A bradykinin receptor active peptide and its use in promoting hair regrowth

Abstract

This invention discloses a bradykinin receptor active peptide and its application in promoting hair regeneration. The invention also discloses compositions comprising the bradykinin receptor active peptide, pharmaceutical preparations, kits, and methods for preparing the bradykinin receptor active peptide. The bradykinin receptor active peptide disclosed in this invention exhibits good hair regeneration effects and safety, and has promising clinical application prospects.

Description

[0001] This application claims priority to Chinese Patent Application No. 202411917455X, filed on December 24, 2024. This application incorporates the entirety of the aforementioned Chinese Patent Application. TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine, in particular to a bradykinin receptor active peptide and its application in promoting hair regeneration. BACKGROUND

[0003] Hair loss is a hair regeneration disorder, which refers to abnormal or excessive hair loss. It can be divided into androgenic alopecia, alopecia areata, traumatic alopecia and nutritional alopecia according to the causes, among which more than 90% are androgenic alopecia.

[0004] Current clinical treatment options for hair loss include the following: ① drug therapy: minoxidil, androgen receptor antagonists and traditional Chinese medicine extracts; ② physical therapy: low-energy laser and microneedle therapy; ③ biological therapy: autologous platelet-rich plasma; ④ surgical treatment: autologous hair transplantation. The above methods have shown certain therapeutic effects on patients with hair loss, but still 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 microneedle still need to be used in combination with drugs. Autologous platelet-rich plasma requires the extraction of the patient's own blood, and the preparation process is relatively cumbersome, with non-uniform quality standards and invasive treatment. Autologous hair transplantation is invasive, with limited hair follicle survival rate, and postoperative drug therapy is still needed to maintain, and the treatment cost is relatively high. Compared with oral drugs, topical drugs produce local effects, reducing systemic drug exposure, and can achieve better safety and patient compliance. In view of this, the development of safe and efficient topical drugs for hair loss is of great significance.

[0005] Hair follicle is the basic unit of hair growth, and the hair papilla cells are located at the bottom of the hair follicle. The capillary blood vessels provide nutrients to the hair follicle through the hair papilla cells. The development and growth of hair follicle is regulated by a fine signal network, involving Wnt, bone morphogenetic protein, transforming growth factor-β, fibroblast growth factor, mitogen-activated protein kinase and phosphatidylinositol 3 kinase / protein kinase B, etc. Among them, the Wnt signal pathway plays a core role in the development and cycle growth of hair follicle. Studies have shown that the key molecule β-catenin of the Wnt signal pathway is an important protein for maintaining the induction activity and proliferation function of hair papilla cells, which can regulate the formation of hair follicle basement, the growth cycle of hair follicle and the proliferation and differentiation of hair follicle stem cells. Hair follicle is a metabolically active organ, and its growth and periodic cycle requires sufficient nutrients and oxygen supply. Vascular endothelial growth factor (VEGF) can promote hair follicle angiogenesis and increase blood supply to hair follicle. In addition, VEGF also has the functions of promoting the proliferation and differentiation of hair follicle stem cells, enhancing the immune defense ability of hair follicle, and regulating the growth cycle of hair follicle. Bradykinin and its analogues can promote the expression of VEGF and promote angiogenesis.

[0006] The bradykinin receptor belongs to the G protein-coupled receptor family, which includes B1 and B2 subtypes. The B1 receptor is almost not expressed in normal tissues, and is only up-regulated after the release of pro-inflammatory factors induced by tissue damage. The B2 receptor is widely present in various tissues of the body. In normal human skin tissue, the bradykinin B2 receptor is highly expressed in keratinocytes, dermal fibroblasts, hair papilla cells, dermal microvascular endothelial cells and dermal lymphatic endothelial cells. From the structural point of view, the active peptides of bradykinin B2 receptor are divided into non-peptide active peptides and peptide active peptides. Due to the similarity in structure with the natural ligand, the peptide active peptides can bind more tightly and specifically to the B2 receptor, showing higher selectivity and affinity. Therefore, the peptide B2 receptor active peptide can play a role in specific biological processes and cell signal transduction, reduce the interference with other receptors, and reduce the risk of side effects. From the perspective of treatment, the peptide B2 receptor active peptide has lower toxicity and lower immunogenicity than small molecule non-peptide B2 receptor active peptide. SUMMARY

[0007] To solve the above technical problems, the present application provides a bradykinin receptor active peptide and its application in promoting hair regeneration.

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

[0009] (I) a polypeptide having 1-6 amino acid residue difference from the amino acid sequence as shown in any one of SEQ ID NOs: 1-15; the difference is deletion, addition or substitution of an amino acid residue; the added or substituted amino acid is a natural amino acid or a non-natural amino acid;

[0010] (II) a polypeptide after amino acid configuration substitution of the amino acid in the polypeptide of (I);

[0011] (III) a polypeptide after amino acid configuration substitution of the amino acid in the polypeptide of (I) and inversion in the direction of C-terminal→N-terminal of the polypeptide;

[0012] (IV) a polypeptide after amino acid modification or cyclization of the polypeptide of (I)-(III), or a bioisosteric polypeptide;

[0013] wherein the amino acid sequence of the polypeptide is not as shown in any one of SEQ ID NOs: 1-6.

[0014] In this context, “amino acid configuration substitution” includes substitution of an L-configuration amino acid with a D-configuration amino acid or substitution of a D-configuration amino acid with an L-configuration amino acid.

[0015] In some embodiments, the polypeptide has 1-5 amino acid residue difference from the amino acid sequence as shown in any one of SEQ ID NOs: 1-15.

[0016] In some embodiments, the polypeptide has 1-4 amino acid residue difference from the amino acid sequence as shown in any one of SEQ ID NOs: 1-15.

[0017] In some embodiments, the polypeptide has 1-3 amino acid residue difference from the amino acid sequence as shown in any one of SEQ ID NOs: 1-15.

[0018] In some embodiments, the polypeptide has 1-3 amino acid residue difference from the amino acid sequence as shown in any one of SEQ ID NOs: 1-12.

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

[0020] (I) a polypeptide having 1-3 amino acid residue difference from the amino acid sequence as shown in any one of SEQ ID NOs: 1-12; the difference is deletion, addition or substitution of an amino acid residue; the added or substituted amino acid is a natural amino acid or a non-natural amino acid;

[0021] (II) a polypeptide after amino acid configuration substitution of the amino acid in the polypeptide of (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) A polypeptide that has undergone amino acid modification or cyclization of the polypeptides described in (I)-(III), or a polypeptide with bioisoelectronic arrangement.

[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 a derivative thereof, a polypeptide or a derivative thereof 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 This refers to the expression of bradykinin B2 receptor in human dermal papilla cells.

[0071] Figure 2 The hair regeneration effect of the polypeptides SEQ ID NO.1~5 in an animal model of androgenic alopecia.

[0072] Figure 3 The hair regeneration effect of the peptides in the animal models of androgenic alopecia (SEQ ID NO. 6 and SEQ ID NO. 7).

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

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

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

[0076] Figure 7 The first image shows the complete blood count (CBC) atlas of SEQ ID NO.1; the second image shows the red blood cell (RBC) count atlas; the third image shows the hemoglobin (HGB) level atlas; the fourth image shows the white blood cell (WBC) count atlas; and the fifth image shows the platelet (PLT) count atlas.

[0077] Figure 8 The following are blood biochemical profiles for SEQ ID NO.1: A is alanine aminotransferase (ALT) level profile; B is aspartate aminotransferase (AST) level profile; C is blood urea nitrogen (BUN) level profile; and D is creatinine (CREA) level profile.

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

[0079] Figure 10 H&E staining pattern of skin in 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: 1g of 2-chlorotriphenylmethyl chloride resin is weighed, swollen with dichloromethane for 15min, 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 10min and 20min 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 300 Å, 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 peptides 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 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]

[0087] Example 2: Computer simulation analysis of peptide and bradykinin B2 receptor protein models

[0088] 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 B2 receptor protein was performed using the molecular docking software Maestro 2018-1. First, the obtained protein structure was preprocessed 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.

[0089] 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 6 Å, thus forming the simulation system. Sufficient Cl was added to the dodecahedral solution box... −To ensure the electroneutrality of the system, the entire system underwent an energy minimization (EM) procedure, allowing the system energy to decrease to below 1000 kJ / mol within 50,000 steps for relaxation. The system was then heated in the NVT ensemble to a 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 over 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, fluctuating around 0.3 Å or 0.35 Å, and all systems essentially reached convergence. 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.

[0090] Table 2. Binding activity of peptides to bradykinin B2 receptor

[0091]

[0092] Example 3: Expression of bradykinin B2 receptor in human dermal papilla cells

[0093] 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 and boiling denaturation in a water bath for 10 min. The samples were then 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 sample was 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 develop and detect protein bands on a gel imaging system. Results are as follows: Figure 1 As shown: human dermal papilla cells show high expression of bradykinin B2 receptor.

[0094] Example 4: Hair regeneration experiment in an animal model of androgenetic alopecia

[0095] 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 4 mice in each group. All groups received a 0.5% (w / v) testosterone solution (ethanol:propylene glycol:water = 56:24:20, v / v) applied topically 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-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) peptide 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.

[0096] Experimental results are as follows Figure 2As shown: 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, 25% of mice in group SEQ ID NO.1 showed gray-black pigmentation on their backs and small areas of short hair shafts. The skin of mice in the minoxidil group and groups SEQ ID NO.1-5 remained pink; ③ On day 21 of drug administration, 50% of mice in group SEQ ID NO.1 showed dense hair growth, with their skin almost completely covering the hair, while the other 50% of mice showed localized pigmentation and short hair shafts. 50% of mice in group SEQ ID NO.2 showed dense hair growth, with their skin partially covering 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 their backs. Groups SEQ ID NO.3 and 5 remained pink. 25% of group SEQ ID NO.4 showed small areas of short hair shafts. On day 28 after administration, 75% of mice in group SEQ ID NO.1 showed dense hair growth and hair coverage on the skin, while the other 25% showed extensive pigmentation and a few short hair shafts. In group 50% of mice in group SEQ ID NO.2, hair growth was dense and hair covered a large area of ​​the skin; the other 50% showed an increase in pigmentation area and short hair shafts compared to before. In the 100% minoxidil group, there was extensive gray-black pigmentation on the back skin, with localized short hair shafts. Groups 25% of mice in groups SEQ ID NO.3–5 showed localized short hair shafts. These experimental results confirm that groups SEQ ID NO.1 and SEQ ID NO.2 showed the best effect in promoting hair regeneration in androgenic alopecia models, significantly superior to the commercially available formulation, Mandi minoxidil tincture, and groups SEQ ID NO.3–5.

[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 two groups: SEQ ID NO.6 and SEQ ID NO.7, with three mice in each group. Both groups received a topical application of 0.5% (w / v) testosterone solution (ethanol:propylene glycol:water = 56:24:20, v / v) at a dose of 25 mg / kg to the hair removal area once daily for 28 days. In addition to the testosterone solution, groups SEQ ID NO.6 and 7 received a topical application of 0.15% (w / v) peptide solution (ethanol:phosphate buffer = 50:50, v / v) at a dose of 7.5 mg / kg to the hair removal area once daily for 28 days. Pigmentation and hair growth in the hair removal area on the backs of the mice were observed and photographed. The experimental results are as follows: Figure 3As shown: ① On day 14 of drug administration, grayish-black pigmentation appeared on the back skin of mice in group SEQ ID NO.6, with small areas showing short hair shafts. Some mice in group SEQ ID NO.7 showed slight pigmentation on their skin; ② On day 21 of drug 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 partial hair coverage. 67% of mice in group SEQ ID NO.7 had dense hair growth, with their skin mostly covered by hair; ③ On day 28 of drug administration, the hair of mice in groups SEQ ID NO.6 and SEQ ID NO.7 showed further growth than before. The above experimental results confirm that both groups SEQ ID NO.6 and SEQ ID NO.7 can promote hair regeneration in androgenic alopecia model animals.

[0098] 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 as follows: Figure 4 As shown, 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.

[0099] 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 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 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 as follows: Figure 5As shown, 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.

[0100] Example 5: Bradykinin receptor-active peptides promote β-catenin expression in skin tissue of an animal model of androgenetic alopecia.

[0101] 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 two groups: a saline group and a SEQ ID NO.1 group. The control group (mice) received no treatment. Each group consisted of three 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 in a tissue fixative. Paraffin-embedded and sectioned tissues were stained with β-catenin antibody using immunofluorescence, and the sections were scanned for observation. Normal mouse skin tissue was used as a control. Experimental results are as follows: Figure 6 As shown: Compared with the blank mouse group, the expression level of β-catenin in the saline group was extremely low; the expression level of β-catenin in the SEQ ID NO.1 group was high, close to the expression level of β-catenin in the skin tissue of blank mice. The above experimental results confirm that the expression level of β-catenin in the skin tissue of 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 androgenic alopecia animal model.

[0102] Example 6: Safety Evaluation

[0103] 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.

[0104] A decrease in red blood cells and an increase in hemoglobin levels suggest that hemolysis may have occurred. Results as follows Figure 7 A and Figure 7 As shown in Figure B, the number of red blood cells and hemoglobin levels in the minoxidil group and SEQ ID NO.1 group were not significantly different from those in the control group, and both were within the normal range. Elevated white blood cell levels suggest a possible inflammatory response in the body. Results are as follows... Figure 7As shown in Figure C, the white blood cell counts in the minoxidil group and SEQ ID NO.1 group were not significantly different from those in the control group, and were all within the normal range. Platelet levels reflect the body's coagulation function. The results are as follows... Figure 7 As shown in Figure D, the platelet counts in both the minoxidil group and the SEQ ID NO.1 group were within the normal range. These experimental results indicate that, with continuous transdermal administration at a dose of 7.5 mg / kg for 28 days, SEQ ID NO.1 did not affect the hematologic system of androgenetic alopecia model mice, demonstrating good hematologic safety.

[0105] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are two important liver function enzymes that reflect the extent of liver damage. When liver cells are damaged, ALT and AST are released into the bloodstream, leading to elevated serum ALT and AST levels. The results are as follows... Figure 8 A and Figure 8 As shown in Figure B, the ALT and AST levels in the minoxidil group and SEQ ID NO.1 group were not significantly different from those in the blank group, and both were within the normal range. Blood urea nitrogen (BUN) and creatinine (CREA) reflect renal excretion and filtration function; elevated BUN and CREA levels suggest renal impairment and decreased glomerular filtration function. Results are as follows... Figure 8 C and Figure 8 As shown in Figure D, the BUN and CREA levels in both the minoxidil group and the SEQ ID NO.1 group were within the normal range. These experimental results indicate that, with continuous transdermal administration at a dose of 7.5 mg / kg for 28 days, SEQ ID NO.1 did not cause liver or kidney damage in androgenic alopecia model mice, demonstrating good hepato-renal safety.

[0106] 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 are as follows: Figure 9As shown: ① Heart section observation showed that the heart tissue structure of mice in all 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 all groups was normal, the hepatocyte nuclei were round, the cytoplasm was abundant, and no necrosis was observed; the hepatic sinusoids were of uniform size and no dilation was observed, the hepatic cords were neatly and tightly arranged; no obvious inflammatory cell infiltration was observed in the tissue; ③ Spleen section observation showed that the spleen nodule structure of mice in all groups was normal, the red and white pulp boundaries were clear, the white pulp lymphocytes were abundant and 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 Observation of kidney sections showed that the glomeruli of mice in all groups were structurally intact, with no dilation of the capsule, rounded cell nuclei without necrosis, and clear capillary loops; no inflammatory cell infiltration or hemorrhage was observed in the interstitium. 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 have adverse effects on the major organs of androgenic alopecia model mice, demonstrating good safety.

[0107] The H&E staining results of pathological sections of mouse skin from the blank control group, saline group, minoxidil group, and SEQ ID NO.1 group are as follows: Figure 10 As shown: 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 tissue. The above 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 having a difference of 1 to 6 amino acid residues from the amino acid sequence shown in any one of SEQ ID NO: 1-15; 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 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; 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 comprises 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.

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