Fusion polypeptide and application thereof

Abstract

The invention belongs to a biopharmacy technology, and particularly relates to a fusion polypeptide and a derivative thereof, and an application of the fusion polypeptide and the derivative in preparation of medicines for treating alopecia, acne, dermatitis and other related diseases. The fusion polypeptide is formed by sequentially connecting an O peptide or a derivative peptide of the O peptide, an I peptide or a derivative peptide of the I peptide and two structural functional domains through a connecting peptide linker or directly connecting the two structural functional domains from the N end to the C end, or the fusion polypeptide is formed by directly connecting the peptide I or the derivative peptide thereof, the peptide O or the derivative peptide thereof and two structural functional domains through a connecting peptide linker in sequence from the N end to the C end; the fusion polypeptide provided by the invention can simultaneously activate GPR158 and / or GPRC6A and / or GPR37 pathways and simultaneously activate an integrin receptor pathway, has the remarkable advantages of high hair growth promotion activity, quick response and long hormone regulation action duration, and provides a new medicine for treating alopecia, acne, dermatitis and other related diseases.

Description

Technical Field

[0001] The present invention belongs to biopharmaceutical technology, and specifically relates to a fusion polypeptide and its derivatives, as well as their use in preparing drugs for treating alopecia, acne, dermatitis and other related diseases. Background Art

[0002] Hair loss, acne, and dermatitis are common conditions caused by a variety of factors, including endocrine and metabolic disorders, aging, and medications. Androgenetic alopecia (AGA) is one of the most common causes of hair loss in both sexes. The onset and progression of AGA depend on multiple factors, including genetic susceptibility, endocrine and metabolic factors, and exogenous factors. Acne and dermatitis are also largely caused by endocrine-related factors. Furthermore, medication-induced hair loss can also be a significant problem for many people.

[0003] The demand for drugs that can improve hair growth and the appearance and condition of the skin has led to a huge industry, but there are still few drugs that can effectively achieve these goals. Summary of the Invention

[0004] In view of the lack of drugs in the prior art for improving hair growth, appearance and skin condition, the present invention proposes the following technical solutions;

[0005] O-peptide is an endogenous peptide found in osteocalcin that can activate GPR158 and / or GPRC6A and / or GPR37. The mouse peptide is composed of 15 amino acids and can improve fat metabolism. Its physiological functions in humans are similar to those of osteocalcin, and it has a long half-life in vivo. O-peptide alone can promote hair growth and treat stress-induced hair follicle atrophy and / or hair loss, seborrheic follicle atrophy and / or hair loss, stress-induced acne and / or dermatitis, and other conditions.

[0006] I-peptide is an endogenous peptide found in irisin and is an agonist that activates the integrin receptor signaling pathway. I-peptide alone can promote hair growth and accelerate the transition from the resting phase to the anagen phase.

[0007] The first aspect of the present invention discloses a fusion polypeptide, wherein the fusion polypeptide is composed of an O peptide or a derivative thereof, and an I peptide or a derivative thereof, from the N-terminus to the C-terminus, and the two structural and functional domains are connected by a linker or directly;

[0008] Or the fusion polypeptide is composed of peptide I or its derivative peptide, peptide O or its derivative peptide from N-terminus to C-terminus, and the two structural and functional domains are directly connected by a linker peptide;

[0009] The O peptide or its derivative peptide is selected from osteocalcin-derived peptides; the osteocalcin-derived peptides include mammalian, reptile, amphibian, bony fish, poultry, and bird osteocalcin-derived peptides; the osteocalcin-derived peptides are conserved fragments of osteocalcin derived from different species;

[0010] The I peptide or its derivative peptide is selected from irisin-derived peptides and homologous polypeptides from different species with irisin as the skeleton; the irisin-derived peptides include irisin-derived peptides from mammals, reptiles, amphibians, bony fish, poultry, and birds; the irisin-derived peptides are conserved fragments of irisin from different species.

[0011] Preferably, the amino acid sequence of the O peptide and its derivative peptides is as shown in SEQ ID NOs. 1 to 8, or has more than 50% homology thereto;

[0012]

[0013] The natural human O-peptide sequence is SEQ ID NO.1. Osteocalcin-derived peptides from different species and mutants may have more than 80% homology with this sequence. SEQ ID NO.2 is a mouse osteocalcin-derived peptide. SEQ ID NO.3 is a livestock-sheep osteocalcin-derived peptide, which is identical to the sequence in otters and badgers. SEQ ID NO.4 is a reptile-lizard osteocalcin-derived peptide. SEQ ID NO.5 is an amphibian-water frog osteocalcin-derived peptide. SEQ ID NO.6 is a bony fish-Wuchang fish osteocalcin-derived peptide. SEQ ID NO.7 is a poultry-chicken osteocalcin-derived peptide. SEQ ID NO.8 is a bird-mandarin duck osteocalcin-derived peptide, which is identical to the sequence in swan geese.

[0014] The amino acid sequences of the peptide I and its derivatives are shown in SEQ ID NOs. 9 to 15, or have more than 50% homology thereto;

[0015]

[0016] The sequence of the natural human I peptide is shown in SEQ ID NO.9. Irisin-derived peptides from different species and mutants may have more than 80% homology with this sequence. SEQ ID NO.10 is a mouse irisin-derived peptide, SEQ ID NO.11 is a marsupial irisin-derived peptide, SEQ ID NO.12 is an avian irisin-derived peptide, SEQ ID NO.13 is a reptile irisin-derived peptide, SEQ ID NO.14 is a teleost-small yellow croaker irisin-derived peptide, and SEQ ID NO.15 is a teleost-eel irisin-derived peptide.

[0017] The polypeptide and its derivatives based on the skeleton can be independently synthesized under general chemical laboratory conditions, can be expressed by genes to produce recombinant proteins containing the polypeptide, can also be industrially synthesized by commercial reagent companies, and use the solid-phase method to synthesize polypeptides, in which different amino acids are directed to synthesize amino acid chains on the resin through condensation reactions, or can be extracts isolated from tissues and organs of different species.

[0018] Many proteins and hormones have a high degree of homology in humans and animals. For example, bovine and porcine insulin can be used to treat diabetic patients; pregnant mare serum gonadotropin (PMSG) has also been used for superovulation in humans and animals; the chemical structure of oxytocin in humans and most mammals is the same; since gonadotropin-releasing hormone (GnRH) was isolated from the brains of pigs and sheep in 1971, there are currently at least 28 types in the GnRH family, 15 of which are from vertebrates and 13 from invertebrates. Except for octopus GnRH (octo GnRH), all GnRH peptides are composed of 10 amino acids, and their molecular length and partial amino acid sequence are very conserved. In view of the homology and functional similarity of polypeptides and hormones in different species, sequences of the same species or with higher homology to the polypeptide may play the same role. In the present invention, the O peptide and I peptide in the fusion polypeptide and fusion polypeptide derivative can be derived from different species of organisms.

[0019] Preferably, the connecting peptide is (Gly-Gly-Gly-Gly-Ser)n, wherein n is an integer from 1 to 10; or the connecting peptide is (Pro-Lys-Pro-Lys-Pro)n, wherein n is an integer from 1 to 10.

[0020] Preferably, the fusion polypeptide comprises O peptide-Linker-I peptide and I peptide-Linker-O peptide, and its structure is as follows:

[0021] I peptide-(Gly-Gly-Gly-Gly-Ser)nO peptide;

[0022] or O peptide-(Gly-Gly-Gly-Gly-Ser)nI peptide;

[0023] or I peptide-(Pro-Lys-Pro-Lys-Pro)nO peptide;

[0024] or O-peptide-(Pro-Lys-Pro-Lys-Pro)nI peptide.

[0025] The second aspect of the present invention discloses derivatives of the above-mentioned fusion polypeptide, characterized in that the derivatives are obtained by conventional modification of the amino acid side chain groups, amino terminus, and carboxyl terminus of the fusion polypeptide; or are products obtained by connecting a tag for polypeptide or protein detection or purification to the fusion polypeptide, products obtained by isotope labeling modification, or extracts isolated from tissues and organs of different species.

[0026] Among them, the conventional modifications include fluorescent group modification, phosphorylation modification, disulfide bond cyclization modification, biotin labeling modification, photosensitizer, azide modification, PEG modification, methylation modification, fluorescence quenching group modification, protein coupling modification, small molecule compound modification, amino modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, esterification modification, and glycosylation modification;

[0027] The fluorescent dye used in the modification of the fluorescent group is selected from AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, and ICG, and the modification can be used for fluorescence detection;

[0028] wherein the phosphorylation modification is selected from a combination of one or more of p-Ser, p-Thr, and p-Tyr;

[0029] wherein the glycosylation modification is selected from a combination of one or more of Ser, Asn, Thr, and Tyr;

[0030] wherein the nitration modification is selected from one or more combinations of Tyr;

[0031] The biotin label is selected from D-biotin, biotin hydrazide, photosensitive biotin and biotin-dUTP.

[0032] Preferably, the conventional modifications of the amino terminus and carboxyl terminus are selected from acetylation modification of the polypeptide N terminus and amination modification of the C terminus.

[0033] Preferably, the conventional modifications of the side chain groups are selected from the modifications of the R groups of the amino acid side chains in polypeptides.

[0034] Preferably, the isotope used in the isotope labeling is selected from one or more combinations of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, and 131I.

[0035] The third aspect of the present invention discloses a polynucleotide encoding the above-mentioned fusion polypeptide or its derivatives.

[0036] The fourth aspect of the present invention discloses a polynucleotide comprising the above-mentioned polynucleotide.

[0037] A fifth aspect of the present invention discloses a host cell transfected with the above-mentioned vector, wherein the vector comprises or is selected from a DNA vector, an RNA vector, a plasmid, a liposome, a particle, a transposon vector, a CRISPR / Cas9 vector, a lentiviral vector, or a viral vector.

[0038] The sixth aspect of the present invention discloses the use of the above-mentioned fusion polypeptide, and / or the above-mentioned fusion polypeptide derivative as a receptor GPR158 and / or GPRC6A and / or GPR37 agonist, and / or integrin agonist.

[0039] Agonists are substances that can bind to receptors and stimulate and / or promote their biological activity, including inorganic ions such as cations / metal ions, anions, and hydrogen ions; organic molecules such as peptides and small molecule compounds; and biological macromolecules such as proteins and enzymes.

[0040] A seventh aspect of the present invention discloses the use of the above-mentioned fusion polypeptide, and / or fusion polypeptide derivative, and / or the combination of the receptor GPR158 and / or GPRC6A and / or GPR37 agonist and the integrin receptor agonist according to claim 14 in the preparation of medical and aesthetic products, cosmetics, health products, foods, additives or medicines for preventing or treating hair follicle decline, hair loss, acne or dermatitis;

[0041] Wherein, the hair loss diseases include stress-induced hair follicle decline and / or hair loss in males and females, androgenic hair follicle decline and / or seborrheic alopecia, and drug-induced hair follicle decline and / or hair loss;

[0042] Wherein, the prevention and / or treatment of hair loss is to promote hair growth;

[0043] Wherein, the acne includes male and female pressure acne and androgenic acne;

[0044] The dermatitis includes male and female stress dermatitis and androgenic dermatitis.

[0045] Preferably, the drug contains one or more pharmaceutically acceptable carriers;

[0046] Preferably, the carrier is a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant or lubricant;

[0047] Preferably, the drug is prepared in the form of tablets, granules, capsules, oral liquids, inhalation liquids, smears, sprays, drops, microneedles or injections;

[0048] Preferably, the drug administration method includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration to the subject;

[0049] Preferably, the administration route includes parenteral, such as intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial; transmucosal, such as buccal, sublingual, palatal, gingival, nasal, vaginal, rectal or transdermal, and also includes the use of liposomal formulations, intravenous infusion, transdermal patch delivery mode.

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

[0051] The fusion polypeptide provided by the present invention can simultaneously activate the GPR158 and / or GPRC6A and / or GPR37 pathways, and simultaneously activate the integrin receptor pathway, and has the significant advantages of high hair growth promoting activity, rapid onset and long duration of hormone regulation, providing a new drug for the treatment of hair loss, acne, dermatitis and other related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The therapeutic effects of polypeptides PY1, PY2, PY3 and PY4 in the androgenic (seborrheic) alopecia model in female mice. Figure 1 Middle A shows the hair growth on the back of the neck of female mice in the control group, DHT group, and DHT+peptide treatment group; Figure 1 Middle B is the skin color scoring result of female mice.

[0053] Figure 2 The results are the quantitative results of skin morphology and hair follicle diameter on the back of the neck of female mice with androgenic (seborrheic) alopecia model. Figure 2 Middle A shows the HE staining results of the skin sections on the back of the neck of female mice in the control group, DHT treatment group, and DHT + peptides PY1, PY2, PY3 and PY4 treatment groups; Figure 2 B is based on Figure 2 Statistical results of hair follicle diameters in the skin after morphological observation in Figure A; Figure 2 C is based on Figure 2 Figure A shows the statistical results of the diameter of sebaceous glands in the skin after morphological observation.

[0054] Figure 3A -C is the RNA-seq sequencing results (upregulated genes) of skin tissues of female mice in the androgenic (seborrheic) alopecia model compared with female mice in the control group. Figure 3A is the heat map of upregulated genes; Figure 3B GO function enrichment results of TOP30 up-regulated differentially expressed genes; Figure 3C KEGG pathway enrichment results of the TOP20 upregulated differentially expressed genes.

[0055] Figure 4A -C is the RNA-seq sequencing results (downregulated genes) of skin tissues of female mice in the androgenic (seborrheic) alopecia model compared with female mice in the control group. Figure 4A is the heat map of downregulated genes; Figure 4B The GO function enrichment results of the TOP30 down-regulated differentially expressed genes; Figure 4C This is the KEGG pathway enrichment result of the TOP20 down-regulated differentially expressed genes.

[0056] Figure 5A -C is the RNA-seq sequencing results (upregulated genes) of the skin tissue of female mice with androgenic (seborrheic) alopecia model after treatment with polypeptide PY1 compared with female mice with model alopecia. Figure 5A is the heat map of upregulated genes; Figure 5B GO function enrichment results of TOP30 up-regulated differentially expressed genes; Figure 5C KEGG pathway enrichment results of the TOP20 upregulated differentially expressed genes.

[0057] Figure 6A -C is the RNA-seq sequencing results (downregulated genes) of the skin tissue of female mice with androgenic (seborrheic) alopecia model after treatment with polypeptide PY1 compared with female mice with model alopecia. Figure 6A is the heat map of downregulated genes; Figure 6B The GO function enrichment results of the TOP30 down-regulated differentially expressed genes; Figure 6C This is the KEGG pathway enrichment result of the TOP20 down-regulated differentially expressed genes.

[0058] Figure 7 The therapeutic effects of polypeptides PY1, PY2, PY3 and PY4 in the androgenic (seborrheic) alopecia model in male rats. Figure 7 Middle A shows the hair growth on the back of the neck of male mice in the control group, androgen group, and androgen + peptide treatment group; Figure 7 Middle B is the skin color scoring result of male mice.

[0059] Figure 8 The results of morphology and quantification of hair follicle diameter of the neck and back skin of male mice with androgenic (seborrheic) alopecia model were presented. Figure 8 Middle A shows the HE staining results of the skin sections on the back of the neck of male mice in the control group, DHT treatment group, and DHT+peptide PY1, PY2, PY3, and PY4 treatment groups; Figure 8 B is based on Figure 8 Statistical results of hair follicle diameters in the skin after morphological observation in Figure A; Figure 8 C is based on Figure 8 Figure A shows the statistical results of the diameter of sebaceous glands in the skin after morphological observation.

[0060] Figure 9A-C is the RNA-seq sequencing results (upregulated genes) of skin tissues of male mice with androgenic (seborrheic) alopecia model compared with male mice in the control group. Figure 9A is the heat map of upregulated genes; Figure 9B GO function enrichment results of TOP30 up-regulated differentially expressed genes; Figure 9C KEGG pathway enrichment results of the TOP20 upregulated differentially expressed genes.

[0061] Figure 10A -C is the RNA-seq sequencing results (downregulated genes) of skin tissues of male mice with androgenic (seborrheic) alopecia model compared with male mice in the control group. Figure 10A is the heat map of downregulated genes; Figure 10B The GO function enrichment results of the TOP30 down-regulated differentially expressed genes; Figure 10C This is the KEGG pathway enrichment result of the TOP20 down-regulated differentially expressed genes.

[0062] Figure 11A -C is the RNA-seq sequencing results (upregulated genes) of skin tissue of male mice with androgenic (seborrheic) alopecia model treated with polypeptide PY1 compared with male mice with model alopecia. Figure 11A is the heat map of upregulated genes; Figure 11B GO function enrichment results of TOP30 up-regulated differentially expressed genes; Figure 11C KEGG pathway enrichment results of the TOP20 upregulated differentially expressed genes.

[0063] Figure 12A -C is the RNA-seq sequencing results (downregulated genes) of skin tissue of male mice with androgenic (seborrheic) alopecia model treated with polypeptide PY1 compared with male mice with model alopecia. Figure 12A is the heat map of downregulated genes; Figure 12B The GO function enrichment results of the TOP30 down-regulated differentially expressed genes; Figure 12C This is the KEGG pathway enrichment result of the TOP20 down-regulated differentially expressed genes.

[0064] Figure 13 The results are a comparison of the therapeutic effects of the fusion polypeptide PY1 and its single O polypeptide and I polypeptide in the androgenic (seborrheic) alopecia model in female mice. Figure 13 A is the hair growth and skin color score results of the neck and back of female mice in the control group, DHT group, DHT+PY1, DHT+O, and DHT+I treatment groups; Figure 13 B is the HE staining results of the skin sections on the back of the neck of female mice in the control group, DHT treatment group, and DHT+PY1, O peptide, and I peptide treatment groups; Figure 13 C is the action position of DHT, O peptide, I peptide, and PY1 fusion peptide in the hair growth cycle.

[0065] Figure 14Identification results of primary human dermal papilla cells. Figure 14 Middle A is a schematic diagram of the dermal papilla cells and their location and bright field morphological observation of cell culture; Figure 14 Middle B is the identification result of human primary dermal papilla cells, and the green color is vimentin.

[0066] Figure 15 This study investigated the rescue effects of peptides PY1, PY2, PY3, and PY4 on apoptosis in human primary dermal papilla cells induced by DHT treatment in vitro. Figure 15 Middle A shows the flow cytometry results of cell apoptosis detection in different treatment groups. The green part in the lower right corner represents apoptotic cells. The larger the green area, the more apoptotic cells there are. Figure 15 B is Figure 15 Quantification results of apoptotic cells in the green area in A.

[0067] Figure 16 This study investigates the alleviating effects of peptides PY1, PY2, PY3, and PY4 on changes in cell-related molecular markers in an in vitro androgen / seborrheic model induced by DHT treatment using primary human dermal papilla cells. Figure 16 A in the middle represents the effect of the peptide on androgen receptor AR and transforming growth factor TGFβ; Figure 16 Middle B represents the effect of the polypeptide on Noggin, Wnt signaling pathway regulatory protein Wnt5a, Wnt signaling pathway regulatory protein β-catenin, fibroblast growth factor FGF7, and hair follicle stem cell marker CD133.

[0068] Figure 17 β-arrestin protein recruitment experiments were performed using HTLA cells (a HEK293 cell line stably expressing a tTA-dependent luciferase reporter protein and a β-arrestin2–TEV peptide). After transfection with a GPCR vector, the peptides PY1, PY2, PY3, and PY4 were added. When the peptides bind to the receptor, β-arrestin2 is recruited, and TEV is cleaved and enters the cell nucleus, where fluorescence is detected. DETAILED DESCRIPTION

[0069] The present invention is further illustrated by way of examples below, but the invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications. The raw materials and equipment used in the examples are well known to those skilled in the art and are all commercially available, easily obtained, or prepared.

[0070] the term

[0071] Seborrheic follicle decline and / or hair loss refers to the excessive secretion of oil by the sebaceous glands caused by androgens, which accumulates around the hair follicles, causing the hair follicles to become smaller and ultimately leading to hair loss.

[0072] Drug-induced hair follicle decline and / or hair loss refers to drugs damaging the growth state of hair follicles, causing accelerated apoptosis of hair follicle cells, growth stagnation, and ultimately leading to hair loss.

[0073] The O-peptide is located in a conserved sequence polypeptide in osteocalcin. The O-peptide-derived peptides include, in addition to the above-mentioned SEQ ID NOs. 1-8, conserved fragments of osteocalcin derived from different species. The other O-peptide-derived peptides are located in the same position as the conserved fragment of the sequence described in any one of SEQ ID NOs. 1-8. The different species of organisms are selected from mammals, reptiles, amphibians, bony fish, poultry, and avian species. The O-peptide-derived peptide has a sequence of X1-X2-...Xn, and the immediately upstream and downstream sequences of the polypeptide-derived peptide in osteocalcin are both invertase cleavage sites, X represents an amino acid, and n is any integer from 8 to 25.

[0074] The I peptide is located in a conserved sequence polypeptide in irisin, and other I peptide-derived peptides are conserved fragments in irisin derived from different species. The other I peptide-derived peptides are located in the same position as the conserved fragment of the sequence described in any one of SEQ ID NOs. 9-15, and the different species of organisms are selected from mammals, reptiles, amphibians, bony fish, poultry and avian species. The polypeptide-derived peptide has a sequence of X1-X2-...Xn, and the immediately upstream and downstream sequences of the polypeptide-derived peptide in irisin are both invertase cleavage sites, X represents an amino acid, and n is any integer from 8 to 25.

[0075] carrier

[0076] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0077] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0078] Example 1: Preparation of polypeptide sequences

[0079] The fusion protein sequence was synthesized by artificial synthesis.

[0080] The fusion protein sequence is as follows:

[0081]

[0082] The fusion polypeptides were synthesized using conventional solid-phase or liquid-phase synthesis methods. The solid-phase peptide synthesis method involves reacting the amino acids from the C-terminus to the N-terminus, followed by resin activation, amino acid linking, elution and protection, and detection, completing the amino acid linking step by step. The peptides were then precipitated and centrifuged with excess ether. The crude peptides were purified by HPLC and analyzed by mass spectrometry, and then freeze-dried in liquid nitrogen for later use.

[0083] Example 2: Promoting anagen hair growth in mice

[0084] Day 49 (7-week-old) telogen mice were used for hair loss and hair regrowth, and this was recorded as day 0. Peptide administration began on day 6 of the growth phase and continued until day 9. The peptide application concentration was 0.162M, and the application volume was 0.1 ml.

[0085] Example 3: Dihydrotestosterone (DHT)-induced androgenic / seborrheic alopecia mouse model and medication effects

[0086] Day 45 C57 mice were pre-treated with 10% ethanol and DHT (2 mg / d) dissolved in 10% ethanol, followed by single administration every other day. Following two DHT pretreatments, day 49 (7-week-old) telogen mice were divided into a control group (10% ethanol), a DHT group (DHT intraperitoneal injection), and a fusion peptide group (DHT intraperitoneal injection + fusion peptide application). Hair depilation was then performed to induce synchronous hair regeneration. DHT (2 mg / d) was administered every other day. The peptide application concentration was 0.162 M, and the application volume was 0.1 ml.

[0087] The experimental results for female mice are shown in Figure 1 - Figure 6:

[0088] pass Figure 1 The comparison of hair growth in female mice in the control group, DHT model group, and DHT+PY1-PY4 peptide application group in A shows that intraperitoneal injection of DHT significantly inhibited the growth of back hair in female mice, while application of peptides significantly alleviated the hair growth inhibition caused by DHT in female mice. Figure 1 B is correct Figure 1 Scoring of mouse back skin color in A quantifies Figure 1 The results in A.

[0089] Furthermore, the skin sections of the back of the neck of female mice in each group were sliced ​​and HE stained ( Figure 2A) It can be seen that after DHT treatment, the number of hair follicles decreased, and after peptide treatment, the number of hair follicles increased significantly; after DHT treatment, the hair follicles atrophied, the diameter decreased significantly, and the sebaceous glands became enlarged, and after peptide treatment, the hair follicle diameter and sebaceous gland size recovered significantly ( Figure 2 B, C)

[0090] from Figure 1-2 It can be seen that the fusion peptides PY1-PY4 can produce significantly different effects on promoting hair growth and restoring hair follicles compared to the DHT group. This shows that although the fusion peptide molecules are larger, administration through the skin does not affect the efficacy of the fusion peptides. The effects can be achieved regardless of the coupling method of O-peptide and I-peptide.

[0091] Furthermore, analysis of RNA-seq sequencing results of the cervical skin of female mice (Figures 3-6) showed that after DHT treatment, genes related to immune and inflammatory responses, cell and chemokine activity in the skin of female mice were upregulated ( Figure 3B ), chemokine and inflammation-related signaling pathways are upregulated ( Figure 3C ); down-regulation of genes related to keratinization, keratin fiber, intermediate filament organization and epidermal structural components ( Figure 4B ), melanin biosynthesis, keratinization, triglyceride catabolism and other pathways are downregulated ( Figure 4C After treatment with polypeptide PY1, genes related to intermediate filament organization, keratinization, keratin fibers, and epidermal structural components were significantly upregulated ( Figure 5B ), melanin biosynthesis, keratinization and other pathways are also upregulated ( Figure 5C ); immune response, extracellular matrix and other related genes were down-regulated ( Figure 6B ), NCAM1, adrenaline receptors, vitamins and other signaling pathways are downregulated ( Figure 6C ). It can be seen that PY1 peptide treatment significantly improved the changes in biological processes, extracellular components, molecular functions, and signaling pathways in the skin of female mice caused by DHT.

[0092] The results of the male mouse experiments are shown in Figure 7-Figure 1 2:

[0093] pass Figure 7 The comparison of hair growth in male mice in the control group, DHT model group, and DHT+PY1-PY4 peptide application group in A shows that intraperitoneal injection of DHT significantly inhibited the growth of back hair in male mice, while application of the fusion peptide was able to alleviate the DHT-induced hair growth blockage in male mice. Figure 7 B is correct Figure 7 Scoring of mouse back skin color in A quantifies Figure 7 The results in A.

[0094] Furthermore, the skin sections of the neck and back of male mice in each group were sliced ​​and HE stained ( Figure 8 A) It can be seen that after DHT treatment, the number of hair follicles decreased, and after peptide treatment, the number of hair follicles increased significantly; after DHT treatment, the hair follicles atrophied, the diameter decreased significantly, and the sebaceous glands became enlarged, and after peptide treatment, the hair follicle diameter and sebaceous gland size recovered significantly ( Figure 8 B, C)

[0095] from Figure 7-8 It can be seen that the fusion peptides PY1-PY4 all produced significantly different effects in promoting hair growth and restoring hair follicles compared to the DHT group. This suggests that despite the increased size of the fusion peptide molecules, transdermal administration did not affect their efficacy, and that the effects were achieved regardless of the coupling method used for peptides O and I. Furthermore, a comparison of the test results in male and female mice showed that the fusion peptides had a positive effect on both male and female subjects, with comparable efficacy.

[0096] Furthermore, analysis of RNA-seq sequencing results of the neck and back skin of male mice (Figures 9-12) showed that after DHT treatment, genes related to steroid metabolism and lipoprotein metabolism, extracellular space, peptidase inhibitor activity, fatty acid binding, and aromatase activity were upregulated in the skin of male mice ( Figure 9B ), chylomicron and lipoprotein-related signaling pathways are upregulated ( Figure 9C ); epithelial cell differentiation, hair follicle development, intermediate filaments and epidermal structural components related genes were down-regulated ( Figure 10B ), keratinization-related pathways are downregulated ( Figure 10C After treatment with polypeptide PY1, keratinocyte differentiation and chemotaxis-related genes were significantly upregulated ( Figure 11B ), chemokine-receptor binding, peptide ligand-receptor binding, GPCR signaling pathway upregulation ( Figure 11C ); down-regulation of genes related to response to polypeptide hormones, keratin fibers, and fatty acid binding ( Figure 12B ), keratinization and other signaling pathways are downregulated ( Figure 12C ). It can be seen that PY1 peptide treatment significantly improved the changes in biological processes, extracellular components, molecular functions, and signaling pathways in the skin of male mice caused by DHT.

[0097] Acne is a chronic inflammatory skin disease of the hair follicles and sebaceous glands that is common in adolescents and adults. Its currently recognized mechanisms are mainly four aspects: excessive keratinization of the epithelium of hair follicles and sebaceous gland ducts; excessive secretion of sebaceous glands; colonization of microorganisms (Propionibacterium acnes and Malassezia); and inflammation and immune response.

[0098] Sequencing results showed that PY1 peptide treatment could improve the damage caused to mouse skin by the DHT model from multiple aspects, including keratin-related structures and pathways, multiple lipid metabolism pathways, chemokines, inflammation and immune responses. It is inferred that the fusion peptide has a certain therapeutic effect on androgenic / seborrheic dermatitis and acne.

[0099] Example 4: Comparison of the effects of fusion polypeptides and single polypeptides in a DHT-induced androgen / seborrheic alopecia mouse model

[0100] 45-day-old male C57 and C57 mice were pre-treated with 10% ethanol and DHT (2 mg / d) dissolved in 10% ethanol, followed by a single dose every other day. After two DHT pretreatments, 49-day-old (7-week-old) telogen mice were divided into a control group (10% ethanol), a DHT group (intraperitoneal injection of DHT), an O-peptide group (intraperitoneal injection of DHT + O-peptide application), an I-peptide group (intraperitoneal injection of DHT + I-peptide application), and a peptide group (intraperitoneal injection of DHT + fusion peptide application). Hair depilation was then performed to induce synchronous hair regeneration. DHT (2 mg / d) was administered every other day. The peptide application concentration was 0.5 mM, and the application volume was 0.1 mL.

[0101] The fusion polypeptide PY is PY1 SEQ ID NO.16, the O peptide is the O peptide SEQ ID NO.1 in PY1, and the I peptide is the I peptide SEQ ID NO.9 in PY1.

[0102] The results showed that in female mice, the therapeutic effect of peptide O was better than that of peptide I, and the therapeutic effect of fusion peptide PY1 was better than both peptide O and peptide I ( Figure 13 A).

[0103] The hair growth cycle is divided into the growth phase, regression phase and resting phase ( Figure 13 C). Through the morphological observation of the skin on the back of the neck of female mice in the androgenic (seborrheic) alopecia model, it can be seen that peptide I plays a role in the early stage of hair growth ( Figure 13 B-DHT+I, improve hair follicles); O peptide plays a role in the middle and late stages of hair growth ( Figure 13 B-DHT+O, hair begins to grow). Therefore, the fusion peptide PY1 can play a role in the entire period of hair growth ( Figure 13 B-DHT+PY, hair follicle improvement + hair growth), plays an anti-hair loss role in both males and females.

[0104] Example 5: Purchase and identification of primary human dermal papilla cells

[0105] Human primary dermal papilla cells (HFDPCs) were purchased from Shanghai Xuanke Biotechnology Co., Ltd. The cells were derived from normal scalp tissue removed by surgery, and the vimentin immunofluorescence staining was positive ( Figure 14 B) The cells are certified to be greater than 90% pure and free of HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi. The culture medium is fibroblast culture medium.

[0106] Example 6: Detection of Early Apoptosis of Human Hair Follicle Papilla Cells (DHT Treatment)

[0107] HFDPCs were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C and 5% CO2, with the medium changed every other day. HFDPCs from the same batch were cultured in 6-well plates and treated with high androgen levels by adding 100 μM DHT to the culture medium (DHT group). Peptides PY1, PY2, PY3, and PY4 were added at a concentration of 1 μM (DHT+peptide group). Early apoptosis was detected after 48 hours of culture.

[0108] Early apoptosis detection (Annexin V): Aspirate the cell culture medium into a centrifuge tube and add an appropriate amount of trypsin cell digestion solution to digest the cells. Incubate at room temperature until the adherent cells are blown off by gently blowing, aspirate the trypsin cell digestion solution and add it to the corresponding centrifuge tube (because DHT can cause cell apoptosis, apoptotic adherent cells will float, so the culture medium cannot be discarded directly). Centrifuge at 1000g for 5 minutes, discard the supernatant, and collect the cells. Resuspend the cells in PBS, centrifuge at 1000g for 5 minutes, discard the supernatant, and collect the cells again. Add 195μl Annexin V-FITC conjugate solution to gently resuspend the cells, and add 5μl Annexin V-FITC and 10μl propidium iodide staining solution, and mix gently. Incubate at room temperature in the dark for 20 minutes, then place in an ice bath, and then use flow cytometry to detect cell scattering and fluorescence.

[0109] like Figure 15 The flow cytometry results showed that the number of apoptotic HFDPCs cells increased significantly after DHT treatment. After the addition of peptides PY1, PY2, PY3 and PY4 ( Figure 15 Only the results of the addition concentration of peptides PY1, PY2, PY3 and PY4 of 1uM are shown in the figure). The number of apoptotic cells was significantly reduced, indicating that peptides PY1, PY2, PY3 and PY4 can significantly alleviate the apoptosis of human primary dermal papilla cells caused by high androgen DHT.

[0110] Example 7: DHT-induced androgen / seborrheic alopecia cell model

[0111] HFDPCs were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C and 5% CO2, with the medium changed every other day. HFDPCs from the same batch were cultured in 6-well plates and treated with high androgen levels by adding 100 μM DHT to the culture medium (DHT group). Peptides PY1, PY2, PY3, and PY4 were added at a concentration of 1 μM (DHT+peptide group). Cellular marker expression was assayed after 48 hours of culture.

[0112] like Figure 16 The results showed that the expression of androgen receptor AR and inflammatory factor TGF-β in cells increased significantly after DHT treatment ( Figure 16 A), while the expression of Wnt signaling pathway regulatory proteins Wnt5a and β-Catenin decreased, the expression of hair follicle stem cell marker CD133 decreased, and the expression of Noggin and Fgf7 also decreased ( Figure 16 B) Peptides PY1, PY2, PY3, and PY4 significantly alleviated these changes. Activation of the Wnt / β-catenin signaling pathway plays a central role in hair follicle regeneration; Fgf7 is a dermal papilla signal that instructs hair germ cells to proliferate and initiate a new hair cycle; and Noggin is a gene that shortens the resting phase and initiates the growth phase of new hair.

[0113] Example 8: β-arrestin protein recruitment experiment

[0114] β-arrestin protein recruitment was measured in HTLA cells (a HEK293 cell line stably expressing a tTA-dependent luciferase reporter protein and a β-arrestin2–TEV peptide) using the PRESTO-Tango (Parallel Receptor Panel Expression and Screening by Transcriptional Output, followed by Transcriptional Activation after Blocked Translocation) system.

[0115] Three GPRs vectors were purchased from Addgene, including GPR37-Tango (#66355), GPRC6A-Tango (#66386), and GPR158-Tango (#66332).

[0116] HTLA cells expressing these vectors were plated in polylysine-coated 96-well clear-bottom white cell culture plates (Greiner Bio-One). The next day, various concentrations of peptides were prepared in filter-sterilized assay buffer and 40 μl was added to each well. After 24 hours, the culture medium was removed from the wells, and a 20-fold dilution of ONE-Glo EX reagent (40 μl / well, Promega) was added to each well. After incubation at 20°C to 25°C for 15 to 20 minutes, the cells were counted in a Synergy H1 Hybrid Multi-Mode Reader (BioTek). Relative luminescence units (rlu) were exported to an Excel spreadsheet, and data were analyzed using GraphPad Prism.

[0117] The results can be seen ( Figure 17 ), polypeptides PY1, PY2, PY3 and PY4 can bind to the receptors GPRC6A / GPR158 / GPR37, β-arrestin is recruited, TEV is cleaved and enters the cell nucleus, and a sharp increase in fluorescence level can be detected.

[0118] The above examples illustrate that the polypeptides of the present invention can be used to alleviate androgenic alopecia, especially androgenic / seborrheic follicle decline or alopecia caused by significantly increased expression of androgen receptor AR, apoptosis of hair follicle cells, abnormally increased expression of inflammatory factors, and hair follicle degeneration, as well as androgenic / seborrheic acne or dermatitis.

[0119] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A fusion polypeptide, characterized in that: The fusion polypeptide is sequentially connected from N-terminus to C-terminus by peptide O or its derivative peptide, peptide I or its derivative peptide, through a linker or directly connected; Or the fusion polypeptide is composed of peptide I or its derivative peptide, peptide O or its derivative peptide, from N-terminus to C-terminus, connected by a linker or directly; The O peptide or its derivative peptide is selected from osteocalcin-derived peptides; the osteocalcin-derived peptides include mammalian, reptile, amphibian, bony fish, poultry, and bird osteocalcin-derived peptides; the osteocalcin-derived peptides are conserved fragments of osteocalcin derived from different species; The I peptide or its derivative peptide is selected from irisin-derived peptides and homologous polypeptides from different species with irisin as the skeleton; the irisin-derived peptides include irisin-derived peptides from mammals, reptiles, amphibians, bony fish, poultry, and birds; the irisin-derived peptides are conserved fragments of irisin from different species.

2. The fusion polypeptide according to claim 1, characterized in that The amino acid sequences of the O peptide and its derivative peptides are shown in SEQ ID NOs. 1 to 8, or have more than 50% homology thereto; wherein SEQ ID NO. 1 is a human osteocalcin-derived peptide, SEQ ID NO. 2 is a mouse osteocalcin-derived peptide, SEQ ID NO. 3 is a livestock-sheep osteocalcin-derived peptide, and the otter and badger have the same sequence, SEQ ID NO. 4 is a reptile-lizard osteocalcin-derived peptide, SEQ ID NO. 5 is an amphibian-water frog osteocalcin-derived peptide, SEQ ID NO. 6 is a bony fish-Wuchang fish osteocalcin-derived peptide, SEQ ID NO. 7 is a poultry-chicken osteocalcin-derived peptide, and SEQ ID NO. 8 is a bird-mandarin duck osteocalcin-derived peptide, and the swan goose has the same sequence.

3. The fusion polypeptide according to claim 1, characterized in that The amino acid sequences of the peptide I and its derivatives are shown in SEQ ID NOs. 9 to 15, or have more than 50% homology thereto; wherein SEQ ID NO. 9 is a human irisin-derived peptide, SEQ ID NO. 10 is a mouse irisin-derived peptide, SEQ ID NO. 11 is a marsupial irisin-derived peptide, SEQ ID NO. 12 is an avian irisin-derived peptide, SEQ ID NO. 13 is a reptile irisin-derived peptide, SEQ ID NO. 14 is a bony fish-small yellow croaker irisin-derived peptide, and SEQ ID NO. 15 is a bony fish-eel irisin-derived peptide.

4. The fusion polypeptide according to claim 1, characterized in that The connecting peptide is (Gly-Gly-Gly-Gly-Ser)n, wherein n is an integer of 1 to 10; or the connecting peptide is (Pro-Lys-Pro-Lys-Pro)n, wherein n is an integer of 1 to 10.

5. The fusion polypeptide according to claim 4, characterized in that The fusion polypeptide includes O peptide-Linker-I peptide and I peptide-Linker-O peptide, and its structure is shown below: I peptide-(Gly-Gly-Gly-Gly-Ser)nO peptide; or O peptide-(Gly-Gly-Gly-Gly-Ser)nI peptide; or I peptide-(Pro-Lys-Pro-Lys-Pro)nO peptide; or O-peptide-(Pro-Lys-Pro-Lys-Pro)nI peptide.

6. The derivative of the fusion polypeptide according to any one of claims 1 to 5, characterized in that: The derivatives are obtained by conventional modification of the amino acid side chain groups, amino terminus, and carboxyl terminus of the fusion polypeptide; or products obtained by connecting a tag for polypeptide or protein detection or purification to the fusion polypeptide, products obtained by isotope labeling modification, or extracts isolated from tissues and organs of different species.

7. The derivative of the fusion polypeptide according to claim 6, characterized in that The conventional modifications include fluorescent group modification, phosphorylation modification, disulfide bond cyclization modification, biotin labeling modification, photosensitizer, azide modification, PEG modification, methylation modification, fluorescence quenching group modification, protein coupling modification, small molecule compound modification, amino modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, esterification modification, and glycosylation modification; The fluorescent dye used in the modification of the fluorescent group is selected from AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, and ICG, and the modification can be used for fluorescence detection; wherein the phosphorylation modification is selected from a combination of one or more of p-Ser, p-Thr, and p-Tyr; wherein the glycosylation modification is selected from a combination of one or more of Ser, Asn, Thr, and Tyr; wherein the nitration modification is selected from one or more combinations of Tyr; The biotin label is selected from D-biotin, biotin hydrazide, photosensitive biotin and biotin-dUTP.

8. The derivative of the fusion polypeptide according to claim 7, characterized in that: The conventional modifications of the amino terminus and the carboxyl terminus are selected from the group consisting of acetylation modification of the polypeptide N terminus and amination modification of the C terminus.

9. The derivative of the fusion polypeptide according to claim 7, characterized in that: Conventional modifications of the side chain groups are selected from modifications of the R groups of the amino acid side chains in polypeptides.

10. The derivative of the fusion polypeptide according to claim 6, wherein the isotope used in the isotope labeling is selected from one or more combinations of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, and 131I.

11. A polynucleotide encoding the fusion polypeptide of claims 1-5 or the derivative of claims 6-10. A vector comprising the polynucleotide according to claim 11 . A host cell transfected with the vector according to claim 12.

14. Use of the fusion polypeptide according to any one of claims 1 to 5, and / or the derivative according to any one of claims 6 to 10 as a receptor agonist of GPR158 and / or GPRC6A and / or GPR37, and / or an integrin agonist.

15. Use of the fusion polypeptide according to any one of claims 1 to 5, and / or the derivative according to any one of claims 6 to 10, and / or the combination of a GPR158 and / or GPRC6A and / or GPR37 receptor agonist and an integrin receptor agonist according to claim 14 in the preparation of medical and aesthetic products, cosmetics, health products, foods, additives, or medicines for preventing or treating hair follicle decline, hair loss, acne, or dermatitis; in, The hair loss diseases include stress-induced hair follicle decline and / or hair loss in males and females, androgenic hair follicle decline and / or seborrheic alopecia, and drug-induced hair follicle decline and / or hair loss; Wherein, the prevention and / or treatment of hair loss is to promote hair growth; Wherein, the acne includes male and female pressure acne and androgenic acne; The dermatitis includes male and female stress dermatitis and androgenic dermatitis.

16. The use according to claim 15, characterized in that The drug contains one or more pharmaceutically acceptable carriers; The carrier is a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption accelerator, adsorption carrier, surfactant or lubricant; The drug is prepared in the form of tablets, granules, capsules, oral liquid, inhalation liquid, smear liquid, spray liquid, drops, microneedle or injection; The drug administration methods include oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration to the subject; Administration routes include parenteral, such as intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial; transmucosal, such as buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal, and also include delivery modes using liposomal formulations, intravenous infusion, and transdermal patches.

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