Fusion polypeptide and use thereof

Abstract

The present invention belongs to a biopharmaceutical technology, and specifically relates to a fusion polypeptide and a derivative thereof, and the use thereof in the preparation of a drug for treating alopecia, acne, dermatitis and other related diseases. Disclosed is a fusion polypeptide, wherein the fusion polypeptide sequentially consists of an O peptide or a derivative peptide thereof, and an I peptide or a derivative peptide thereof from the N-terminus to the C-terminus, and the two structural functional domains are connected via a linker peptide linker or directly; or the fusion polypeptide sequentially consists of an I peptide or a derivative peptide thereof, and an O peptide or a derivative peptide thereof from the N-terminus to the C-terminus, and the two structural functional domains are directly connected via a linker peptide. The provided fusion polypeptide can simultaneously activate GPR158 and / or GPRC6A and / or GPR37 pathways, and simultaneously activate the integrin receptor pathway, has the remarkable advantages of high activity in promoting hair growth, rapid onset of action and long action duration in regulating hormone, thereby providing a new drug for treating alopecia, acne, dermatitis and other related diseases.

Description

A fusion polypeptide and its application 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 currently few drugs are effective for these purposes. 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] YLYQWLGAPVPYPDPLEPR SEQ ID No.1

[0013] YLGASVPSPDPLEPT SEQ ID No.2

[0014] LDPGLGAPAPYPDPLEPR SEQ ID No.3

[0015] NYNRFYNARAAPTPDPLEPL SEQ ID No.4

[0016] SNLRNAVFGTPVRDPLESK SEQ ID No.5

[0017] AGAVTAADLSLTQLESL SEQ ID No.6

[0018] YAQDSGVAGAPPNPLEAQ SEQ ID No.7

[0019] SDFYER YFMHYKTPMEQM SEQ ID No.8

[0020] 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.

[0021] 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;

[0022] HVQAISIQGQSPASEPVL SEQ ID No.9

[0023] HVQAISIQGQSPASEPLL SEQ ID No.10

[0024] HVQAISIQGQSPASEPVT SEQ ID No.11

[0025] HVQSISIQGQSPASEPVL SEQ ID No.12

[0026] HVQSISIQGQSPASEPVH SEQ ID No.13

[0027] HVQSISMSGTSPLSEPLR SEQ ID No.14

[0028] HVQSISMSGMSPLSEPLR SEQ ID No.15

[0029] 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.

[0030] 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.

[0031] Many proteins and hormones share high homology in humans and animals. For example, bovine and porcine insulin can be used to treat diabetes; 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 identical; and since gonadotropin-releasing hormone (GnRH) was isolated from pig and sheep brains in 1971, the GnRH family has grown to include at least 28 types, 15 from vertebrates and 13 from invertebrates. With the exception of octopus GnRH, all GnRH peptides consist of 10 amino acids, with highly conserved length and partial amino acid sequences. Given the homology and functional similarities between polypeptides and hormones across species, sequences from the same species or with higher homology to the polypeptide may exert the same effects. In the present invention, the O and I peptides in fusion polypeptides and fusion polypeptide derivatives can be derived from different species.

[0032] 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.

[0033] Preferably, the fusion polypeptide comprises O peptide-Linker-I peptide and I peptide-Linker-O peptide, and its structure is as follows:

[0034] I peptide-(Gly-Gly-Gly-Gly-Ser)nO peptide;

[0035] or O peptide-(Gly-Gly-Gly-Gly-Ser)nI peptide;

[0036] or I peptide-(Pro-Lys-Pro-Lys-Pro)n- O peptide;

[0037] or O peptide-(Pro-Lys-Pro-Lys-Pro)n-I peptide.

[0038] 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.

[0039] 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;

[0040] 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;

[0041] wherein the phosphorylation modification is selected from a combination of one or more of p-Ser, p-Thr, and p-Tyr;

[0042] wherein the glycosylation modification is selected from a combination of one or more of Ser, Asn, Thr, and Tyr;

[0043] wherein the nitration modification is selected from one or more combinations of Tyr;

[0044] The biotin label is selected from D-biotin, biotin hydrazide, photosensitive biotin and biotin-dUTP.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] The third aspect of the present invention discloses a polynucleotide encoding the above-mentioned fusion polypeptide or its derivatives.

[0049] The fourth aspect of the present invention discloses a polynucleotide comprising the above-mentioned polynucleotide.

[0050] A fifth aspect of the present invention discloses a host cell transfected with the aforementioned vector. The vector includes 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.

[0051] 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.

[0052] 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.

[0053] 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;

[0054] 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;

[0055] Wherein, the prevention and / or treatment of hair loss is to promote hair growth;

[0056] Wherein, the acne includes male and female pressure acne and androgenic acne;

[0057] The dermatitis includes male and female stress dermatitis and androgenic dermatitis.

[0058] Preferably, the drug contains one or more pharmaceutically acceptable carriers;

[0059] Preferably, the carrier is a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant or lubricant;

[0060] Preferably, the drug is prepared in the form of tablets, granules, capsules, oral liquids, inhalation liquids, smears, sprays, drops, microneedles or injections;

[0061] 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;

[0062] 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.

[0063] The present invention has the following significant advantages and effects compared to the prior art:

[0064] 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

[0065] Figure 1 shows the therapeutic effects of peptides PY1, PY2, PY3, and PY4 in a female mouse model of androgenic (seborrheic) alopecia. Figure 1A shows hair growth on the back of the necks of female mice in the control, DHT, and DHT+peptide treatment groups; Figure 1B shows skin color scores for the female mice.

[0066] Figure 2 shows the morphological and quantified results of hair follicle diameter in the cervical and dorsal skin of female mice in an androgenic (seborrheic) alopecia model. Figure 2A shows HE staining of cervical and dorsal skin sections of female mice in the control, DHT-treated, and DHT+peptide PY1, PY2, PY3, and PY4-treated groups. Figure 2B shows the statistical results of hair follicle diameter in the skin based on the morphological observation in Figure 2A. Figure 2C shows the statistical results of sebaceous gland diameter in the skin based on the morphological observation in Figure 2A.

[0067] Figures 3A-C show the RNA-seq results (upregulated genes) of skin tissue from female mice with androgenic (seborrheic) alopecia compared to female mice in the control group. Figure 3A shows a heatmap of upregulated genes; Figure 3B shows the GO function enrichment results for the top 30 differentially upregulated genes; and Figure 3C shows the KEGG pathway enrichment results for the top 20 differentially upregulated genes.

[0068] Figures 4A-C show the RNA-seq results (downregulated genes) of skin tissue from female mice with androgenic (seborrheic) alopecia compared to female mice in the control group. Figure 4A shows a heatmap of downregulated genes; Figure 4B shows the GO function enrichment results for the top 30 differentially downregulated genes; and Figure 4C shows the KEGG pathway enrichment results for the top 20 differentially downregulated genes.

[0069] Figures 5A-C show the RNA-seq results (upregulated genes) of skin tissue from female mice with androgenic (seborrheic) alopecia treated with peptide PY1 compared to female mice with normal alopecia treated with peptide PY1. Figure 5A shows a heatmap of upregulated genes; Figure 5B shows the GO function enrichment results for the top 30 differentially upregulated genes; and Figure 5C shows the KEGG pathway enrichment results for the top 20 differentially upregulated genes.

[0070] Figures 6A-C show the RNA-seq results (downregulated genes) of skin tissue from female mice with androgenic (seborrheic) alopecia treated with peptide PY1 compared to female mice with normal alopecia treated with peptide PY1. Figure 6A shows a heatmap of downregulated genes; Figure 6B shows the GO function enrichment results for the top 30 differentially downregulated genes; and Figure 6C shows the KEGG pathway enrichment results for the top 20 differentially downregulated genes.

[0071] Figure 7 shows the therapeutic effects of peptides PY1, PY2, PY3, and PY4 in a male rat model of androgenic (seborrheic) alopecia. Figure 7A shows hair growth on the back of the necks of male rats in the control, androgen, and androgen + peptide treatment groups; Figure 7B shows skin color scores of male rats.

[0072] Figure 8 shows the morphological and quantified results of hair follicle diameter in the cervical and dorsal skin of male rats in an androgenic (seborrheic) alopecia model. Figure 8A shows HE staining of cervical and dorsal skin sections of male rats in the control, DHT-treated, and DHT+peptide PY1, PY2, PY3, and PY4-treated groups; Figure 8B shows the statistical results of hair follicle diameter in the skin based on the morphological observation in Figure 8A; Figure 8C shows the statistical results of sebaceous gland diameter in the skin based on the morphological observation in Figure 8A.

[0073] Figures 9A-C show the RNA-seq results (upregulated genes) of skin tissue from male mice with androgenic (seborrheic) alopecia compared to control mice. Figure 9A shows a heatmap of upregulated genes; Figure 9B shows the GO function enrichment results for the top 30 differentially upregulated genes; and Figure 9C shows the KEGG pathway enrichment results for the top 20 differentially upregulated genes.

[0074] Figures 10A-C show the RNA-seq results (downregulated genes) of skin tissue from male mice with androgenic (seborrheic) alopecia compared to male mice in the control group. Figure 10A shows a heatmap of downregulated genes; Figure 10B shows the GO function enrichment results for the top 30 differentially downregulated genes; and Figure 10C shows the KEGG pathway enrichment results for the top 20 differentially downregulated genes.

[0075] Figures 11A-C show the RNA-seq results (upregulated genes) of skin tissue from male mice with androgenic (seborrheic) alopecia treated with peptide PY1 compared to male mice with normal alopecia treated with peptide PY1. Figure 11A shows a heatmap of upregulated genes; Figure 11B shows the GO function enrichment results for the top 30 differentially upregulated genes; and Figure 11C shows the KEGG pathway enrichment results for the top 20 differentially upregulated genes.

[0076] Figures 12A-C show the RNA-seq results (downregulated genes) of skin tissue from male mice with androgenic (seborrheic) alopecia treated with peptide PY1 compared to male mice with normal alopecia treated with peptide PY1. Figure 12A shows a heatmap of downregulated genes; Figure 12B shows the GO function enrichment results for the top 30 differentially downregulated genes; and Figure 12C shows the KEGG pathway enrichment results for the top 20 differentially downregulated genes.

[0077] Figure 13 compares the therapeutic effects of the fusion peptide PY1 and its single peptides O and I in a female mouse model of androgenic (seborrheic) alopecia. Figure 13A shows the hair growth and skin color scores of the cervical dorsum of female mice in the control, DHT, DHT+PY1, DHT+O, and DHT+I treatment groups. Figure 13B shows HE staining of cervical dorsum skin sections of female mice in the control, DHT-treated, and DHT+PY1, O, and I peptide treatment groups. Figure 13C shows the locations of action of DHT, O peptide, I peptide, and PY1 fusion peptide in the hair growth cycle.

[0078] Figure 14 shows the identification results of primary human dermal papilla cells. Figure 14A shows a schematic diagram of dermal papilla cells and their location, along with bright-field observation of cell culture morphology. Figure 14B shows the identification results of primary human dermal papilla cells, with vimentin (green) representing the protein.

[0079] Figure 15 shows the rescue effects of peptides PY1, PY2, PY3, and PY4 on apoptosis in human primary dermal papilla cells induced by DHT in vitro. Figure 15A shows the flow cytometry results of apoptosis assays for different treatment groups. The green area in the lower right corner represents apoptotic cells, with larger green areas indicating more apoptotic cells. Figure 15B shows the quantification of apoptotic cells in the green area in Figure 15A.

[0080] Figure 16 shows the mitigating effects of peptides PY1, PY2, PY3, and PY4 on changes in cell-related molecular markers following DHT-induced androgen / seborrheic model in human primary dermal papilla cells in vitro. Figure 16A shows the effects of the peptides on androgen receptor AR and transforming growth factor TGFβ; Figure 16B shows the effects of the peptides on Noggin, Wnt signaling pathway regulator Wnt5a, Wnt signaling pathway regulator β-catenin, fibroblast growth factor FGF7, and hair follicle stem cell marker CD133.

[0081] Figure 17 shows a β-arrestin protein recruitment experiment. Using HTLA cells (a HEK293 cell line stably expressing a tTA-dependent luciferase reporter protein and a β-arrestin2–TEV peptide), the GPCR vector was transfected and then 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

[0082] 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.

[0083] the term

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] carrier

[0089] 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.

[0090] 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.

[0091] Example 1: Preparation of polypeptide sequences

[0092] The fusion protein sequence was synthesized by artificial synthesis.

[0093] The fusion protein sequence is as follows:

[0094] PY1: I peptide-(Gly‑Gly‑Gly‑Gly‑Ser)n- O peptide SEQ ID NO.16

[0095] PY2: O peptide-(Gly‑Gly‑Gly‑Gly‑Ser)n-I peptide SEQ ID NO.17

[0096] PY3:I peptide-(Pro-Lys-Pro-Lys-Pro)n-O peptide SEQ ID NO.18

[0097] PY4: O peptide-(Pro-Lys-Pro-Lys-Pro)n- I peptide SEQ ID NO.19

[0098] 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.

[0099] Example 2: Promoting anagen hair growth in mice

[0100] Day 49 (7-week-old) telogen mice were used for hair loss and hair regrowth, designated 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.

[0101] Example 3: Dihydrotestosterone (DHT)-induced androgenic / seborrheic alopecia mouse model and medication effects

[0102] Day 45 C57 mice were pre-treated with 10% ethanol and DHT (2 mg / day) dissolved in 10% ethanol, followed by a single dose every other day. Following these two DHT pretreatments, day 49 (7-week-old) telogen mice were divided into a control group (10% ethanol), a DHT group (intraperitoneal injection of DHT), and a fusion peptide group (intraperitoneal injection of DHT plus application of the fusion peptide). Hair depilation was then performed to induce synchronous hair regrowth. DHT (2 mg / day) was administered every other day. The peptide concentration was 0.162 M, and the application volume was 0.1 ml.

[0103] The experimental results of female mice are shown in Figures 1 to 6:

[0104] From the comparison results of hair growth in female mice in the control group, DHT model group, and DHT+PY1-PY4 polypeptide application group in Figure 1A, it can be seen that intraperitoneal injection of DHT significantly inhibited the growth of hair on the back of female mice, and polypeptide application was able to significantly alleviate the hair growth obstruction of female mice caused by DHT. Figure 1B is the score of the skin color on the back of the mice in Figure 1A, which quantifies the results in Figure 1A.

[0105] Furthermore, through the sections and HE staining results of the neck and back skin of female mice in each group (Figure 2A), it can be seen that after DHT treatment, the number of hair follicles decreased, while after polypeptide treatment, the number of hair follicles increased significantly; after DHT treatment, the hair follicles atrophied, the diameter decreased significantly, and the sebaceous glands hypertrophied, while the hair follicle diameter and sebaceous gland size were significantly restored after polypeptide treatment (Figure 2B, C).

[0106] As shown in Figures 1-2, the fusion peptides PY1-PY4 all produced significantly different effects in promoting hair growth and restoring hair follicles compared to the DHT group. This indicates that although the fusion peptide molecules are larger, administration through the skin does not affect the efficacy of the fusion peptides, and the effects can be achieved regardless of the coupling method used for peptide O and peptide I.

[0107] Further, RNA-seq analysis of the dorsal skin of female mice (Figures 3-6) revealed that DHT treatment upregulated genes related to immune and inflammatory responses, cellular and chemokine activity, and chemokine signaling pathways in female mouse skin (Figure 3B), as well as chemokine and inflammatory signaling pathways (Figure 3C). Genes associated with keratinization, keratin fiber organization, intermediate filament organization, and epidermal structural components were downregulated (Figure 4B), as were pathways such as melanin biosynthesis, keratinization, and triglyceride catabolism (Figure 4C). Treatment with the peptide PY1 significantly upregulated genes related to intermediate filament organization, keratinization, keratin fiber organization, and epidermal structural components (Figure 5B), as well as pathways such as melanin biosynthesis and keratinization (Figure 5C). Genes associated with immune responses and the extracellular matrix were downregulated (Figure 6B), as were signaling pathways such as NCAM1, adrenergic receptors, and vitamins (Figure 6C). Thus, PY1 peptide treatment significantly ameliorated DHT-induced changes in biological processes, extracellular components, molecular functions, and signaling pathways in female mouse skin.

[0108] The experimental results of male mice are shown in Figures 7 to 12:

[0109] From the comparison results of hair growth in male mice in the control group, DHT model group, and DHT+PY1-PY4 polypeptide application group in Figure 7A, it can be seen that intraperitoneal injection of DHT significantly inhibited the growth of hair on the back of male mice, and the application of the fusion polypeptide was able to alleviate the hair growth inhibition of male mice caused by DHT. Figure 7B is the score of the skin color on the back of the mice in Figure 7A, which quantifies the results in Figure 7A.

[0110] Furthermore, through the sections and HE staining results of the neck and back skin of male mice in each group (Figure 8A), it can be seen that after DHT treatment, the number of hair follicles decreased, while after polypeptide treatment, the number of hair follicles increased significantly; after DHT treatment, the hair follicles atrophied, the diameter decreased significantly, and the sebaceous glands became hypertrophic, while the hair follicle diameter and sebaceous gland size were significantly restored after polypeptide treatment (Figure 8B, C).

[0111] Figures 7-8 show 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 molecular size of the fusion peptides, 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 results from the studies in male and female mice revealed that the fusion peptides demonstrated comparable efficacy in both male and female subjects.

[0112] Further, RNA-seq analysis of male mouse dorsum skin revealed that DHT treatment upregulated genes involved in steroid metabolism, lipoprotein metabolism, extracellular space, peptidase inhibitor activity, fatty acid binding, and aromatase activity (Figure 9B), as well as chylomicron and lipoprotein signaling pathways (Figure 9C). Genes involved in epithelial cell differentiation, hair follicle development, intermediate filaments, and epidermal structural components were downregulated (Figure 10B), as were pathways associated with keratinization (Figure 10C). Furthermore, treatment with the peptide PY1 significantly upregulated genes involved in keratinocyte differentiation and chemotaxis (Figure 11B), chemokine receptor binding, peptide ligand-receptor binding, and GPCR signaling pathways (Figure 11C). Genes involved in responses to peptide hormones, keratin fibers, and fatty acid binding were downregulated (Figure 12B), as were pathways associated with keratinization (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.

[0113] 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.

[0114] 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.

[0115] Example 4: Comparison of the effects of fusion polypeptides and single polypeptides in a DHT-induced androgen / seborrheic alopecia mouse model

[0116] Day 45 C57 male and female mice were pretreated with 10% ethanol and DHT (2 mg / day) dissolved in 10% ethanol, followed by a single dose every other day. Following these two DHT pretreatments, day 49 (7-week-old) telogen mice were divided into three groups: a control group (10% ethanol), a DHT group (intraperitoneal injection of DHT), an O-peptide group (intraperitoneal injection of DHT plus O-peptide application), an I-peptide group (intraperitoneal injection of DHT plus I-peptide application), and a peptide group (intraperitoneal injection of DHT plus fusion peptide application). Hair depilation was then performed to induce synchronous hair regrowth. DHT (2 mg / day) was administered every other day. The peptide application concentration was 0.5 mM, and the application volume was 0.1 mL.

[0117] 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.

[0118] 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 13A ).

[0119] The hair growth cycle is divided into the anagen phase, the catagen phase, and the telogen phase (Figure 13C). Morphological observation of the skin on the nape of female mice in a model of androgenic (seborrheic) alopecia revealed that peptide I exerts its effects in the early anagen phase (Figure 13B - DHT + I, hair follicle improvement); peptide O exerts its effects in the middle and late anagen phase (Figure 13B - DHT + O, hair growth begins). Therefore, the fusion peptide PY1 can exert its effects throughout the entire anagen phase (Figure 13B - DHT + PY, hair follicle improvement + hair growth), exerting an anti-hair loss effect in both males and females.

[0120] Example 5: Purchase and identification of primary human dermal papilla cells

[0121] Primary human dermal papilla cells (HFDPCs) were purchased from Shanghai Xuanke Biotechnology Co., Ltd. They were derived from surgically resected normal scalp tissue and were immunofluorescently positive for vimentin (Figure 14B). The cells were identified to be >90% pure and free of HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi. Fibroblast culture medium was used for culture.

[0122] Example 6: Detection of Early Apoptosis of Human Hair Follicle Papilla Cells (DHT Treatment)

[0123] HFDPCs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C and 5% CO2, with medium exchanged 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.

[0124] Early apoptosis detection (Annexin V): Aspirate the cell culture medium into a centrifuge tube and add an appropriate amount of trypsin to digest the cells. Incubate at room temperature until adherent cells are dislodged by gentle pipetting. Aspirate the trypsin and transfer the culture medium to the appropriate centrifuge tube (because DHT can cause apoptosis, apoptotic adherent cells will float, so the culture medium should not 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. Gently resuspend the cells in 195μl of Annexin V-FITC conjugate solution, and add 5μl of Annexin V-FITC and 10μl of propidium iodide staining solution, and mix gently. Incubate at room temperature in the dark for 20 minutes, then place in an ice bath. Cell scatter and fluorescence detection are performed using a flow cytometer.

[0125] As shown in the flow cytometry results in Figure 15 , the number of apoptotic HFDPCs cells increased significantly after DHT treatment. After the addition of polypeptides PY1, PY2, PY3 and PY4 (Figure 15 only shows the results of polypeptides PY1, PY2, PY3 and PY4 added at a concentration of 1uM), the number of apoptotic cells decreased significantly, indicating that polypeptides PY1, PY2, PY3 and PY4 can significantly alleviate the apoptosis of human primary dermal papilla cells caused by high androgen DHT.

[0126] Example 7: DHT-induced androgen / seborrheic alopecia cell model

[0127] HFDPCs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C and 5% CO2, with medium exchanged 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 assessed after 48 hours of culture.

[0128] As shown in Figure 16, DHT treatment significantly increased the expression of the androgen receptor (AR) and the inflammatory factor TGF-β (Figure 16A), while the expression of Wnt signaling pathway regulators Wnt5a and β-Catenin decreased, as did the hair follicle stem cell marker CD133, Noggin, and Fgf7 (Figure 16B). Peptides PY1, PY2, PY3, and PY4 significantly mitigated 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.

[0129] Example 8: β-arrestin protein recruitment experiment

[0130] 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.

[0131] Three GPRs vectors were purchased from Addgene, including GPR37-Tango (#66355), GPRC6A-Tango (#66386), and GPR158-Tango (#66332).

[0132] HTLA cells expressing these constructs 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.

[0133] The results showed (Figure 17) that the peptides PY1, PY2, PY3 and PY4 were able to bind to the receptors GPRC6A / GPR158 / GPR37, β-arrestin was recruited, and TEV was cleaved and entered the cell nucleus, where a sharp increase in fluorescence level was detected.

[0134] 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.

[0135] 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)n- O peptide; or O peptide-(Pro-Lys-Pro-Lys-Pro)n-I 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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