Transdermal assembly polypeptides and uses thereof

Abstract

The application provides a transdermal assembly polypeptide and application thereof, and belongs to the technical field of biological medicine. The transdermal assembly polypeptide comprises any one of GFFY, GFYF, GYFF, GYYF, GYFY, GFYY, GYYY and GFFF, and a transdermal absorption promoter can be prepared by using the transdermal assembly polypeptide. The transdermal assembly polypeptide has a modifiable active site and can load various exogenous active molecules. The transdermal assembly peptide-emodin conjugate comprising emodin and a transdermal assembly peptide unit is prepared by using the transdermal assembly polypeptide. The obtained conjugate is covalently coupled by the assembly peptide with the optimal transdermal efficiency screened and the emodin, the conjugate can self-assemble to form a nano vesicle, significantly improves the solubility of the emodin, retains the anti-inflammatory activity and reduces the toxicity, simultaneously improves the skin retention rate after percutaneous administration, and can be used for treating inflammatory skin diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a transdermal assembly polypeptide and its application. Background Technology

[0002] Topical transdermal drug delivery delivers medication directly to the site of skin lesions, making it an important method of local treatment for skin diseases. However, the stratum corneum, as the body's primary physical barrier against invading exogenous substances, has a dense structure that significantly limits the translayer penetration of drug molecules. Furthermore, the drug's molecular weight, solubility, and lipid solubility, among other physicochemical properties, collectively determine its transdermal behavior, becoming key factors restricting efficacy. Therefore, overcoming these barriers and improving transdermal drug delivery is a pressing technical challenge in this field.

[0003] Existing transdermal delivery systems mainly fall into two categories: physical transdermal technologies and chemical transdermal technologies. Physical transdermal enhancement technologies include microneedling, iontophoresis, electroporation, ultrasound-guided delivery, and laser ablation. While these physical methods can weaken the stratum corneum barrier and broaden the transdermal drug spectrum, they still suffer from drawbacks such as damaging the stratum corneum and skin barrier, complex operation, and high cost. Existing chemical transdermal technologies mainly include chemical penetration enhancers, penetrating peptides, and nanocarriers. However, chemical penetration enhancers often use substances such as alcohols and azones, which can easily cause skin redness, stinging, and barrier damage with long-term use; penetrating peptides are mostly rich in positive charges, which can disrupt cell membrane integrity and induce cytotoxicity at high concentrations; and nanocarriers such as liposomes and nanoemulsions have poor stability and potential toxicity. Therefore, there is an urgent need to develop novel transdermal delivery strategies.

[0004] Self-assembling peptides are a class of peptides that can spontaneously assemble into highly ordered nanostructures through non-covalent interactions under specific conditions, demonstrating unique potential in drug delivery. Utilizing the self-assembly properties of self-assembling peptides can form nanoparticles, improving the stability and solubility of poorly soluble drugs and reducing systemic toxicity. However, there are few reports on the role of self-assembling peptides in promoting transdermal drug absorption. Developing self-assembling peptide delivery systems with skin penetration capabilities could provide a new strategy to address the problems of low transdermal efficiency and high systemic toxicity of poorly soluble drugs.

[0005] Celastrol (Cel) is a hydrophobic small molecule with anti-inflammatory activity and shows potential in the treatment of inflammatory skin diseases. However, its poor water solubility, low bioavailability, and strong toxic side effects severely limit its clinical translation. Therefore, developing self-assembled peptides with skin penetration capabilities and using them as carriers to conjugate with celastrol is of great significance for achieving effective transdermal delivery of the poorly soluble drug celastrol and safe treatment of inflammatory skin diseases. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a transdermal assembly polypeptide and its application. The conjugate obtained by this invention is formed by covalently coupling the transdermal assembly polypeptide with the best transdermal efficiency obtained through screening with triptolide. It can self-assemble into nanovesicles, significantly improving the solubility of triptolide, retaining its anti-inflammatory activity and reducing toxicity, while improving the skin retention rate after transdermal administration, and can be used for the treatment of inflammatory skin diseases.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a transdermal assembly polypeptide, wherein the transdermal assembly polypeptide includes any one of GFFY, GFYF, GYFF, GYYF, GYFY, GFYY, GYYY and GFFF.

[0008] Preferably, F and Y in the transdermal assembly polypeptide are D-configuration amino acids.

[0009] Preferably, the transdermal assembly peptide can be covalently coupled with an exogenous active molecule; the exogenous active molecule includes any one of small molecule natural compounds, peptides, proteins, and nucleic acids.

[0010] Preferably, the exogenous active molecule is triptolide, which is covalently coupled to obtain a transdermal assembled peptide-triptolide conjugate.

[0011] This invention provides the application of the aforementioned transdermal assembly peptide in the preparation of transdermal absorption enhancers.

[0012] This invention provides a transdermal assembly peptide-tripterin conjugate, wherein the transdermal assembly peptide-tripterin conjugate comprises tripterin and transdermal assembly peptide units; The transdermal assembly peptide unit is the transdermal assembly polypeptide.

[0013] As a preferred method, the screening method for transdermal assembly peptide units conjugated with triptolide is as follows: by measuring the skin retention rate of the transdermal assembly peptide, the assembly peptide with the highest transdermal efficiency is screened, which is the transdermal assembly peptide unit that can be conjugated with triptolide.

[0014] This invention provides a method for preparing the transdermal assembled peptide-tripterin conjugate, comprising the following steps: (1) Transdermal assembly peptide units were synthesized using solid-phase synthesis; (2) The triptolide and the coupling agent are coupled with the transdermal assembly peptide unit and cleaved to obtain the transdermal assembly peptide-triptolide conjugate.

[0015] Preferably, the transdermal assembled peptide unit synthesized in step (1) retains the N-terminal free amino group.

[0016] Preferably, the coupling agent is PyBOP.

[0017] The present invention also provides a bioactive solution containing the transdermal assembly peptide-tripterin conjugate, wherein the concentration of the transdermal assembly peptide-tripterin conjugate in the bioactive solution is 1~3000 μmol / L.

[0018] The present invention also provides the use of the transdermal assembled peptide-tripterin conjugate, the transdermal assembled peptide-tripterin conjugate obtained according to the preparation method, or the bioactive solution thereof in the preparation of drugs for treating inflammatory skin diseases.

[0019] Compared with existing technologies, the present invention has the following beneficial effects: The transdermal assembled peptide-triptyline conjugate provided by the present invention can significantly improve the solubility and transdermal absorption efficiency of triptyline. While retaining the anti-inflammatory activity of triptyline, it reduces cytotoxicity. Topical administration of the transdermal assembled peptide-triptyline conjugate provided by the present invention can effectively improve the symptoms of atopic dermatitis and psoriasis, and reduce systemic toxicity. Attached Figure Description

[0020] Figure 1 The images show the high performance liquid chromatography and mass spectra of Cel-Gfyf, the transdermal peptide-tripterin conjugate synthesized in Example 1. The first and second images are high performance liquid chromatograms, and the last image is a primary mass spectrum. Figure 2 A quantitative graph showing the skin retention of transdermal assembled peptides; Figure 3 Optical images of the solution and transmission electron microscopy images of Cel-Gfyf; Figure 4 A quantitative map of skin retention of Cel and Cel-Gfyf; Figure 5 A representative image of a frozen section of skin tissue; Figure 6 The graph shows the inhibition rate of cell viability as evaluated by the CCK-8 assay. Figure 7 Western blot representation and quantitative diagram of Iκκβ, p-Iκκα / β, IκBα, and p-IκBα proteins in the NF-κB inflammatory signaling pathway by Cel-Gfyf; Figure 8 This is a graph showing the changes in body weight of mice during treatment for atopic dermatitis. Figure 9 To detect the expression levels of cytokines IL-13 and IL-4 in skin homogenates from mice with atopic dermatitis using ELISA; Figure 10H&E sections of skin in different groups of mice in a specific dermatitis model and statistical graphs of epidermal / dermal thickness; Figure 11 This is a graph showing the changes in body weight in mice in a psoriasis model. Figure 12 PASI score in mice in a psoriasis model; Figure 13 H&E sections of skin from different groups of mice in a psoriasis model and statistical graphs of epidermal / dermal thickness. Detailed Implementation

[0021] This invention provides a transdermal assembly polypeptide, which includes any one of GFFY, GFYF, GYFF, GYYF, GYFY, GFYY, GYYY and GFFF, preferably GFYF. In the transdermal assembly polypeptide, F and Y are D-configured amino acids, that is, the transdermal assembly polypeptide is preferably Gfyf.

[0022] In this invention, the transdermal assembly peptide can be covalently coupled with an exogenous active molecule; the exogenous active molecule includes any one of small molecule natural compounds, peptides, proteins, and nucleic acids; the exogenous active molecule is preferably triptolide, and covalent coupling yields a transdermal assembly peptide-triptolide conjugate.

[0023] This invention provides the application of the aforementioned transdermal assembly peptide in the preparation of transdermal absorption enhancers.

[0024] This invention provides a transdermal assembly peptide-tripterin conjugate, wherein the transdermal assembly peptide-tripterin conjugate comprises tripterin and transdermal assembly peptide units; The transdermal assembly peptide unit is the transdermal assembly polypeptide, preferably Gfyf.

[0025] In this invention, the structural formula of the transdermal assembled peptide-tripterin conjugate is shown in Formula I: Formula I.

[0026] In this invention, the screening method for transdermal assembly peptide units used for coupling with triptolide is as follows: by measuring the skin retention rate of the transdermal assembly peptide, the assembly peptide with the highest transdermal efficiency is screened, which is the transdermal assembly peptide unit that can be used for coupling with triptolide. The transdermal assembly peptide is preferably a transdermal assembly peptide labeled with Cy5.5.

[0027] This invention provides a method for preparing the transdermal assembled peptide-tripterin conjugate, comprising the following steps: (1) Transdermal assembly peptide units were synthesized using solid-phase synthesis; (2) The triptolide and the coupling agent are coupled with the transdermal assembly peptide unit and cleaved to obtain the transdermal assembly peptide-triptolide conjugate.

[0028] In this invention, the transdermal assembly peptide unit synthesized in step (1) retains the N-terminal free amino group.

[0029] In this invention, the coupling agent is PyBOP, and the purpose of the cleavage is to remove resin and purify the transdermal assembled peptide-tripterin conjugate.

[0030] The present invention also provides a bioactive solution containing the transdermal assembly peptide-tripterin conjugate, wherein the concentration of the transdermal assembly peptide-tripterin conjugate in the bioactive solution is 1~3000 μmol / L, preferably 300~1000 μmol / L, and more preferably 500 μmol / L.

[0031] In this invention, the solvent of the bioactive solution is PBS or a PBS-glycerol mixture, and the bioactive solution is applied to the skin.

[0032] The present invention also provides the use of the transdermal assembled peptide-tripterin conjugate, the transdermal assembled peptide-tripterin conjugate obtained according to the preparation method, or the bioactive solution thereof in the preparation of drugs for treating inflammatory skin diseases.

[0033] In this invention, the inflammatory skin disease is atopic dermatitis, psoriasis, contact dermatitis, or seborrheic dermatitis.

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1: Synthesis of transdermal peptide-tripterin conjugate

[0036] Transdermal peptide-tripterin conjugates were synthesized using the Fmoc-solid-phase synthesis method. Taking Cel-Gfyf as an example, the specific steps are as follows: (1) Weigh 0.5 mmol of 2-Cl-Trt resin into a solid phase synthesis tube, add 10 mL of DCM to swell for 10 min, and then squeeze out the liquid completely with a syringe. (2) Coupling of the first amino acid: Dissolve 0.75 mmol Fmoc-D-phenylalanine (Fmoc-D-Phe-OH) in 10 mL DCM containing 1.5 mmol DIPEA, mix well, add to the solid-phase synthesis tube above, and react at room temperature on a shaker for 1 h. (3) Blocking: Blow out the reaction solution with a rubber bulb, wash with DCM 3 times (10mL / time), blow out the washing solution, add 15mL of blocking solution (DCM:DIPEA:MeOH=17:1:2, volume ratio), and react at room temperature for 15min; (4) Deprotection: Blow out the blocking solution, wash 3 times with DCM (10 mL / time), then wash 3 times with DMF (10 mL / time), blow out the washing solution, add 15 mL of DMF solution containing 20% ​​piperidine (piperidine to DMF volume ratio is 1:4), react at room temperature for 30 min to remove the Fmoc protecting group, and then wash 3 times with DMF (10 mL / time). (5) Coupling of amino acids: Weigh 1 mmol each of the following amino acids: Fmoc-O-tert-butyl-D-tyrosine (Fmoc-D-Tyr(tBu)-OH) and HATU, dissolve them completely in 10 mL of DMF containing 2 mmol DIPEA, and add them to the solid-phase synthesis tube above. React at room temperature for 1 h. (6) Repeat steps (4) and (5) to add amino acids sequentially until the last amino acid (glycine) is coupled and deprotected. Weigh 0.75 mM each of triptolide and coupling agent PyBOP, add them to 10 mL of DMF containing 2 mmol DIPEA and dissolve them completely. Add the solution to a solid-phase synthesis tube and react overnight. Then wash the tube three times with DMF and DCM respectively and blow out all the washing solution. (7) Add 10 mL of lysis buffer (95% TFA, 2.5% TIS, 2.5% H2O, volume ratio) to the solid-phase synthesis tube, react at room temperature for 1 h, collect the lysis buffer in a round-bottom flask, wash the solid-phase tube three times with DCM (10 mL / time) and collect the DCM wash buffer in a round-bottom flask; then remove the organic solvent by rotary evaporation, precipitate the peptide with ice-cold ether, collect the crude product by filtration, then separate and purify by high performance liquid chromatography, freeze dry to obtain the transdermal assembled peptide-tripterpinen conjugate Cel-Gfyf, structural formula shown in Formula I. The purity and molecular weight were identified by high performance liquid chromatography and mass spectrometry, and the results are shown in [Figure 1]. Figure 1 .

[0037] Formula I.

[0038] Depend on Figure 1 It can be seen that the method of the present invention can successfully synthesize polypeptides with a purity greater than 95%.

[0039] Example 2

[0040] Gffy, Gyff, Gyyf, Gyfy, Gfyy, Gyyy and Gfff peptides were constructed using the method in Example 1 for subsequent screening of the optimal transdermal assembly peptide units.

[0041] Experimental Example 1: Determination of Skin Retention Rate of Assembled Peptides using the Franz Diffusion Cell Method

[0042] Fresh, hairless mouse back skin was fixed between the supply and receiving chambers of a Franz diffusion cell. 100 μL of 100 μM Cy5.5-labeled peptides (Gffy, Gfyf, Gyff, Gyyf, Gyfy, Gfyy, Gyyy, and Gfff) prepared with PBS buffer was added to the supply chamber and incubated with the skin for 12 h. The skin surface was then gently rinsed three times with PBS buffer and dried to remove residual drug. The treated skin was collected and placed in a glass homogenizer. 600 μL of acetonitrile-water (80:20, v / v) solution was added to thoroughly homogenize the skin tissue to extract the compounds. All solutions were collected in 1.5 mL centrifuge tubes. The mixture was centrifuged at 10000 rpm for 5 min, and the supernatant was collected for fluorescence intensity determination using a multi-well microplate reader. A standard curve of fluorescence intensity versus drug concentration was established to quantitatively analyze the compound content in the skin extract. Results are shown below. Figure 2 .

[0043] Depend on Figure 2 It was found that the skin retention of the assembled peptide Cy5.5-Gfyf was significantly higher than that of the other candidate peptides. Therefore, the assembled peptide Gfyf was selected to be conjugated with triptolide.

[0044] Experiment Example 2: Dissolution Test and Transmission Electron Microscopy Experiment

[0045] Weigh 1.00 mg each of the conjugate Cel-Gfyf and triptolide synthesized in Example 1, add an appropriate amount of PBS solution to achieve a theoretical concentration of 2 mM, heat and cool, prepare the sample, photograph and record the dissolution state, and observe the microstructure of the Cel-Gfyf assembly using transmission electron microscopy. The results are shown in [Figure number missing]. Figure 3 .

[0046] like Figure 3 As shown in Figure A, the transdermal assembly peptide-tripterbin conjugate Cel-Gfyf prepared in Example 1 can be self-assembled via a heating-cooling method. This assembly system significantly enhances the dispersibility of tripterbin, remaining in solution at a concentration of 2 mM, while the active pharmaceutical ingredient tripterbin is almost insoluble in PBS buffer. This indicates that the covalent coupling and assembly characteristics of the transdermal assembly peptide Gfyf greatly improve the solubility of tripterbin. Furthermore, Figure 3 The B-transmission electron microscopy results show that Cel-Gfyf can self-assemble into nanovesicle structures after heating and cooling.

[0047] Experimental Example 3: Determination of the transdermal transdermal activity of Cel-Gfyf

[0048] Fresh, hairless mouse back skin was fixed between the supply and receiving chambers of a Franz diffusion cell. 200 μL of 500 μM Cel-Gfyf was prepared using PBS buffer and PBS buffer containing 30% ethanol, respectively, and added to the supply chamber for 12 h. The skin was then gently rinsed three times with PBS buffer and dried to remove any residual drug. The treated skin was collected and placed in a glass homogenizer. 1.5 mL of acetonitrile-water (80:20, v / v) solution was added to thoroughly homogenize the skin tissue to extract the compound. All the homogenized solution was collected in a 1.5 mL centrifuge tube and centrifuged at 10,000 rpm for 5 min. The supernatant was analyzed by LC-MS. The results are shown below. Figure 4 As shown.

[0049] Depend on Figure 4 It can be seen that, compared with free triptolide, Cel-Gfyf has a significantly higher skin retention rate in PBS buffer and PBS solution containing 30% ethanol.

[0050] Simultaneously, the spatial distribution of nanomedicines in the epidermis and dermis was determined using a Franz diffusion cell combined with cryofluorescence sectioning. Cy5.5-Gfyf and Cel-Gfyf were dissolved in PBS buffer at a molar ratio of 1:10, and co-assembled after heating and cooling to obtain an assembly mixture, which was then fluorescently labeled with Cel-Gfyf. This assembly mixture was added to the supply chamber of the diffusion cell system, and after 6 hours of contact with skin, skin samples were taken for cryosectioning. The spatial distribution of nanomedicines in the epidermis and dermis was observed using fluorescence imaging. The results are as follows: Figure 5 As shown.

[0051] Depend on Figure 5 It can be seen that the free fluorescent molecule Cy5.5 aggregates in the epidermis and fails to effectively penetrate the skin barrier; while obvious fluorescent signals can be observed in both the epidermis and dermis of the skin treated with Cel-Gfyf, indicating that Cel-gfyf can effectively penetrate the stratum corneum barrier and reach the active epidermis and dermis to exert its effects.

[0052] Experimental Example 4: Detection of Cytotoxicity by CCK-8 Assay

[0053] HaCaT cells were divided into 5×10 3The culture medium was seeded at a density of / wells into 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. The medium was discarded, and DMEM medium with concentration gradients of 0, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8 μM Cel and 0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 μM Cel-Gfyf was added, and the plates were incubated for another 24 hours. The supernatant was discarded, and DMEM medium containing 10% CCK-8 solution was added and incubated for 1 hour. The absorbance was measured at 450 nm using a microplate reader. The results are as follows: Figure 6 As shown.

[0054] Depend on Figure 6 It was found that when Cel-Gfyf was co-incubated with HaCaT cells at concentrations of 10 μM and below for 24 h, the cell viability remained above 80%, and no obvious cytotoxicity was observed. Figure 6 In contrast, free triptolide at a concentration of 2 μM reduced the average cell viability to 23.65%, exhibiting severe cytotoxicity. Figure 6 (B in the text). The above results indicate that modification with peptide Gfyf significantly reduced the cytotoxicity of triptolide.

[0055] Experimental Example 5: Detection of the effect of peptides on the NF-κB signaling pathway using Western blotting (WB) technique

[0056] Three groups were set up: Blank group, Model group, and Cel-Gfyf group. HaCaT cells were administered at a rate of 1×10⁻⁶. 5Cells were seeded at a density of / wells in six-well plates. Once cells reached 80% confluence, the Cel-Gfyf group was pretreated with 10 μM Cel-Gfyf for 2 h, while the other groups were treated with an equal volume of fresh culture medium. After 2 h, the Model and Cel-Gfyf groups were treated with 10 ng / mL TNF-α for 5 min. The culture medium was immediately discarded, and the cells were washed three times with PBS. 100 μL of RIPA lysis buffer containing a mixture of phosphatase and protease inhibitors was added to each well, and the cells were lysed on ice for 10 min. The cell lysates were collected in 1.5 mL EP tubes and centrifuged at 14000 rpm for 15 min at 4 °C. Protein concentration was quantified using a BCA kit. Then, 5× loading buffer was added at a 4:1 ratio of cell lysate to 5× loading buffer, and the plates were heated in a metal bath at 100 °C for 10 min to obtain protein samples. SDS-PAGE with polyacrylamide gel electrophoresis was performed using Beyotime precast gels. The protein loading was 30 μg, the electrophoresis voltage was 160 V, and the time was 50 min. After electrophoresis, the gel was cut, and the protein was transferred to a PVDF membrane. The PVDF membrane was washed once with 1×TBST, incubated with rapid blocking buffer at room temperature for 15 min, and washed three times with 1×TBST for 10 min each time. The membrane was cut according to the marker instructions, incubated overnight at 4°C with primary antibody, washed three times with 1×TBST for 10 min each time, incubated for 2 h with secondary antibody at room temperature, and washed three times with 1×TBST for 10 min each time. After washing, the membrane was exposed and photographed. The results are shown below. Figure 7 As shown.

[0057] Depend on Figure 7 It was found that, compared with the control group, the protein expression levels of p-Iκκα / β and p-IκBα were significantly increased in the model group after TNF-α stimulation, indicating that TNF-α induced the activation of the upstream NF-κB signaling pathway. However, the relative protein expression levels of p-Iκκα / β and p-IκBα in the Cel-Gfyf pretreatment group were significantly lower than those in the model group, but not significantly different from the control group, indicating that Cel-Gfyf can inhibit TNF-α-induced activation of the upstream NF-κB signaling pathway and preserve the anti-inflammatory activity of triptolide.

[0058] Experimental Example 6: Treatment of Atopic Dermatitis in Mice

[0059] Four groups were set up: Blank group (no model established), Model group (model established but no drug administered), Cel-Gfyf group, and Cel group. Six-week-old female BALB / c mice were used, and the hair on their backs was completely removed using a small animal shaver and depilatory cream; this was recorded as day 0. On day 1, 200 μL of 1% 2,4-dinitrochlorobenzene (DNCB) was applied to the exposed skin on the backs of the mice. Sensitization with 1% DNCB was repeated on day 4. After successful model establishment (day 8), the mice were randomly assigned to other groups and treated with 200 μL of 0.5% DNCB continuously for 3 weeks (3 times a week). The control group received a solvent (acetone: olive oil, 3:1, v / v). Starting from day 9, approximately 4 hours after treatment with 0.5% DNCB, all groups were treated with 200 μL of 500 μM drug (PBS:glycerol = 3:1, v / v) applied evenly according to their experimental groups. The Model group received the same solution (PBS:glycerol = 3:1, v / v). One day after the last administration (day 29), mice were sacrificed, and skin from the affected area on the back was harvested for H&E staining to assess skin damage. Simultaneously, another portion of the affected skin from the back was ground on ice to extract the homogenate, which was used for ELISA to analyze the secretion levels of cytokines IL-4 and IL-13 in the skin. Changes in mouse body weight were monitored throughout the experiment, and the results are shown below. Figures 8-10 .

[0060] Depend on Figures 8-10 It can be seen that the body weight of mice in the Cel-Gfyf group did not change significantly during the treatment period, which preliminarily verified its safety. Figure 8 ELISA results of the skin abrasive fluid showed that, compared with the Model group and the Cel group, the levels of cytokines IL-13 and IL-4 in the affected skin of mice in the Cel-Gfyf group were significantly decreased. Figure 9 Furthermore, H&E sectioning and statistical results showed that the epidermal and dermal thickness of the affected skin in the Cel-Gfyf treatment group was significantly reduced compared to the Model group and the Cel group, significantly alleviating the specific dermatitis-like lesions in the mice. Figure 10 ).

[0061] Experiment 7: Treatment of Psoriasis in Mice

[0062] Four groups were set up: Blank group (no modeling), Model group (modeling without drug administration), Cel-Gfyf group, and Cel group. Six-week-old female BALB / c mice were used. The hair on the backs of the mice was completely removed using a small animal shaver and depilatory cream, marked as day 0. Mice were then randomly assigned to groups of six. Starting from day 1, except for the Blank group, mice in the other groups received 62.5 mg of imiquimod cream on their backs daily for five consecutive days to establish a mouse psoriasis model. The Cel-Gfyf and Cel groups were treated daily from day 1 to day 5 with 200 μL of 500 μM drug (solvent: PBS: glycerol = 3:1, v / v), while the Model group received the solvent (PBS: glycerol = 3:1, v / v). On day 6, the mice were sacrificed, and skin from the affected areas on the backs was collected for H&E staining to assess skin damage. During the experiment, the mice's body weight was monitored daily, and PASI scores (0-4) were performed to assess the severity of skin thickness, scaling, and erythema at the affected areas. Results are shown below. Figures 11-13 .

[0063] Depend on Figures 11-13 It was found that compared to the Blank group, the body weight of mice in the Model group, Cel-Gfyf group, and Cel group all decreased significantly. However, compared to the Model group, the body weight of mice in the Cel-Gfyf group did not change significantly. This indicates that the decrease in mouse body weight was caused by the toxicity of the modeling agent imiquimod cream and was unrelated to Cel-Gfyf. Figure 11 The PASI scores of mice showed that, compared with the Model group, the PASI scores of mice in the Cel-Gfyf group were significantly lower. Figure 12 Furthermore, statistical analysis of mouse epidermal thickness in H&E sections showed that treatment with Cel-Gfyf significantly reduced mouse epidermal thickness. Figure 13 This further illustrates the effectiveness of Cel-Gfyf in treating psoriasis.

[0064] Therefore, this invention provides a transdermal assembly peptide-tripterbin conjugate and its application in the treatment of inflammatory skin diseases. The transdermal assembly peptide-tripterbin conjugate comprises tripterbin and transdermal assembly peptide units. The transdermal assembly peptide-tripterbin conjugate provided by this invention retains the anti-inflammatory activity of tripterbin while reducing its cytotoxicity, significantly improving the solubility and transdermal absorption efficiency of tripterbin. When administered topically through the skin, it demonstrates excellent efficacy in the treatment of atopic dermatitis and psoriasis.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A transdermal assembly polypeptide, characterized in that, The transdermal assembly peptide includes any one of GFFY, GFYF, GYFF, GYYF, GYFY, GFYY, GYYY, and GFFF.

2. The transdermal assembly polypeptide according to claim 1, characterized in that, In the transdermal assembly polypeptide, F and Y are D-configuration amino acids.

3. The transdermal assembly polypeptide according to claim 1 or 2, characterized in that, The transdermal assembly peptide can be covalently coupled with an exogenous active molecule; the exogenous active molecule includes any one of small molecule natural compounds, peptides, proteins, and nucleic acids.

4. The transdermal assembly peptide according to claim 3, characterized in that, The exogenous active molecule is triptolide, which is covalently coupled to obtain a transdermal assembled peptide-triptolide conjugate.

5. The use of the transdermal assembly polypeptide according to any one of claims 1 to 4 in the preparation of a transdermal absorption enhancer.

6. A transdermal peptide-tripterin conjugate, characterized in that, The transdermal assembly peptide-tripterin conjugate includes tripterin and transdermal assembly peptide units; The transdermal assembly peptide unit is the transdermal assembly polypeptide according to claim 1.

7. The transdermal peptide-tripterin conjugate according to claim 6, characterized in that, The screening method for transdermal assembly peptide units conjugated with triptolide is as follows: by measuring the skin retention rate of the transdermal assembly peptide, the assembly peptide with the highest transdermal efficiency is screened, which is the transdermal assembly peptide unit that can be conjugated with triptolide.

8. The method for preparing the transdermal assembled peptide-tripterin conjugate according to claim 6 or 7, characterized in that, Includes the following steps: (1) Transdermal assembly peptide units were synthesized using solid-phase synthesis; (2) The triptolide and the coupling agent are coupled with the transdermal assembly peptide unit and cleaved to obtain the transdermal assembly peptide-triptolide conjugate.

9. The preparation method according to claim 8, characterized in that, The transdermal assembly peptide unit synthesized in step (1) retains the N-terminal free amino group.

10. The preparation method according to claim 8, characterized in that, The coupling agent is PyBOP.

11. A bioactive solution containing the transdermal assembled peptide-tripterin conjugate of claim 6 or 7, characterized in that, The concentration of the transdermal assembled peptide-tripterin conjugate in the bioactive solution is 1~3000 μmol / L.

12. The use of the transdermal assembled peptide-tripterin conjugate of claim 6 or 7, the transdermal assembled peptide-tripterin conjugate obtained by the preparation method according to any one of claims 8 to 10, or the bioactive solution of claim 11 in the preparation of a drug for treating inflammatory skin diseases.

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