inflammatory skin disease that affects 2% - 5% of the world's population. The World Health Organization defines it as a chronic, inflammatory, recurrent and incurable non-communicable disease, and it is listed as one of the most serious non-communicable diseases because complications occur during its course and it affects multiple organ systems. Since psoriasis is a heterogeneous disease, genetic susceptibility loci and environmental factors such as skin trauma, infection, and stress are important factors that induce or affect the development of psoriasis. Its clinical types can be divided into vulgaris, arthropathic, pustular, palmoplantar pustulosis, erythrodermic, etc., and the vulgaris type, which accounts for 85% - 90%, shows skin erythema with silver raised scales. The specific manifestations of pathological tissues are epidermal keratinocyte hyperproliferation, parakeratosis, thickening of the epidermal spinous layer, dilation of superficial dermal microvessels, and infiltration of inflammatory cells in the dermis. Research shows that the quality of life and social mental health of psoriasis patients are usually greatly negatively affected. Compared with ordinary people, most psoriasis patients are more likely to have an inferiority complex and even suffer from depression.

[0003] In view of the increasing trend of the incidence of psoriasis year by year and it has become one of the key research diseases in the current dermatology field, currently, a variety of treatment options have been developed. For moderate to severe psoriasis that cannot be controlled by topical medications, oral medications such as methotrexate, cyclosporine, and vitamin A analogs are usually used, but these oral medications also show side effects such as hepatotoxicity and gastrointestinal discomfort. Although the biological agents developed for targeted immunotherapy of psoriasis have good efficacy and safety, the increased risk of infection and their high medication costs mentioned in relevant reports still limit the application of such drugs. As the first-line medication for local treatment with the best patient medication compliance, vitamin D derivatives have definite curative effects, but long-term use is likely to cause side effects such as skin dryness and redness, and increased blood calcium. Glucocorticoids, which are also widely used, are likely to cause adverse reactions such as local skin atrophy, thinning of the stratum corneum, and pigmentation after long-term use, and are prone to recurrence or rebound after discontinuation. Therefore, it is important to seek an external medication for treating psoriasis with low toxicity and side effects and good curative effects. Summary of the Invention

[0004] The primary object of the present invention is to overcome the deficiencies of the prior art and provide a small molecule polypeptide for treating psoriasis.

[0005] Another object of the present invention is to provide the above-mentioned pharmaceutical composition for treating psoriasis.

[0006] Another object of the present invention is to provide a method for preparing the pharmaceutical composition for treating psoriasis.

[0007] Another object of the present invention 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 invention 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] Use of the small molecule polypeptide for treating psoriasis in the preparation of a medicament for treating psoriasis.

[0011] The medicament can be made into various dosage forms according to needs, such as ointments (cream preparations), etc.

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

[0013] Preferably, the small molecule polypeptide accounts for 0.005% - 0.01% of the total mass of the pharmaceutical composition.

[0014] The excipient includes at least one of a transdermal absorption agent, a fat-soluble matrix, an emulsifier, a humectant, a pH regulator, and an antioxidant.

[0015] Preferably, the transdermal absorption agent is one or a combination of more of azone, urea, turpentine, and polyethylene glycol; more preferably azone.

[0016] Preferably, the fat-soluble matrix is one or a combination of more of stearic acid, glyceryl monostearate, hydrogenated oil, vegetable oil, beeswax, paraffin wax, liquid paraffin, and white petrolatum; more preferably glyceryl monostearate and white petrolatum.

[0017] Preferably, the emulsifier is one or a combination of more of acrylamide dimethyltaurate / VP copolymer, cetyltrimethylammonium bromide, sodium laurate, sodium dodecyl sulfate, cetyl alcohol, lecithin, and cholesterol; more preferably acrylamide dimethyltaurate / VP copolymer, lecithin, and cholesterol.

[0018] Preferably, the humectant is one or a combination of more of glycerol, propylene glycol, betaine, 1,2 - hexanediol, and p - hydroxyacetophenone; more preferably glycerol, 1,2 - hexanediol, and p - hydroxyacetophenone.

[0019] Preferably, the pH regulator is one or a combination of more of triethanolamine, citric acid, isobutanolamine, and potassium hydroxide; more preferably triethanolamine.

[0020] The antioxidant mentioned above is preferably vitamin E.

[0021] The pharmaceutical composition for treating psoriasis preferably comprises the following components: by mass percentage, small molecule polypeptide 0.005% - 0.01%, transdermal absorbent 0% - 6% (preferably 0.4% - 6%), liposoluble matrix 1% - 18%, emulsifier 0.15% - 13%, humectant 3% - 15%, pH regulator 0.1% - 0.2%, antioxidant 0 - 0.4%, and the balance is water; the pH of the finally prepared ointment is controlled between 6.9 and 7.1.

[0022] The pharmaceutical composition for treating psoriasis is further preferably composed of the following components: by mass percentage, small molecule polypeptide 0.005% - 0.01%, transdermal absorbent 0 - 1%, liposoluble matrix 12%, emulsifier 0.15% - 0.57%, humectant 4.1%, pH regulator 0.15%, antioxidant 0 - 0.4%, and the balance is water; the pH of the finally prepared ointment is controlled between 6.9 and 7.1.

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

[0024] (1) The pharmaceutical composition of medicated cream A

[0025] By mass percentage, small molecule polypeptide 0.01%, azone 1%, glyceryl monostearate 2%, white petrolatum 10%, acrylamide dimethyltaurate / VP copolymer 0.15%, glycerol 3%, 1,2 - hexanediol 0.6%, hydroxyacetophenone 0.5%, triethanolamine 0.15%, and water 82.59%;

[0026] (2) The pharmaceutical composition of medicated cream B

[0027] By mass percentage, small molecule polypeptide 0.005%, lecithin 0.32%, cholesterol 0.1%, glyceryl monostearate 2%, white petrolatum 10%, acrylamide dimethyltaurate / VP copolymer 0.15%, glycerol 3%, 1,2 - hexanediol 0.6%, hydroxyacetophenone 0.5%, triethanolamine 0.15%, vitamin E 0.4%, and water 82.775%.

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

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

[0030] S1. Prepare medicated cream A

[0031] (a) Pretreatment of small molecule polypeptide: Take a part of water and dissolve the small molecule polypeptide at room temperature to obtain a small molecule polypeptide solution;

[0032] (b) Preparation of oil phase: Heat and melt white petrolatum and glyceryl monostearate at 80 ± 5 °C, then cool to 70 ± 5 °C and maintain this temperature. Use the obtained mixture as the oil phase;

[0033] (c) Preparation of water phase: Heat the remaining water, triethanolamine, laurocapram, glycerol, 1,2 - hexanediol, p - hydroxyacetophenone, and acrylamidodimethyltaurate / VP copolymer to 85 ± 5 °C for melting, then cool to 70 ± 5 °C and maintain this temperature. Use the obtained mixture as the water phase;

[0034] (d) Emulsification: Under the conditions of a temperature of 70 ± 5 °C and stirring, slowly add the water phase (solution) to the oil phase. After homogenization treatment, stir to cool and defoam;

[0035] (e) Adding small molecule polypeptide: When the temperature of the emulsified system drops to 30 ± 5 °C, add the small molecule polypeptide solution, continue to stir and mix evenly, and cool to room temperature to obtain drug cream A;

[0036] S2. Preparation of drug cream B:

[0037] (i) Preparation of small molecule polypeptide liposome: Dissolve the small molecule polypeptide, lecithin, and cholesterol in chloroform. Then, under the condition of 35 ± 5 °C, rotate and evaporate to remove most of the organic solvents to form a uniform transparent film. Then, under room temperature conditions, vacuum dry to remove the residual organic solvents. Then add PBS buffer solution to the product, and carry out constant temperature hydration at 40 ± 5 °C. After ultrasonic treatment, centrifuge, filter through a membrane, and resuspend in the liposome dispersion to obtain small molecule polypeptide liposomes;

[0038] (ii) Preparation of oil phase: Heat and melt white petrolatum and glyceryl monostearate at 80 ± 5 °C, then cool to 70 ± 5 °C and maintain this temperature. Use the obtained mixture as the oil phase;

[0039] (iii) Preparation of water phase: Heat water, triethanolamine, glycerol, 1,2 - hexanediol, p - hydroxyacetophenone, acrylamidodimethyltaurate / VP copolymer, and vitamin E to 85 ± 5 °C for melting, then cool to 70 ± 5 °C and maintain this temperature. Use the obtained mixture as the water phase;

[0040] (iv) Emulsification: Under the conditions of a temperature of 70 ± 5 °C and stirring, slowly add the water phase (solution) to the oil phase. After homogenization treatment, stir to cool and defoam;

[0041] (v) Adding small molecule polypeptide liposome

[0042] When the temperature of the emulsified system drops to 30 ± 5 °C, small molecule polypeptide liposomes are added. After homogenization and stirring, it is cooled to room temperature to obtain drug cream B.

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

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

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

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

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

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

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

[0050] The present invention has the following advantages and effects compared with the prior art:

[0051] (1) Since the pH value of the skin affected by psoriasis is acidic, in order to ensure the stability and effectiveness of the polypeptide in the microenvironment of the affected skin, the elution conditions in the peptide library screening of the present invention are reduced to 5.2, and polypeptide S1 that can stably bind to cell receptors in psoriatic skin is screened out. So far, it has not been reported that this polypeptide can be used for the treatment of psoriasis.

[0052] (2) In the present invention, small molecule polypeptides are used as the drug active substances in the drug composition. Their screening conditions are more suitable for the microenvironment of the affected skin, and they are screened from a natural amino acid peptide library, with natural sources, low toxicity and side effects, and have the potential to prepare excellent drugs for the treatment of psoriasis.

[0053] (3) The small molecule polypeptide in the present invention can inhibit the proliferation of human keratinocytes. However, when the small molecule polypeptide is simply added to the cream matrix to prepare the drug cream, the transdermal effect is relatively poor. Therefore, the present invention uses the transdermal absorbent azone to improve the transdermal ability of the ointment, and the transdermal absorption amount of the drug is greatly increased. In addition, the present invention also adds the small molecule polypeptide to the cream matrix in the form of liposomes, which significantly improves the stability of the drug composition. The transdermal absorption amount of the drug almost linearly increases with time, and it can achieve a significant therapeutic effect on psoriasis at a relatively low drug concentration (0.005%).

[0054] (4) The present invention provides a drug composition for treating psoriasis with low toxicity and side effects and good curative effect. The drug composition is composed of an active ingredient and a medically acceptable excipient. The formulation is scientific, the preparation process is simple, and the drug composition can inhibit the excessive proliferation of keratinocytes, significantly relieve the phenotype of psoriasis in mice, increase the number of Tregs cell populations in the diseased skin lesions, and has low toxicity and side effects, a wide range of applicable populations, good transdermal permeability, and can continuously act on the treatment site. The small molecule polypeptide can effectively treat psoriasis. Description of the Drawings

[0055] Figure 1 It is the CCK-8 experimental result graph of the inhibition of the proliferation of human keratinocytes by the small molecule polypeptide S1 ("****" indicates that P is less than 0.0001).

[0056] Figure 2 It is the CCK-8 detection result graph of the growth inhibitory effect of the drug creams prepared in Examples 2 and 3 on human keratinocytes (HaCaT) (the comparison between the blank cream matrix group and the groups of Example 2 and Example 3 is indicated by "*", and "***" indicates that P is less than 0.001).

[0057] Figure 3 It is the Edu experimental result graph of the inhibition of DNA synthesis of human keratinocytes by the drug creams prepared in Examples 2 and 3.

[0058] Figure 4 It is the statistical result graph of the Edu positive rate of the inhibition of DNA synthesis of human keratinocytes by the drug creams prepared in Examples 2 and 3 (the comparison between the blank cream matrix group and the groups of Example 2 and Example 3 is indicated by "*", and "***" indicates that P is less than 0.001).

[0059] Figure 5 It is the clone formation experimental result graph of the inhibition of the growth of human keratinocytes (HaCaT) stimulated by inflammatory factors by the drug creams prepared in Examples 2 and 3.

[0060] Figure 6It is a statistical result graph of the relative percentages of clone colonies in each group in the clone formation experiment of inhibiting the growth of human keratinocytes (HaCaT) stimulated by inflammatory factors by the drug creams prepared in Examples 2 and 3 (the comparison between the blank cream matrix group and the model control group is indicated by "#", and the comparison between the blank cream matrix group and the groups of Example 2 and Example 3 is indicated by "*"; "##" indicates that P is less than 0.01, and "***" indicates that P is less than 0.001).

[0061] Figure 7 It is a graph of the psoriatic-like skin lesion symptoms of each group of mice after drug administration treatment.

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

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

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

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

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

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

[0068] “####” indicates P less than 0.0001, and “***” indicates P less than 0.001). DETAILED DESCRIPTION

[0069] The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0070] Example 1

[0071] 1. Screening of small molecule peptides

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

[0073] First, add an appropriate amount of FGFR2 protein (Sino Biological, F05-11G) to the polypropylene microplate to cover the bottom of the microwells, and incubate with slight shaking at 4°C overnight. After aspirating the protein solution, fill the wells with blocking solution (formula: 0.1M NaHCO3 (pH8.6), 5mg / ml BSA, 0.02% NaN3) to block for 2h. The phage peptide library (Ph.D. TM -C7C phage display peptide library kit, Catalog #: E8120) and the fixed protein were gently shaken at room temperature for 1 hour, and then washed 10 times with TBST buffer (pH 5.2) to remove unbound phages. Finally, the bound phages were dissociated using elution buffer (TBST buffer) and infected with Escherichia coli ER2738 (BNCC359223, Shangcheng Beina Chuanglian Biotechnology Co., Ltd.) for amplification. After 5 rounds of screening, the screened phages were cloned and sequenced to determine the sequences of the peptides displayed by these phages. The peptides finally screened out were verified to determine their functions. After sequencing, the small molecule polypeptide consists of 9 amino acid residues and is a cyclic peptide derived from the sequence CNAGQRSEC, which is named S1.

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

[0075] Cell experiment: The CCK-8 method was used to detect the growth inhibitory effect of the small molecule polypeptide solution on human keratinocytes (HaCaT). Among them, the human keratinocyte HaCaT was purchased from Cyagen Biosciences (Shanghai) Inc.

[0076] HaCaT cells in the logarithmic growth phase were seeded in 96-well plates at a density of 3000 cells per well. After attachment, the cells were starved for 24 hours. Appropriate amounts of sterile-filtered small molecule polypeptide S1 (the small molecule polypeptide was dissolved in sterile distilled water in advance) 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 their final concentrations 0, 0.00256, 0.0128, 0.064, 0.32, 1.6, 8, 40, 200 μM. The cells were incubated with the mixture for 48 hours, and then CCK-8 reagent was added. After being placed in a 37°C incubator for 1 hour, the OD values of each well were measured with an enzyme-linked immunosorbent assay (ELISA) reader. Cell survival rate (%) = absorbance value of the experimental group / absorbance value of the control group × 100%. The experiment was set up with three replicates. The results are as Figure 1 shown: Small molecule polypeptide S1 can significantly inhibit the proliferation of human keratinocytes.

[0077] Example 2

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

[0079] By mass percentage, small molecule polypeptide S1 is 0.01% (synthesized by Suzhou Qiangyao Biotechnology Co., Ltd.), laurocapram is 1% (Shanghai Aladdin Biochemical Technology Co., Ltd., 59227-89-3), glyceryl monostearate is 2% (Aladdin Biochemical Technology Co., Ltd., G196240), white petrolatum is 10% (Aladdin Biochemical Technology Co., Ltd., V105023), acrylamide dimethyltaurate / VP copolymer is 0.15% (Nanjing Apras Chemical Co., Ltd., APF3651C), glycerol is 3% (Aladdin Biochemical Technology Co., Ltd., G358402), 1,2-hexanediol is 0.6% (Aladdin Biochemical Technology Co., Ltd., H108067), p-hydroxyacetophenone is 0.5% (CATO Corporation, USA, CCHM700219), triethanolamine is 0.15% (Aladdin Biochemical Technology Co., Ltd., T108154), and purified water is 82.59%.

[0080] The specific preparation process is as follows:

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

[0082] (2) Preparation of the oil phase: Melt white petrolatum and glyceryl monostearate at 80°C, then cool to 70°C and maintain this temperature. This mixture serves as the oil phase;

[0083] (3) Preparation of the aqueous phase: Heat the remaining purified water, triethanolamine, laurocapram, glycerol, 1,2 - hexanediol, hydroxyacetophenone, and acrylamidodimethyltaurate / VP copolymer to 85°C until melted, then cool to 70°C and maintain this temperature. This mixture serves as the aqueous phase;

[0084] (4) Emulsification: At a temperature of 70°C and a stirring speed of 300 - 700 r / min, slowly add the aqueous phase solution to the oil phase, start homogenization for 6 - 10 min (rotation speed 20000 r / min), keep warm for 5 min, then stir and cool to remove bubbles;

[0085] (5) When the emulsified system cools to 30°C, add the pretreated small molecule polypeptide, continue stirring for 5 - 15 min, and then cool 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 is first prepared into a liposome solution and then mixed with the cream matrix. 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% (Aladdin Biochemical Technology Co., Ltd., L105732), cholesterol 0.1% (Shanghai Macklin Biochemical Co., Ltd., C804519), glyceryl monostearate 2% (Aladdin Biochemical Technology Co., Ltd., G196240), white petrolatum 10% (Aladdin Biochemical Technology Co., Ltd., V105023), acrylamidodimethyltaurate / VP copolymer 0.15% (Nanjing Apras Chemical Co., Ltd., APF3651C), glycerol 3% (Aladdin Biochemical Technology Co., Ltd., G358402), 1,2 - hexanediol 0.6% (Aladdin Biochemical Technology Co., Ltd., H108067), hydroxyacetophenone 0.5% (CATO Corporation, USA, CCHM700219), triethanolamine 0.15% (Aladdin Biochemical Technology 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 liposomes: Dissolve small molecule polypeptides, lecithin, and cholesterol in an appropriate amount of chloroform solution. Then, place the mixture in a rotary evaporator and remove most of the organic solvents under reduced pressure at 35°C to form a uniform transparent film. Finally, dry it overnight under vacuum at room temperature to remove the residual organic solvents. The next day, add PBS buffer solution (pH 6.5) to the product, hydrate it at a constant temperature of 40°C for 2 hours, centrifuge it after ultrasonic treatment, and finally filter it through a filter membrane with a pore size of 0.8 μm and resuspend it in liposome dispersion (Guangzhou Chuangsai Biomedical Materials Co., Ltd., CS-202407) to obtain the product.

[0091] (2) Preparation of the oil phase: Melt white petrolatum and glyceryl monostearate at 80°C, then cool it to 70°C and maintain this temperature. This mixture serves as the oil phase.

[0092] (3) Preparation of the aqueous phase: Heat purified water, triethanolamine, acrylamidopropyl dimethyltaurate / VP copolymer, glycerol, 1,2 - hexanediol, hydroxyacetophenone, and vitamin E to 85°C until melted, then cool it to 70°C and maintain this temperature. This mixture serves as the aqueous phase.

[0093] (4) Emulsification: At a temperature of 70°C and a stirring speed of 300 - 700 r / min, slowly add the aqueous phase solution to the oil phase, start homogenization for 6 - 10 min (rotation speed 20000 r / min), keep warm for 5 min, and then stir and cool to remove bubbles.

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

[0095] Example 4

[0096] 1. Cell experiments

[0097] 1.1 Detection of the growth inhibitory effect of the drug creams prepared in Example 2 and Example 3 on human keratinocytes (HaCaT) by the CCK - 8 method. Among them, human keratinocytes HaCaT were purchased from Cyagen (Shanghai) Biotechnology Co., Ltd.

[0098] Take HaCaT cells in the logarithmic growth phase, seed them in a 96-well plate at a density of 3000 cells per well, and after they adhere, starve them for 24 hours. Then, take appropriate amounts of the cream after filtration sterilization and mix it with DMEM medium (gibco, C11995500BT) containing pentafactor (M5, containing 10 μg / L of IL-1α, IL-17, IL-22, TNF-α, Oncostatin M) so that the final concentration of the cream is 50 μg / ml. Incubate the cells with it for 48 hours, then add CCK-8 reagent, and after placing it in a 37°C incubator for 1 hour, measure the OD value of each well with an enzyme-linked immunosorbent assay (ELISA) reader. Cell survival rate (%) = absorbance value of the experimental group / absorbance value of the control group × 100%. The experiment is set up with three replicates. The blank cream matrix is the remaining cream system without adding small molecule polypeptides and small molecule polypeptide liposomes, and the remaining preparation steps are the same as in Example 3, which is used as the blank control group of the experiment for comparison.

[0099] The results are as Figure 2 shown: The creams in Example 2 group and Example 3 group can significantly inhibit the proliferation of human keratinocytes.

[0100] 1.2 Edu experiment was used to determine the growth inhibitory effect of the drug creams 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. During cell proliferation, Edu will be taken up into the nucleus to form an Edu-DNA complex. Based on the specific reaction between EdU and Apollo fluorescent dye, the DNA replication activity is detected, and detecting the EdU label can accurately reflect the cell proliferation situation. Referring to the above step 1.1, add a drug medium mixture with the same concentration as in the CCK8 experiment to incubate the cells for 48 hours, fix and stain them, and observe under a fluorescence microscope. The experiment is set up with three replicates.

[0102] The results are as Figure 3 and Figure 4 shown: The creams in Example 2 group and Example 3 group can significantly inhibit the DNA synthesis of human keratinocytes.

[0103] 1.3 Colony formation experiment was used to determine the growth inhibitory effect of the drug creams prepared in Example 2 and Example 3 on human keratinocytes (HaCaT).

[0104] Digest HaCaT cells in the logarithmic growth phase with trypsin and pipette them into single cells. After diluting them to a certain multiple, inoculate them in 6-well plates (1.5×10 3(cells / well), and gently rotate in a figure-eight motion to evenly disperse the cells. While stimulating HaCaT cells with DMEM medium (gibco, C11995500BT) containing five cytokines (M5, containing 10 μg / L of IL-1α, IL-17, IL-22, TNF-α, Oncostatin M), appropriate amounts of the sterilized-by-filtration cream were added to the wells to a final concentration of 50 μg / ml. The cells were incubated for 2 to 3 weeks, and after discarding the medium, the cells were fixed and stained with crystal violet. Photographs were taken for observation and the number of cell clone clusters was counted. The experiment was set up with three replicates.

[0105] The results are as Figure 5 and Figure 6 shown: The experimental results show that both drug creams can significantly inhibit the growth of human keratinocytes (HaCaT) stimulated by inflammatory factors. It is worth noting that in this long-term cell experiment, although the drug polypeptide content in the drug composition in the Example 3 group is half less than that in the Example 2 group, the Example 3 group shows a better inhibitory effect, proving that in the cream containing liposomes, the encapsulated polypeptide drug has more stable activity and more persistent efficacy.

[0106] 2. Animal experiments

[0107] 2.1 Experimental grouping and drug administration

[0108] Twenty-four male BalB / c mice (Guangdong Medicilon Biotechnology Co., Ltd.) weighing about 18 - 22 g were randomly divided into 4 groups, namely the blank control group, the model control group, the Example 2 group, and the 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: among them, the model control group was smeared with the blank cream matrix (without adding small molecule polypeptide and small molecule polypeptide liposome, the same as above 1.1) every afternoon, and the Example 2 group and the Example 3 group were smeared with the drug creams prepared in Examples 2 and 3 respectively, 0.1 g each time.

[0109] 2.2 Establishment of a psoriasis mouse model and drug administration treatment

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

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

[0112] The skin lesions of psoriasis mice were photographed regularly, and the severity of infiltration (I), erythema (E), and epidermal desquamation / scale (D) at the skin lesions of the mice was scored according to the Psoriasis Area and Severity Index (PASI) scoring standard. The total score was obtained by adding the scores of the three items. The PASI scoring standard is: asymptomatic: 0; mild: 1; moderate: 2; severe: 3; extremely severe: 4. After the mice were sacrificed, the spleen index of each group was calculated, and the calculation formula was: Spleen Index = [mouse spleen weight (mg) / mouse body weight (g)] × 1000.

[0113] The results are as Figure 7 and Figure 8 shown. It can be seen that compared with the blank control group, the back skin lesions of the model control group had obvious erythema, thickening, and scales, indicating that the application of imiquimod cream successfully induced a psoriasis model. Compared with the model control group, the groups of Example 2 and Example 3 showed significant alleviation of symptoms such as erythema, scales, and epidermal thickening. In addition, Figure 9 the statistical results of the spleen index of the mice in showed that the drugs in Example 2 and Example 3 could effectively inhibit the splenomegaly induced by imiquimod, indicating that the drug compositions in Example 2 and Example 3 could effectively treat psoriasis.

[0114] 2.4 Histopathology of mouse skin lesions

[0115] The paraffin-embedded tissues were cut into 5-μm sections and baked in an oven at 65 °C for 1 hour. After deparaffinization, hematoxylin staining was performed for 10 min, followed by blueing in a blueing solution for 3 min after rinsing with tap water. After rinsing with tap water again, eosin staining was performed for 2 min, followed by dehydration, clearing, and mounting with neutral gum. The thickness of the skin epidermis was observed, photographed, and calculated under a microscope.

[0116] In the immunohistochemical staining experiment, first, the paraffin sections were deparaffinized and then treated with 3% hydrogen peroxide at room temperature for 10 min. Then, microwave repair was performed. The sections were placed in 0.01 M citrate buffer and heated to boiling at maximum power, repeated 2 times. After the sections were naturally cooled to room temperature, they were washed 3 times with PBS and blocked with 5% (v / v) bovine serum albumin (BSA) for 2 hours. The primary antibody was added and incubated overnight. After washing 3 times with PBS, the secondary antibody was added, followed by washing 3 times with PBS. The streptavidin-biotin-peroxidase (SABC) complex was added and incubated at room temperature for 2 hours. After washing 3 times with PBS again, 3,3'-diaminobenzidine (DAB) chromogenic solution was added and reacted for about 5 min. Then, it was rinsed thoroughly with tap water, counterstained with hematoxylin, dehydrated, cleared, and mounted with neutral gum. Observation and photography were performed under a microscope.

[0117] The results are as Figure 10 and Figure 11As shown, from the HE staining micrographs of the skin lesions on the backs of each group of mice, it can be seen that compared with the blank control group, the epidermis of the model control group was significantly thickened, scales were produced, and inflammatory cells were significantly infiltrated. The psoriasis phenotype and pathological phenotype of the groups of Example 2 and Example 3 were significantly alleviated, and at the same time, the infiltration of inflammatory cells and the production of scales were reduced. Further measurement of the thickness of the epidermis in the HE-stained sections found that the epidermal thickness of the two examples was significantly reduced and statistically significant. In addition, Figure 12 The results of immunohistochemical experiments showed that the expression of Ki67 in the basal cells of the epidermis in the groups of Example 2 and Example 3 was effectively inhibited. It shows that the cream preparations 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 the spleen of mice

[0119] After sacrificing the mice in each experimental group, the spleens were taken out, ground and enzymatically digested, and then passed through a sieve to make a cell suspension. After centrifugation, the supernatant was aspirated. After lysing the red blood cells therein, the precipitate was washed twice with cold PBS. After cell counting, 10 6 cells were resuspended in 110 μl PBS, and then anti-CD4-FITC antibody (Absin Bioscience Inc., abs180002) was added according to the instructions and incubated at room temperature in the dark for 30 min. Subsequently, the cells were fixed and permeabilized, and then anti-Foxp3-APC antibody (Abbiotec Medical Technology (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 Figure 13 shown. According to the flow cytometry results of the skin cells on the backs of the mice in each experimental group, the number of Tregs cells in the model control group was significantly reduced, while the groups of Example 2 and Example 3 could significantly inhibit the reduction in the number of Tregs cells induced by imiquimod, proving that the cream preparations prepared in Example 2 and Example 3 can reverse the trend of the decline in the immunosuppressive function in mice, thereby inhibiting the activation of the inflammatory response in mice, and finally achieving the purpose of alleviating the psoriasis symptoms of 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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

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

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

3. A pharmaceutical composition for treating psoriasis, characterized in that: The invention comprises the small molecule polypeptide according to claim 1 and medically acceptable excipients.

4. The pharmaceutical composition for treating psoriasis according to claim 3, characterized in that: The small molecule 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, characterized in that: The auxiliary materials include at least one of a transdermal absorbent, a fat-soluble matrix, an emulsifier, a moisturizer, a pH regulator and an antioxidant; The transdermal absorbent is one or more combinations of laurocapram, urea, turpentine and polyethylene glycol; The fat-soluble matrix is ​​one or more combinations of stearic acid, glyceryl monostearate, hydrogenated oil, vegetable oil, beeswax, paraffin, liquid paraffin and white vaseline; The emulsifier is one or more combinations of acrylamide dimethyl taurate ammonium / VP copolymer, hexadecyl trimethyl ammonium bromide, sodium laurate, sodium lauryl sulfate, palmityl alcohol, lecithin and cholesterol; The moisturizing agent is one or more combinations of glycerin, propylene glycol, betaine, 1,2-hexanediol and p-hydroxyacetophenone; The pH regulator is one or more combinations of triethanolamine, citric acid, isobutanolamine and potassium hydroxide; The antioxidant is vitamin E.

6. The pharmaceutical composition for treating psoriasis according to claim 3, characterized in that: Calculated by mass percentage, the small molecule polypeptide is 0.005% to 0.01%, the transdermal absorbent is 0% to 6%, the fat-soluble matrix is ​​1% to 18%, the emulsifier is 0.15% to 13%, the moisturizer is 3% to 15%, the pH adjuster is 0.1% to 0.2%, the antioxidant is 0 to 0.4%, and water is the balance.

7. The pharmaceutical composition for treating psoriasis according to claim 6, characterized in that: Calculated by mass percentage, the small molecule polypeptide is 0.005% to 0.01%, the transdermal absorbent is 0 to 1%, the fat-soluble matrix is ​​12%, the emulsifier is 0.15% to 0.57%, the moisturizer is 4.1%, the pH adjuster is 0.15%, the antioxidant is 0 to 0.4%, and the balance is water.

8. The pharmaceutical composition for treating psoriasis according to claim 7, characterized in that: The pharmaceutical composition is any one of the following compositions: (1) Pharmaceutical composition of medicated cream A By mass percentage, small molecule polypeptide 0.01%, laurocapram 1%, glyceryl monostearate 2%, white vaseline 10%, acrylamide dimethyl ammonium taurate / VP copolymer 0.15%, glycerol 3%, 1,2-hexanediol 0.6%, p-hydroxyacetophenone 0.5%, triethanolamine 0.15%, water 82.59%; (2) Pharmaceutical composition of medicated cream B By mass percentage, it includes 0.005% small molecule polypeptide, 0.32% lecithin, 0.1% cholesterol, 2% glyceryl monostearate, 10% white vaseline, 0.15% acrylamide dimethyl taurate ammonium / VP copolymer, 3% glycerol, 0.6% 1,2-hexanediol, 0.5% p-hydroxyacetophenone, 0.15% triethanolamine, 0.4% vitamin E and 82.775% water.

9. A medicinal cream for treating psoriasis, characterized in that: The method is prepared by using the raw material of the pharmaceutical composition for treating psoriasis according to any one of claims 6 to 8, and the specific steps are as follows: S1. Preparation of Medicinal Cream A (a) Pretreatment of small molecule polypeptides: taking a portion of water to dissolve the small molecule polypeptides at room temperature to obtain a small molecule polypeptide solution; (b) preparing the oil phase: heating white vaseline and glyceryl monostearate to melt at 80±5°C, cooling to 70±5°C and maintaining the temperature, and using the obtained mixture as the oil phase; (c) preparing the aqueous phase: heating the remaining water, triethanolamine, laurocapram, glycerol, 1,2-hexanediol, p-hydroxyacetophenone, and acrylamide dimethyl taurate ammonium / VP copolymer to 85±5°C to melt, cooling to 70±5°C and maintaining the temperature, and using the obtained mixture as the aqueous phase; (d) Emulsification: slowly add the water phase to the oil phase at a temperature of 70±5°C and stirring, and after homogenization, stir to cool and defoam; (e) Adding small molecule polypeptide: When the temperature of the emulsified system is lowered to 30±5° C., add the small molecule polypeptide solution, continue to stir and mix evenly, and cool to room temperature to obtain drug cream A; S2. Preparation of medicated cream B: (i) preparing small molecule polypeptide liposomes: dissolving small molecule polypeptide, lecithin and cholesterol in chloroform, then removing most of the organic solvent by rotary evaporation at 35±5°C to form a uniform transparent film, then vacuum drying at room temperature to remove the residual organic solvent, adding PBS buffer solution to the product, hydrating at 40±5°C, centrifuging after ultrasonic treatment, filtering with a filter membrane, and resuspending in a liposome dispersion to obtain small molecule polypeptide liposomes; (ii) preparing the oil phase: heating white vaseline and glyceryl monostearate to melt at 80±5°C, cooling to 70±5°C and maintaining the temperature, and using the obtained mixture as the oil phase; (iii) preparing the aqueous phase: heating water, triethanolamine, glycerol, 1,2-hexanediol, p-hydroxyacetophenone, ammonium acrylamide dimethyltaurate / VP copolymer and vitamin E to 85±5°C to melt, then cooling to 70±5°C and maintaining the temperature, and using the obtained mixture as the aqueous phase; (iv) Emulsification: slowly add the water phase to the oil phase at a temperature of 70±5°C and stirring, homogenize, then stir, cool and defoam; (v) Adding small molecule peptide liposomes When the emulsified system is cooled to 30±5° C., small molecule polypeptide liposomes are added, and after homogenization and stirring, the system is cooled to room temperature to obtain drug cream B.

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

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