Bradykinin receptor active peptide and use thereof in promoting hair regeneration

Abstract

Provided are a bradykinin receptor active peptide and a use thereof in promoting hair regeneration. Further provided are a composition comprising the bradykinin receptor active peptide, a pharmaceutical preparation, a kit, and a preparation method for the bradykinin receptor active peptide. The bradykinin receptor active peptide has a good hair regeneration effect and safety, and has clinical application prospects.

Description

A bradykinin receptor-active peptide and its application in promoting hair regeneration

[0001] This application claims priority to Chinese patent application 202411917455X, filed on 2024 / 12 / 24. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedicine, specifically to a bradykinin receptor active peptide and its application in promoting hair regeneration. Background Technology

[0003] Hair loss is a hair regeneration disorder that refers to abnormal or excessive hair loss. Based on its causes, it can be classified into androgenetic alopecia, neurogenic alopecia, traumatic alopecia, and nutritional alopecia, among which more than 90% are androgenetic alopecia.

[0004] Current clinical treatment options for hair loss include: ① Drug therapy: minoxidil, androgen receptor antagonists, and traditional Chinese medicine extracts, etc.; ② Physical therapy: low-energy laser and microneedling; ③ Biological therapy: autologous platelet-rich plasma; ④ Surgical treatment: autologous hair transplantation. All of these methods have shown some therapeutic effect on hair loss patients, but they all have certain limitations. Drug therapy is often accompanied by varying degrees of side effects, such as local contact dermatitis, hirsutism, sexual dysfunction, and heart disease. Physical therapies such as low-energy laser and microneedling still need to be used in combination with medication. Autologous platelet-rich plasma requires extracting the patient's own blood, the preparation process is complicated, quality standards are not uniform, and it is an invasive treatment. Autologous hair transplantation is invasive, the survival rate of hair follicles is limited, postoperative drug treatment is still required for maintenance, and the treatment cost is relatively high. Compared with oral medications, topical medications produce local efficacy, reduce systemic drug exposure, and can achieve better safety and patient compliance. Therefore, the development of safe and effective topical medications for hair loss is of great significance.

[0005] Hair follicles are the basic units of hair growth. Dermal papilla cells are located in the depression at the base of the hair follicle, and capillaries supply nutrients to the follicle through these cells. Hair follicle development and growth are regulated by a sophisticated signaling network involving multiple signaling pathways, including Wnt, bone morphogenetic protein, transforming growth factor-β, fibroblast growth factor, mitogen-activated protein kinase, and phosphatidylinositol 3-kinase / protein kinase B. Among these, the Wnt signaling pathway plays a central role in hair follicle development and growth cycle. Studies have shown that β-catenin, a key molecule in the Wnt signaling pathway, is an important protein for maintaining the induced activity and proliferative function of dermal papilla cells. It can regulate the formation of the hair follicle substrate, the hair follicle growth cycle, and the proliferation and differentiation of hair follicle stem cells. Hair follicles are metabolically active organs, and their growth and cyclical processes require a sufficient supply of nutrients and oxygen. Vascular endothelial growth factor (VEGF) can promote hair follicle angiogenesis and increase blood supply to the follicle. Furthermore, VEGF also promotes the proliferation and differentiation of hair follicle stem cells, enhances the immune defense capabilities of hair follicles, and regulates the hair follicle growth cycle. Bradykinin and its analogues can promote VEGF expression and angiogenesis.

[0006] Bradykinin receptors belong to the G protein-coupled receptor family, comprising two subtypes, B1 and B2. B1 receptors are almost not expressed in normal tissues; their expression is only upregulated after injury, when pro-inflammatory factors are released. B2 receptors, on the other hand, are ubiquitous in various tissues. In normal human skin tissue, bradykinin B2 receptors are highly expressed in keratinocytes, dermal fibroblasts, dermal papilla cells, dermal microvascular endothelial cells, and dermal lymphatic endothelial cells. Structurally, bradykinin B2 receptor bioactive peptides are classified into non-peptide bioactive peptides and peptide bioactive peptides. Due to their structural similarity to natural ligands, peptide bioactive peptides typically bind more tightly to the B2 receptor, exhibiting higher selectivity and affinity. Therefore, peptide bioactive peptides can play a role in specific biological processes and cell signaling, reducing interference with other receptors and lowering the risk of side effects. From a therapeutic perspective, peptides with active B2 receptors have lower toxicity and lower immunogenicity than small molecule non-peptide peptides with active B2 receptors. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a bradykinin receptor active peptide and its application in promoting hair regeneration.

[0008] Specifically, the present invention provides a polypeptide and its derivatives having bradykinin receptor binding activity, wherein the polypeptide is selected from the following:

[0009] (I) A polypeptide that differs from the amino acid sequence shown in any one of SEQ ID NO:1-15 by 1-6 amino acid residues; said difference being the deletion, addition, or substitution of amino acid residues; said added or substituted amino acid being a natural or non-natural amino acid;

[0010] (II) A polypeptide obtained by replacing the amino acid configuration in the polypeptide described in (I);

[0011] (III) A polypeptide in which the amino acid configuration of the polypeptide described in (I) is replaced and flipped in the direction of C-terminus → N-terminus;

[0012] (IV) Peptides that have undergone amino acid modification or cyclization of the peptides described in (I)-(III), or bioisoelectronic peptides;

[0013] The amino acid sequence of the polypeptide is not shown in any of SEQ ID NO:1-6.

[0014] In this article, "amino acid configuration substitution" includes replacing an L-configuration amino acid with a D-configuration amino acid or vice versa.

[0015] In some embodiments, the polypeptide differs from the amino acid sequence shown in any one of SEQ ID NO:1-15 by 1-5 amino acid residues.

[0016] In some embodiments, the polypeptide differs from the amino acid sequence shown in any one of SEQ ID NO:1-15 by 1-4 amino acid residues.

[0017] In some embodiments, the polypeptide differs from the amino acid sequence shown in any one of SEQ ID NO:1-15 by 1-3 amino acid residues.

[0018] In some embodiments, the polypeptide differs from the amino acid sequence shown in any one of SEQ ID NO:1-12 by 1-3 amino acid residues.

[0019] In some embodiments, the polypeptide is selected from the following:

[0020] (I) A polypeptide that differs from the amino acid sequence shown in any one of SEQ ID NO:1-12 by 1-3 amino acid residues; said difference being the deletion, addition, or substitution of amino acid residues; said added or substituted amino acid being a natural or non-natural amino acid;

[0021] (II) A polypeptide obtained by replacing the amino acid configuration in the polypeptide described in (I);

[0022] (III) A polypeptide in which the amino acid configuration of the polypeptide described in (I) is replaced and flipped in the direction of C-terminus → N-terminus;

[0023] (IV) Peptides that have undergone amino acid modification or cyclization of the peptides described in (I)-(III), or bioisoelectronically arranged peptides.

[0024] In this article, "amino acid residue differences" includes amino acid residue differences caused by amino acid mutations.

[0025] In some embodiments, the amino acid sequence of the polypeptide is not shown in any of SEQ ID NO:1-6.

[0026] In some embodiments, the polypeptide is selected from the following:

[0027] (i) A polypeptide with an amino acid sequence as shown in any one of SEQ ID NO:7-15;

[0028] (ii) A polypeptide obtained by replacing the amino acids in the polypeptide described in (i) with amino acid configurations;

[0029] (iii) A polypeptide in which the amino acid configuration of the polypeptide described in (i) is replaced and flipped in the direction of C-terminus → N-terminus;

[0030] (iv) A polypeptide that has undergone amino acid modification or cyclization of the polypeptides described in (i)-(iii), or a polypeptide with bioisoelectronic arrangement.

[0031] In some embodiments, the polypeptide is selected from the following:

[0032] (i) A polypeptide with an amino acid sequence as shown in any one of SEQ ID NO:7-12;

[0033] (ii) A polypeptide obtained by replacing the amino acids in the polypeptide described in (i) with amino acid configurations;

[0034] (iii) A polypeptide in which the amino acid configuration of the polypeptide described in (i) is replaced and flipped in the direction of C-terminus → N-terminus;

[0035] (iv) A polypeptide that has undergone amino acid modification or cyclization of the polypeptides described in (i)-(iii), or a polypeptide with bioisoelectronic arrangement.

[0036] In some embodiments, the amino acid modification includes C-terminal modification, N-terminal modification, intermediate residue modification, and non-natural amino acid modification.

[0037] In some embodiments, the modification types of the C-terminal modification, N-terminal modification, and intermediate residue modification are selected from one or more of acylation modification, amidation modification, esterification modification, glycosylation modification, fatty acid modification, non-natural amino acid modification, external group modification, and pseudopeptide modification; the external group modification is preferably cholesterol modification or polyethylene glycol modification.

[0038] In some specific embodiments, the polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NO:7-15.

[0039] In some specific embodiments, the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO:7-15.

[0040] In some specific embodiments, the polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NO:7-12.

[0041] In some specific embodiments, the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO:7-12.

[0042] In some specific implementations, the bradykinin receptor is bradykinin receptor B2.

[0043] In some specific embodiments, the polypeptide and its derivatives have hair regeneration-promoting activity.

[0044] The present invention also provides a composition comprising the aforementioned polypeptide or a derivative thereof, and at least one of a pharmaceutically acceptable carrier, excipient, or diluent.

[0045] The present invention also provides a pharmaceutical formulation comprising the aforementioned polypeptide or its derivative or the aforementioned composition.

[0046] In some embodiments, the pharmaceutical preparation is a topical preparation that promotes hair growth.

[0047] In some embodiments, the topical preparation is a transdermal preparation.

[0048] In some embodiments, the topical preparation is a foam, liniment, gel, patch, spray, cream, emulsion, solution, tincture, or ointment.

[0049] The present invention also provides a pillbox kit, the pillbox kit comprising pillbox A, wherein pillbox A comprises one or more of the aforementioned polypeptide or its derivative, the aforementioned composition, or the aforementioned pharmaceutical preparation; and,

[0050] Kit B contains one or more of the following: milodil, prostaglandin derivatives, autologous platelet-rich plasma, botulinum toxin, growth factors, androgen receptor antagonists, and antibody drugs.

[0051] In some specific implementations, the androgen receptor antagonist includes finasteride.

[0052] The present invention also provides a method for preparing polypeptides and their derivatives with bradykinin receptor binding activity, the method comprising directly synthesizing the polypeptides and their derivatives using solid-phase polypeptide synthesis technology.

[0053] The present invention also provides the use of the aforementioned polypeptides and their derivatives, polypeptides and their derivatives with amino acid sequences as shown in any one of SEQ ID NO:1-6, the aforementioned compositions, the aforementioned pharmaceutical preparations, or the aforementioned kits in the preparation of products that promote hair regeneration.

[0054] The present invention also provides a method for promoting hair regeneration, the method comprising administering to a subject in need an effective amount of the said polypeptide or its derivative, a polypeptide or its derivative with an amino acid sequence as shown in any one of SEQ ID NO:1-6, the said composition, the said pharmaceutical preparation, or the said kit.

[0055] In some specific implementations, the method is a non-diagnostic or therapeutic method.

[0056] The present invention also provides a method for preventing and / or treating hair loss, the method comprising administering to a subject in need an effective amount of the said polypeptide or its derivatives, a polypeptide or its derivatives with an amino acid sequence as shown in any one of SEQ ID NO:1-6, the said composition, the said pharmaceutical preparation, or the said kit.

[0057] In some implementations, the hair loss is either non-scarring alopecia or scarring alopecia.

[0058] In some embodiments, the non-scarring alopecia preferably includes androgenetic alopecia, alopecia areata, telogen effluvium, and chemotherapy-induced alopecia.

[0059] In some specific implementations, the hair loss is androgenetic alopecia.

[0060] In some implementations, the method further includes the use of one or more of physical therapy, biological therapy, and surgical treatment.

[0061] In some specific implementations, the physical therapy includes low-energy laser therapy and microneedling therapy; the biological therapy includes autologous platelet-rich plasma therapy, antibody drug therapy, growth factor drug therapy, botulinum toxin therapy, and gene therapy; and the surgical treatment includes autologous hair transplantation.

[0062] The present invention also provides the aforementioned polypeptide or its derivatives, polypeptides and their derivatives with amino acid sequences as shown in any one of SEQ ID NO:1-6, the aforementioned composition, the aforementioned pharmaceutical preparation, or the aforementioned kit for the prevention and / or treatment of hair loss.

[0063] In some implementations, the hair loss is either non-scarring alopecia or scarring alopecia.

[0064] In some embodiments, the non-scarring alopecia preferably includes androgenetic alopecia, alopecia areata, telogen effluvium, and chemotherapy-induced alopecia.

[0065] In some specific implementations, the hair loss is androgenetic alopecia.

[0066] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0067] The reagents and raw materials used in this invention are all commercially available.

[0068] The positive and progressive effects of this invention are as follows:

[0069] The polypeptides and their derivatives with bradykinin receptor binding activity of the present invention exhibit good hair regeneration effects and safety when administered transdermally, and have promising clinical application prospects. Attached Figure Description

[0070] Figure 1 shows the expression of bradykinin B2 receptor in human hair papilla cells.

[0071] Figure 2 shows the hair regeneration effect of the SEQ ID NO.1-5 polypeptides in an animal model of androgen-induced alopecia.

[0072] Figure 3 shows the hair regeneration effect of the peptides SEQ ID NO.6 and SEQ ID NO.7 in the animal model of androgenic alopecia.

[0073] Figure 4 shows the hair regeneration effect of the SEQ ID NO.8~12 polypeptide in an animal model of androgenic alopecia.

[0074] Figure 5 shows the hair regeneration effect of the SEQ ID NO.13~15 polypeptide in the animal model of androgen-induced alopecia.

[0075] Figure 6 shows the expression of β-catenin in skin tissue of an animal model of androgenetic alopecia induced by the polypeptide of SEQ ID NO.1.

[0076] Figure 7 shows the complete blood count atlas of SEQ ID NO.1; A is the red blood cell (RBC) count atlas; B is the hemoglobin (HGB) level atlas; C is the white blood cell (WBC) count atlas; and D is the platelet (PLT) count atlas.

[0077] Figure 8 shows the blood biochemistry profile of SEQ ID NO.1; A is the alanine aminotransferase (ALT) level profile; B is the aspartate aminotransferase (AST) level profile; C is the blood urea nitrogen (BUN) level profile; and D is the creatinine (CREA) level profile.

[0078] Figure 9 shows the H&E staining patterns of the heart, liver, spleen, lungs, and kidneys in SEQ ID NO.1 (scale bar = 100 μm).

[0079] Figure 10 shows the skin H&E staining pattern of SEQ ID NO.1 group (scale bar = 100 μm). Detailed Implementation

[0080] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0081] Example 1: Synthesis and Characterization of Bradykinin Receptor Active Peptides

[0082] Using solid-phase peptide synthesis technology, the first amino acid residue at the C-terminus of any peptide in SEQ ID NO. 1–12 (Table 1) is first coupled to a solid-phase support. Then, following the sequence of any peptide in SEQ ID NO. 1–7, the corresponding amino acids are coupled to the solid-phase resin according to the C-terminus to N-terminus solid-phase synthesis strategy. The specific method is as follows: 1 g of 2-chlorotriphenylmethyl chloride resin is weighed, swollen with dichloromethane for 15 min, washed with dimethylformamide (DMF), and then deprotected with a deprotecting agent (piperidine:DMF = 1:4, v / v) to remove Fmoc protection. Deprotection is performed twice, for 10 min and 20 min respectively. Next, an excess of 8 times the amount of amino acids and an appropriate amount of 1-hydroxybenzotriazole were dissolved in a DMF solution of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate. Diisopropylethylamine was added, and the reaction was allowed to proceed for 45 min. After the reaction, the resin was washed with DMF, and then the reaction was checked using ninhydrin to determine whether the terminal amino groups of the amino acids had completely reacted. The above steps were repeated until all amino acids were successfully linked. After the reaction, the resin was dried with dichloromethane and methanol, and then cleaved with dichloromethane containing 1% trifluoroacetic acid (TFA). After removing the organic solvent under reduced pressure, a small amount of acetonitrile was added for dispersion, and the peptides were precipitated with an appropriate amount of water. The crude peptide solution was obtained by rotary evaporation under reduced pressure.

[0083] Preparative HPLC was used to purify the peptides. The chromatographic conditions were as follows: Column: Symmetry C-18 column (300 mm × 19 mm); mobile phase A: 10% acetonitrile solution (containing 0.1% TFA), mobile phase B: 50% acetonitrile solution (containing 0.1% TFA); gradient elution: 0–60 min, 100% A–100% B; flow rate: 10 mL / min; detection wavelength: UV 214 nm. The eluent was collected and freeze-dried to obtain a white solid. The purity of the peptide was determined and identified by HPLC and mass spectrometry. The chromatographic conditions were as follows: Column: YMC C-18 column (4.6 mm × 150 mm, 5 μm); Mobile phase A: water (containing 0.1% TFA), Mobile phase B: acetonitrile (containing 0.1% TFA); Gradient elution: 0–1 min, 5% B, 2–32 min, 5%–65% B, 33–37 min, 90% B, 38–45 min, 5% B; Flow rate: 0.7 mL / min; Detection wavelength: UV 214 nm. HPLC and ESI-MS results showed that T... SEQ ID NO.1 =18.11min, MW:1059.6; T SEQ ID NO.2 =18.76min, MW:1172.7; T SEQ ID NO.3 =18.12min, MW:1059.6; TSEQ ID NO.4 =17.24min, MW:1187.7; T SEQ ID NO.5 =20.12min, MW:1172.7; T SEQ ID NO.6 =17.38min, MW:1187.7; T SEQ ID NO.7 =17.48min, MW:1059.6; T SEQ ID NO.8 =18.79min, MW:1075.6; T SEQ ID NO.9 =14.47min, MW:1196.7; T SEQ ID NO.10 =17.96min, MW:1029.6; T SEQ ID NO.11 =13.56min, MW:1040.6; T SEQ ID NO.12 =16.88min, MW:1159.7; T SEQ ID NO.13 =15.44min, MW:1072.2; T SEQ ID NO.14 =18.29min, MW:1005.1; T SEQ ID NO.15 =17.67min, MW:988.1.

[0084] The above results indicate that the purity of SEQ ID NO.1-SEQ ID NO.15 is all above 95%, and the mass spectra show that the molecular ion peak m / z (M+H) + The measured molecular weights of SEQ ID NO.1-SEQ ID NO.15 are 1060.8, 1173.9, 1060.8, 1188.9, 1173.9, 1188.9, 1060.8, 1076.7, 1197.8, 1030.6, 1041.7, 1160.7, 1072.6, 1005.6, and 988.6, respectively. The theoretical molecular weights of SEQ ID NO.1-SEQ ID NO.15 are 1059.6, 1172.7, 1059.6, 1187.7, 1172.7, 1187.7, 1059.6, 1075.6, 1196.7, 1029.6, 1040.6, 1159.7, 1072.2, 1005.1, and 988.1, respectively, and the measured values ​​are consistent with the theoretical values.

[0085] Table 1: Amino acid sequences of bradykinin receptor active peptides

[0086] Example 2: Computer simulation analysis of peptide and bradykinin B2 receptor protein models

[0087] The cryo-electron microscopy structure of the bradykinin receptor b2 (BDKRB2) protein was obtained from the RCSB Protein Data Bank (PDB ID: 7F6I). Docking between the peptide and the bradykinin receptor b2 protein was performed using the molecular docking software Maestro 2018-1. First, the obtained protein structure was pre-processed using the Protein Preparation Wizard module to complete any missing side chains and loop regions, and the system energy was minimized. The peptide was prepared using the LigPrep tool to generate different conformations for subsequent docking. The Receptor Grid Generation module was used to specify the docking sites between the peptide and the receptor protein to generate docking boxes. Docking between the peptide and the receptor protein was performed in the Ligand docking module, and the calculated conformations were scored and ranked using the Glide standard precision (SP) scoring function.

[0088] Molecular dynamics simulations of the peptide-bradykinin B2 receptor protein complex were performed using the GROMACS software package (version 2020.6) with a CHARMM36 force field and a TIP3P water model. The complex was dissolved in a dodecahedral box containing approximately 185,032 water molecules, and the minimum distance between the dodecahedral box and the complex was set to [value missing]. This constitutes a simulation system. By adding sufficient Cl to the dodecahedral solution chamber... - To ensure the electroneutrality of the system, the entire system underwent an energy minimization (EM) procedure, relaxing the system to below 1000 kJ / mol within 50,000 steps. Then, the system was heated in the NVT ensemble to the final temperature of 300 K within 100 ps, ​​and equilibrated in the NPT ensemble for 100 ps at a pressure of 1 bar. Finally, molecular dynamics simulations were performed for each different peptide for 100 ns. Trajectory analysis following the molecular dynamics simulations was performed using the GROMACS software package (version 2020.6), and the convergence of the system was evaluated by detecting the root mean square deviation (RMSD) of the backbone atoms in the simulation system. The results showed that the complexes of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7 with the bradykinin B2 receptor remained relatively stable during the simulation. or The system experienced fluctuations, and all systems essentially reached a convergent state. To calculate the binding free energy between the peptide and the bradykinin B2 receptor, 500 structural snapshots were extracted within 30 ns, and the binding free energy between the bradykinin B2 receptor and the peptide molecule was calculated. The results are shown in Table 2. SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7 all showed strong affinity for the bradykinin B2 receptor.

[0089] Table 2. Binding activity of peptides to bradykinin B2 receptor

[0090] Example 3: Expression of bradykinin B2 receptor in human dermal papilla cells

[0091] Human dermal papilla cells (HDPCs) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. HDPCs were washed twice with pre-cooled PBS, and total cell protein was extracted according to the instructions of the total cell protein extraction kit. Protease inhibitors were added to the protein extraction reagents immediately before use to prevent protein degradation. The total protein concentration was determined using a BCA kit, followed by the addition of protein loading buffer. The samples were then boiled in a water bath for 10 min to denature the protein, and stored at -20°C for later use. Samples containing 25 μg of protein were injected through the wells into a 10% polyacrylamide gel. After the bands separated, the samples were transferred to a nitrocellulose membrane. The membrane was blocked in 5% skim milk at room temperature for 1 h, and then hybridized overnight at 4°C with a recombinant rabbit monoclonal antibody against bradykinin B2 receptor. The membrane was washed three times with Tris buffer containing 0.1% Tween-20, and then incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 1 h followed by three washes. Following the instructions, the enhanced chemiluminescent protein blot assay kit was used to visualize and detect protein bands on a gel imaging system. The results are shown in Figure 1: human dermal papilla cells showed high expression of bradykinin B2 receptor.

[0092] Example 4: Hair regeneration experiment in an animal model of androgenetic alopecia

[0093] C57 mice were anesthetized with 8% chloral hydrate. A 2cm × 2cm area was shaved off along the spine, and depilatory cream was evenly applied. After 1 minute, the cream was washed off with water to confirm that the hair growth was in the resting phase (the skin was pink). 24 hours after hair removal, the animals were divided into four groups: saline group, minoxidil group, SEQ ID NO.1 group, SEQ ID NO.2 group, SEQ ID NO.3 group, SEQ ID NO.4 group, and SEQ ID NO.5 group, with four mice in each group. All groups received a 0.5% (w / v) testosterone solution (ethanol:propylene glycol:water = 56:24:20, v / v) at a dose of 25 mg / kg, applied topically to the depilated area once daily for 28 days. In addition to the administration of testosterone solution, the drug administration regimens for each experimental group were as follows: The saline group received topical application of saline solution to the hair-removed area once daily for 28 days; the minoxidil group received topical application of commercially available 5% minoxidil solution (250 mg / kg) to the hair-removed area once daily for 28 days; and groups SEQ ID NO. 1–5 received topical application of 0.15% (w / v) polypeptide solution (ethanol: phosphate buffer = 50:50, v / v) to the hair-removed area at a dose of 7.5 mg / kg once daily for 28 days. Pigmentation and hair growth in the hair-removed area on the backs of the mice were observed and photographed.

[0094] The experimental results are shown in Figure 2: ① During days 0-28, the skin of mice in the saline group remained pink, and their hair was in the resting phase, indicating that the androgenic alopecia animal model was successfully established; ② On day 14 of drug administration, gray-black pigmentation and small areas of short hair shafts appeared on the back skin of mice in the 25% SEQ ID NO.1 group. The skin of mice in the minoxidil group and SEQ ID NO.1-5 groups remained pink; ③ On day 21 of drug administration, the hair of mice in the 50% SEQ ID NO.1 group grew densely, and the skin almost completely covered the hair, while the other 50% of mice showed localized pigmentation and short hair shafts. The hair of mice in the 50% SEQ ID NO.2 group grew densely, and the skin partially covered the hair, while the other 50% of mice showed localized short hair shafts. The 75% minoxidil group showed localized gray-black pigmentation and a few short hair shafts on the back skin. SEQ ID NO.3 and 5 groups remained pink. 25% of SEQ ID NO.4 group showed small areas of short hair shafts; ④ On day 28 after administration, 75% of SEQ ID NO.1 group showed dense hair growth and skin covered with hair, while the other 25% showed large areas of pigmentation and a few short hair shafts. 50% of SEQ ID NO.2 group mice showed dense hair growth and skin covered with hair, while the other 50% of mice showed an increase in pigmentation area and short hair shafts compared to before. 100% minoxidil group showed large areas of gray-black pigmentation on the back skin, with localized short hair shafts. 25% of SEQ ID NO.3–5 groups showed localized short hair shafts. The above experimental results confirm that SEQ ID NO.1 and SEQ ID NO.2 groups have the best effect on promoting hair regeneration in androgenic alopecia model animals, significantly better than the commercially available preparation, Mandi minoxidil tincture, and SEQ ID NO.3–5 groups.

[0095] The animal model of androgenetic alopecia was established using the same method as above. 24 hours after hair removal, the animals were divided into SEQ ID NO.6 and SEQ ID NO.7 groups, with 3 mice in each group. Both groups received a 0.5% (w / v) testosterone solution (ethanol:propylene glycol:water = 56:24:20, v / v) at a dose of 25 mg / kg topically applied to the hair-removed area once daily for 28 days. In addition to the testosterone solution, groups SEQ ID NO.6 and SEQ ID NO.7 received a 0.15% (w / v) polypeptide solution (ethanol:phosphate buffer = 50:50, v / v) at a dose of 7.5 mg / kg topically applied to the hair-removed area once daily for 28 days. Pigmentation and hair growth in the hair-removed area on the backs of the mice were observed and photographed. The experimental results are shown in Figure 3: ① On day 14 of administration, grayish-black pigmentation appeared on the back skin of mice in group SEQ ID NO.6, with small areas showing short hair shafts. ① In group SEQ ID NO.7, some mice showed slight skin pigmentation; ② On day 21 of administration, 67% of mice in group SEQ ID NO.6 had dense hair growth, with their skin almost completely covered by hair, while the other 33% of mice had only partial hair coverage. In group SEQ ID NO.7, 67% of mice had dense hair growth, with their skin mostly covered by hair; ③ On day 28 of administration, the hair of mice in groups SEQ ID NO.6 and SEQ ID NO.7 showed further growth than before. These experimental results confirm that both groups SEQ ID NO.6 and SEQ ID NO.7 can promote hair regeneration in androgenic alopecia model animals.

[0096] The animal model of androgenetic alopecia was established using the same method as above. Twenty-four hours after hair removal, the animals were divided into four groups: SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, with four mice in each group. All five groups received a 0.5% (w / v) testosterone solution (ethanol:propylene glycol:water = 56:24:20, v / v) applied topically to the hair-removed area at a dose of 25 mg / kg once daily for 28 days. In addition to the testosterone solution, groups SEQ ID NO.8–12 received a 0.15% (w / v) polypeptide solution (ethanol:phosphate buffer = 50:50, v / v) applied topically to the hair-removed area at a dose of 7.5 mg / kg once daily for 28 days. Two weeks after drug withdrawal, the pigmentation and hair growth in the hair-removed area on the backs of the mice were photographed and recorded. The experimental results are shown in Figure 4: Groups SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 showed better effects in promoting hair regeneration in androgenic alopecia model animals.

[0097] The animal model of androgenetic alopecia was established using the same method as above. Twenty-four hours after hair removal, the animals were divided into three groups: SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15, with three mice in each group. All three groups received a 0.5% (w / v) testosterone solution (ethanol:propylene glycol:water = 56:24:20, v / v) applied topically to the hair-removed area at a dose of 25 mg / kg once daily for 28 days. In addition to the testosterone solution, groups SEQ ID NO.13–15 received a 0.05% (w / v) polypeptide solution (diethylene glycol monoethyl ether:Tween 20:water = 15:5:80, v / v) applied topically to the hair-removed area at a dose of 2.5 mg / kg once daily for 28 days. Two weeks after drug withdrawal, the pigmentation and hair growth in the hair-removed area on the backs of the mice were photographed and recorded. The experimental results are shown in Figure 5: SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15 groups showed better effects in promoting hair regeneration in androgenic alopecia model animals.

[0098] Example 5: Bradykinin receptor-active peptides promote β-catenin expression in skin tissue of an animal model of androgenetic alopecia.

[0099] The animal model of androgenetic alopecia was established using the same method as above. 24 hours after hair removal, the animals were divided into two groups: a saline group and a SEQ ID NO.1 group. The control group (mice) received no treatment. Each group consisted of 3 mice. Both groups received a 0.5% (w / v) testosterone solution (ethanol:propylene glycol:water = 56:24:20, v / v) applied topically to the hair-removed area at a dose of 25 mg / kg once daily for 28 days. In addition to the testosterone solution, the following administration regimens were used in both groups: the saline group received a topical application of saline solution to the hair-removed area once daily for 28 days; the SEQ ID NO.1 group received a 0.15% (w / v) polypeptide solution (ethanol:phosphate buffer = 50:50, v / v) applied topically to the hair-removed area at a dose of 7.5 mg / kg once daily for 28 days. Animals were euthanized, and skin tissue was collected and preserved by immersion in tissue fixative. Paraffin-embedded and sectioned tissues were stained with β-catenin antibody and scanned for observation. Normal mouse skin tissue was used as a control. The experimental results are shown in Figure 6: Compared with the blank mouse group, the β-catenin expression level in the saline group was extremely low; the β-catenin expression level in the SEQ ID NO.1 group was high, close to the β-catenin expression level in the skin tissue of the blank mouse group. These experimental results confirm that the expression level of β-catenin in the skin tissue of the androgenic alopecia animal model is significantly downregulated, and the bradykinin B2 receptor active peptide SEQ ID NO.1 can promote the normalization of β-catenin expression in the skin tissue of the androgenic alopecia animal model.

[0100] Example 6: Safety Evaluation

[0101] The animal experiments were divided into four groups: a blank group (Blank), a saline group (Saline), a minoxidil group (Minoxidil), and a SEQ ID NO.1 group, with three mice in each group. The blank group mice received no treatment. C57 mice were anesthetized with 8% chloral hydrate. A 2cm × 2cm area was shaved off along the spine, and depilatory cream was evenly applied. After 1 minute, the depilatory cream was washed off with water to confirm that the hair growth was in the resting phase (the skin was pink). 24 hours after hair removal, the saline group, minoxidil group, and SEQ ID NO.1 group all applied 0.5% (w / v) testosterone solution (ethanol:propylene glycol:water = 56:24:20, v / v) to the depilated area at a dose of 25 mg / kg once daily for 28 days. In addition to the administration of testosterone solution, the drug administration regimens for each experimental group were as follows: The saline group received topical application of saline solution to the hair-removing area once daily for 28 days; the minoxidil group received topical application of commercially available 5% minoxidil solution (250 mg / kg) to the hair-removing area once daily for 28 days; and the SEQ ID NO.1 group received topical application of 0.15% (w / v) peptide solution (ethanol: phosphate buffer = 50:50, v / v) to the hair-removing area at a dose of 7.5 mg / kg once daily for 28 days. After drug administration, the hair and debris around the mouse's eye sockets were cleaned, and blood was collected from the eye sockets into anticoagulant tubes containing EDTA-K2. After sampling, the tubes were gently inverted several times to thoroughly mix the blood with the anticoagulant, and the resulting whole blood samples were stored at 4°C. Blood was collected from the orbital region of mice into 1.5 mL EP tubes. The whole blood was allowed to stand at room temperature for 1 hour to allow clotting. After clotting, the blood was centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant serum was transferred to another clean EP tube, and the serum sample was stored at -80°C. Complete blood count and blood biochemistry analyses were performed on whole blood and serum samples from each group of mice to assess the blood safety and effects on liver and kidney function of SEQ ID NO.1 during continuous drug administration. Finally, the heart, liver, spleen, lungs, kidneys, and skin of each group of mice were removed and placed in 4% paraformaldehyde fixative for embedding, sectioning, and H&E staining. Pathological sections of the major organs and skin of each group of mice were observed and analyzed.

[0102] Decreased red blood cell count and elevated hemoglobin levels suggest possible hemolysis. Results are shown in Figures 7A and 7B. The red blood cell count and hemoglobin levels in the minoxidil group and SEQ ID NO.1 group were not significantly different from the control group, and both were within the normal range. Elevated white blood cell levels suggest a possible inflammatory response. Results are shown in Figure 7C. The white blood cell count in the minoxidil group and SEQ ID NO.1 group was not significantly different from the control group, and both were within the normal range. Platelet levels reflect the body's coagulation function. Results are shown in Figure 7D. The platelet count in both the minoxidil group and SEQ ID NO.1 group were within the normal range. These experimental results indicate that continuous transdermal administration of SEQ ID NO.1 at a dose of 7.5 mg / kg for 28 days did not affect the blood system of androgenetic alopecia model mice, demonstrating good blood safety.

[0103] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are two important liver function enzymes that reflect liver damage. When hepatocytes are damaged, ALT and AST are released into the bloodstream, leading to elevated serum ALT and AST levels. As shown in Figures 8A and 8B, the ALT and AST levels in the minoxidil group and the SEQ ID NO.1 group were not significantly different from the control group and were within the normal range. Blood urea nitrogen (BUN) and creatinine (CREA) reflect renal excretion and filtration function. Elevated BUN and CREA levels indicate renal impairment and decreased glomerular filtration rate. As shown in Figures 8C and 8D, the BUN and CREA levels in the minoxidil group and the SEQ ID NO.1 group were within the normal range. These experimental results indicate that continuous transdermal administration of SEQ ID NO.1 at a dose of 7.5 mg / kg for 28 days did not cause liver and kidney damage in androgenic alopecia model mice, demonstrating good hepato-renal safety.

[0104] Figure 9 shows the H&E staining results of pathological sections of major organs from mice in the blank control group, saline group, minoxidil group, and SEQ ID NO.1 group: ① Heart section observation showed that the cardiac tissue structure of mice in the above groups was basically normal, the myocardial fibers were neatly and tightly arranged without breakage; the myocardial cell nuclei were clear, the cytoplasm was abundant, and no necrosis or degeneration was observed; no inflammatory cell infiltration or fibrosis was observed in the tissue; ② Liver section results showed that the liver tissue structure of mice in the above groups was normal, the hepatocyte nuclei were round, the cytoplasm was abundant, and no necrosis was observed; the hepatic sinusoids were of uniform size without dilation, and the hepatic cords were neatly and tightly arranged; no obvious inflammation was observed in the tissue. ③ Spleen section observation showed that the splenic nodule structure of mice in all groups was normal, the red and white pulp boundaries were clear, the white pulp was rich in lymphocytes, tightly arranged, and no necrosis was observed; ④ Lung section results showed that the lung tissue structure of mice in all groups was basically normal, the bronchial epithelial cells were neatly and tightly arranged, and no inflammatory cell infiltration was observed in the tissue; ⑤ Kidney section observation showed that the glomeruli of mice in all groups were intact, the capsule cavity was not dilated, the cell nuclei in the glomeruli were rounded, no necrosis was observed, and the capillary loop structure was clear; no inflammatory cell infiltration or hemorrhage was observed in the interstitium. The above experimental results indicate that continuous transdermal administration at a dose of 7.5 mg / kg for 28 days did not have adverse effects on the major organs of androgenic alopecia model mice and has good safety.

[0105] Figure 10 shows the H&E staining results of skin sections from mice in the blank control group, saline group, minoxidil group, and SEQ ID NO.1 group. The skin tissue structure of mice in each group was basically normal, with an intact epidermal layer and uniform spinous layer thickness. The collagen fibers in the dermis were neatly and tightly arranged and abundant. No inflammatory cell infiltration was observed in the tissues. These experimental results indicate that continuous transdermal administration of SEQ ID NO.1 at a dose of 7.5 mg / kg for 28 days did not adversely affect the skin tissue morphology of androgenetic alopecia model mice, demonstrating good skin safety.

Claims

1. A polypeptide and its derivatives having bradykinin receptor binding activity, characterized in that, The polypeptide is selected from the following: (I) A polypeptide that differs from the amino acid sequence shown in any one of SEQ ID NO:1-15 by 1-6 amino acid residues; said difference being the deletion, addition, or substitution of amino acid residues; said added or substituted amino acid being a natural or non-natural amino acid; (II) A polypeptide obtained by replacing the amino acid configuration in the polypeptide described in (I); (III) A polypeptide in which the amino acid configuration of the polypeptide described in (I) is replaced and flipped in the direction of C-terminus → N-terminus; (IV) Peptides that have undergone amino acid modification or cyclization of the peptides described in (I)-(III), or bioisoelectronic peptides; The amino acid sequence of the polypeptide is not shown in any of SEQ ID NO:1-6.

2. The polypeptide and its derivatives as described in claim 1, characterized in that, The polypeptide differs from the amino acid sequence shown in any one of SEQ ID NO:1-15 by 1-3 amino acid residues; Preferably, the polypeptide differs from the amino acid sequence shown in any one of SEQ ID NO:1-12 by 1-3 amino acid residues.

3. The polypeptide and its derivatives as described in claim 1, characterized in that, The polypeptide is selected from the following: (i) A polypeptide with an amino acid sequence as shown in any one of SEQ ID NO:7-15; (ii) A polypeptide obtained by replacing the amino acids in the polypeptide described in (i) with amino acid configurations; (iii) A polypeptide in which the amino acid configuration of the polypeptide described in (i) is replaced and flipped in the direction of C-terminus → N-terminus; (iv) Peptides that have undergone amino acid modification or cyclization of the peptides described in (i)-(iii), or bioisoelectronic peptides; Preferably, the amino acid modification includes C-terminal modification, N-terminal modification, intermediate residue modification, and non-natural amino acid modification; the modification type of the C-terminal modification, N-terminal modification, and intermediate residue modification is preferably selected from one or more of acylation modification, amidation modification, esterification modification, glycosylation modification, fatty acid modification, non-natural amino acid modification, external group modification, and pseudopeptide modification; the external group modification is preferably cholesterol modification or polyethylene glycol modification.

4. The polypeptide and its derivatives as described in any one of claims 1-3, characterized in that, The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO:7-15; And / or, the bradykinin receptor is bradykinin receptor B2; And / or, the polypeptide and its derivatives have hair regeneration-promoting activity.

5. A composition, characterized in that, The composition comprises a polypeptide or a derivative thereof as described in any one of claims 1-4, and at least one of a pharmaceutically acceptable carrier, excipient, or diluent.

6. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises a polypeptide or a derivative thereof as described in any one of claims 1-4 or a composition as described in claim 5; The pharmaceutical preparation is a topical preparation that promotes hair growth, and the topical preparation is preferably a transdermal preparation, such as a foam, liniment, gel, patch, spray, cream, emulsion, solution, tincture or ointment.

7. A medicine box set, characterized in that, The kit includes a pillbox A, wherein pillbox A comprises one or more of the polypeptide or derivative thereof as described in any one of claims 1-4, the composition as described in claim 5, or the pharmaceutical preparation as described in claim 6; and, Kit B contains one or more of the following: milodil, prostaglandin derivatives, autologous platelet-rich plasma, botulinum toxin, growth factors, androgen receptor antagonists, and antibody drugs.

8. A method for preparing polypeptides and their derivatives with bradykinin receptor binding activity, characterized in that, The method includes directly synthesizing the polypeptides and their derivatives as described in any one of claims 1-4 using solid-phase polypeptide synthesis technology.

9. The use of a polypeptide or derivative thereof comprising an amino acid sequence as shown in any one of SEQ ID NO: 1-6, a polypeptide or derivative thereof as described in any one of claims 1-4, a composition as described in claim 5, a pharmaceutical preparation as described in claim 6, or a kit as described in claim 7 in the preparation of a product for promoting hair regeneration.

10. A method for promoting hair regeneration, characterized in that, The method includes administering to a subject in need an effective amount of a polypeptide or a derivative thereof comprising an amino acid sequence as shown in any one of SEQ ID NO: 1-6, a polypeptide or a derivative thereof as described in any one of claims 1-4, a composition as described in claim 5, a pharmaceutical preparation as described in claim 6, or a kit as described in claim 7; The method described is neither a diagnostic nor a treatment method.

11. A method for preventing and / or treating hair loss, characterized in that, The method includes administering to a subject in need an effective amount of a polypeptide or a derivative thereof comprising an amino acid sequence as shown in any one of SEQ ID NO: 1-6, a polypeptide or a derivative thereof as described in any one of claims 1-4, a composition as described in claim 5, a pharmaceutical preparation as described in claim 6, or a kit as described in claim 7; Preferably, the hair loss is non-scarring hair loss or scarring hair loss, and the non-scarring hair loss preferably includes androgenetic alopecia, alopecia areata, telogen effluvium and chemotherapy-induced hair loss; More preferably, the hair loss is androgenetic alopecia.

12. The method as described in claim 11, characterized in that, The method also includes the use of one or more of physical therapy, biological therapy and surgical treatment; Preferably, the physical therapy includes low-energy laser therapy and microneedling therapy; the biological therapy includes autologous platelet-rich plasma therapy, antibody drug therapy, growth factor drug therapy, botulinum toxin therapy, and gene therapy; and the surgical treatment includes autologous hair transplantation.

13. A polypeptide or a derivative thereof comprising an amino acid sequence as shown in any one of SEQ ID NO: 1-6, a polypeptide or a derivative thereof as described in any one of claims 1-4, a composition as described in claim 5, a pharmaceutical preparation as described in claim 6, or a kit as described in claim 7, for promoting hair regeneration or for preventing and / or treating hair loss; Preferably, the hair loss is non-scarring hair loss or scarring hair loss, and the non-scarring hair loss preferably includes androgenetic alopecia, alopecia areata, telogen effluvium and chemotherapy-induced hair loss; More preferably, the hair loss is androgenetic alopecia.

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