Polypeptides and related substances for combating hair loss and dermatitis, and their applications.

Abstract

This invention discloses polypeptides and related substances for combating hair loss and dermatitis, and their applications. Specifically, it discloses the use of osteocalcin-inducing peptides and homologous polypeptides, polypeptide-inducing peptides, or polypeptide and polypeptide-inducing peptide derivatives derived from different species that form its backbone, in the preparation of drugs for treating stress-induced hair follicle aging and / or hair loss, seborrheic hair follicle aging and / or hair loss, stress-induced acne and / or dermatitis, and seborrheic acne and / or dermatitis. It also discloses the use of substances that can bind to and function with the GPR158 receptor in the preparation of drugs, medical and cosmetic products, cosmetics, health foods, foods, and additives for combating, preventing and / or treating hair follicle aging, hair loss, acne, and dermatitis, as well as methods for preparing relevant hair loss models based on the discovery of the GPR158 receptor.

Description

Technical Field

[0001] The present invention belongs to the field of medical biology and relates to polypeptides and related substances for combating problems such as hair loss and their applications.

Background Art

[0002] Hair loss refers to the phenomenon of hair shedding and is classified into physiological hair loss and pathological hair loss. Here, physiological hair loss refers to the normal shedding of hair during the telogen and resting phases to maintain a normal number of hairs. On the other hand, pathological hair loss refers to abnormal or excessive hair shedding.

[0003] According to the data of the "China Hair Loss Group Survey Report" in 2017, the current Chinese hair loss group is about 200 million, and it is shown that it is increasing at a rate of at least 15% annually. The majority of the hair loss group is male, about 130 million.

[0004] There are many causes of hair loss, including genetic factors, immune factors, excessive mental stress, staying up late, drugs (such as hair loss caused by chemotherapy), environmental factors, endocrine disorders, etc.

[0005] Hair loss can be classified into stress-induced hair loss, androgenetic (seborrheic) hair loss, pathological hair loss, physical / chemical hair loss, nutritional hair loss, obesity-related hair loss, genetic hair loss, etc. according to the etiology. Currently, there are two types of drugs for treating hair loss approved by the US Food and Drug Administration (FDA), minoxidil and finasteride. Here, minoxidil is a topical scalp application drug, has an "exacerbation phase", has a long effect, and has certain side effects. Finasteride is an oral drug, but it has a significant risk to female fertility and is ineffective for postmenopausal women, so it is not recommended for women to take. Clinically, there is a need to develop more safe, effective and intervention drugs suitable for diverse populations and diverse hair loss types.

[0006] Androgenic alopecia (AGA) is divided into male pattern baldness (MPHL) and female pattern baldness (FPHL), and is caused by genetic factors and the action of androgens. Due to differences in sex hormones between men and women, male and female alopecia differ significantly in their etiology, diagnosis, and treatment. In men, androgen levels in the body are relatively high, and after testosterone reaches the hair follicles, it is converted to dihydrotestosterone (DHT) by 5α-reductase, causing atrophy of the hair follicles, gradually thinning the hair, and in men with a genetic predisposition, this leads to hair loss, mainly showing a backward shift of the hairline, M-shaped alopecia, and eventually appearing as thinning hair. In women, androgen levels in the body are relatively low, and estrogen can inhibit androgen, but if the body's hormonal balance is disrupted or estrogen decreases after middle age, insufficient estrogen secretion / excess androgen can cause hair loss, mainly showing progressive hair loss, clinically characterized by progressive miniaturization of hair follicles, and the hair in the center of the crown gradually thins and becomes sparse.

[0007] While the role of androgens in the pathogenesis of male pattern androgenic alopecia is generally clear, the mechanism of action in female pattern androgenic alopecia is not yet clear. In addition to androgens, non-androgen-dependent pathways such as chronic low-grade scalp inflammation, androgen insensitivity, estrogen deficiency, thyroid dysfunction, and metabolic syndrome may also be involved (see "China Expert Consensus on the Diagnosis and Treatment of Female Androgenic Alopecia (2022 Edition)"). [Overview of the project]

[0008] Regarding the bottleneck problem in the treatment of hair loss provided in the background technology described above, the object of the present invention is to provide the application of polypeptides and related products that counteract problems such as hair loss.

[0009] One aspect of the present invention provides the use of polypeptides, polypeptide-derived peptides, or derivatives of polypeptides and polypeptide-derived peptides in the preparation of drugs for treating stress-induced hair follicle aging and / or hair loss, seborrheic hair follicle aging and / or hair loss, stress-induced acne and / or dermatitis, seborrheic acne and / or dermatitis, The polypeptides described above were selected from osteocalcin-inducible peptides and homologous polypeptides from different species that use them as a backbone, and their sequences are as follows: YLYQWLGAPVPYPDPLEPR SEQ ID No.1 YLGASVPSPDPLEPT SEQ ID No.2 LDPGLGAPAPYPDPLEPR SEQ ID No.3 NYNRFYNARAAPTPDPLEPL SEQ ID No.4 SNLRNAVFGTPVRDPLESK SEQ ID No.5 AGAVTAADLSLTQLESL SEQ ID No.6 YAQDSGVAGAPPNPLEAQ SEQ ID No.7 SDFYER YFMHYKTPMEQM SEQ ID No.8 Here, SEQ ID NO: 1 is human osteocalcin-inducing peptide, SEQ ID NO: 2 is mouse osteocalcin-inducing peptide, SEQ ID NO: 3 is domesticated sheep osteocalcin-inducing peptide (the sequence of this part is the same for otters and badgers), SEQ ID NO: 4 is reptile lizard osteocalcin-inducing peptide, SEQ ID NO: 5 is amphibian aquatic frog osteocalcin-inducing peptide, SEQ ID NO: 6 is bony fish Wuchang fish osteocalcin-inducing peptide, SEQ ID NO: 7 is avian chicken osteocalcin-inducing peptide, and SEQ ID NO: 8 is avian osidiosteocalcin-inducing peptide (the sequence of this part is the same for swan goose).

[0010] In light of the functional similarities between osteocalcin-inducing peptides of different species, the above-mentioned osteocalcin-inducing peptides include mammalian, reptile, amphibian, bony fish, bird, and avian osteocalcin-inducing peptides.

[0011] The polypeptide-derived peptide described above is a conserved fragment in osteocalcin derived from a different species of organism, and the polypeptide-derived peptide is at the same position as the conserved fragment where the sequence described in any one of SEQ ID NOs: 1-8 is located, and the different species of organism described above are selected from mammals, reptiles, amphibians, bony fish, birds and birds, and the polypeptide-derived peptide is the sequence X1-X2- ...... X n Furthermore, the immediate upstream and downstream sequences of the polypeptide-derived peptide in osteocalcin are both conversion enzyme cleavage sites, where X represents an amino acid and n is any integer between 8 and 25. The above derivatives are products obtained after modification of the terminal or side chain of the polypeptide or polypeptide-derived peptide, or products obtained by attaching a tag used for the detection or purification of polypeptides or proteins, or products obtained by isotopic labeling modification. The products obtained by the above modifications are selected from products obtained by fluorescent group modification, or products obtained by phosphorylation modification, or products obtained by cyclization modification based on disulfide bonds, or products obtained by biotin labeling modification, or products obtained by photosensitizer modification, or products obtained by azide modification, or products obtained by PEG modification, or products obtained by methylation modification, or products obtained by fluorescent quenching group modification, or products obtained by protein coupling modification, or products obtained by small molecule compound modification, or products obtained by amination modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, esterification modification, or glycosylation modification.

[0012] Furthermore, the above-mentioned terminal modifications are selected from acetylation modifications at the N-terminus and amination modifications at the C-terminus of the polypeptide.

[0013] Furthermore, the above-mentioned side chain modifications are selected from modifications at the R group of the amino acid side chain in the polypeptide.

[0014] Furthermore, the fluorescent dye used for the modification of the fluorescent group is selected from AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, ICG, and this modification can be used for fluorescence detection.

[0015] Furthermore, the phosphorylation modification is selected from one or a combination of more than one of p-Ser, p-Thr, and p-Tyr.

[0016] Furthermore, the glycosylation modification is selected from one or a combination of more than one of Ser, Asn, Thr, and Tyr.

[0017] Furthermore, the nitration modification is selected from one or a combination of more than one of Tyr.

[0018] Furthermore, regarding the labeling of the above biotin, the biotin is selected from D-biotin, biotin hydrazide, photosensitive biotin, and biotin-dUTP.

[0019] Furthermore, the photosensitizer modification can be used for the preparation of photosensitive preparations.

[0020] Furthermore, the azide modification can be used for secondary binding reactions.

[0021] Furthermore, the PEG modification can be used for the preparation of drug carriers.

[0022] Furthermore, the isotope used for the isotope labeling is selected from one or a combination of more than one of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, and 131I.

[0023] The above-mentioned polypeptide and derivatives having it as a backbone can be independently synthesized under general chemical laboratory conditions. Recombinant proteins containing the above-mentioned polypeptide obtained by gene expression can also be industrially synthesized by a commercial reagent company. The polypeptide is synthesized by adopting the solid-phase method, and different amino acids in the resin are used to directionally synthesize an amino acid chain through a condensation reaction, or are extracts separated from different tissues and organs. The derivative of the polypeptide is further labeled with a modifying group after the bonding of the amino acids is completed.

[0024] Furthermore, the hair follicle aging and / or hair loss caused by the above stress is hair follicle aging and / or hair loss caused by stress in a female or male subject, Seborrheic hair follicle aging and / or hair loss is seborrheic hair follicle aging and / or hair loss in a male, Acne and / or dermatitis caused by stress is acne and / or dermatitis caused by stress in a female or male, Seborrheic acne and / or dermatitis is seborrheic acne and / or dermatitis in a male, Another aspect of the present invention provides an action in which the above-mentioned polypeptide binds to its receptor and exhibits a function.

[0025] Furthermore, the above receptor mainly refers to GPR158.

[0026] Another aspect of the present invention provides the use of a substance that can bind to the GPR158 receptor and exhibit a function in the preparation of drugs for hair follicle aging and / or hair loss caused by stress, seborrheic hair follicle aging and / or hair loss, acne and / or dermatitis caused by stress, and seborrheic acne and / or dermatitis.

[0027] Furthermore, the substance that can bind to the GPR158 receptor and exhibit a function is the above-mentioned polypeptide.

[0028] Furthermore, substances that can bind to and function with the GPR158 receptor include, in addition to the polypeptides mentioned above, biomacromolecules such as inorganic ions including cations / metal ions, anions, and hydrogen ions; organic molecules such as polypeptides and small molecule compounds; enzymes; peptides that bind to the screened receptor; antibodies; siRNAs; shRNAs; antisense RNA and DNA enzymes or combinations thereof; and expression vectors containing siRNAs, shRNAs, and antisense molecules.

[0029] Another aspect of the present invention provides a method for preparing an animal model of hair loss, the method comprising knocking out the GPR158 receptor gene in an animal or treating the animal with a GPR158 receptor inhibitor.

[0030] Another aspect of the present invention provides the use of the above polypeptides and their derivatives, as well as substances that can bind to and function with the GPR158 receptor, in the preparation of medical and cosmetic products, cosmetics, health foods, foods, and additives for the countermeasures, prevention and / or treatment of hair follicle aging, hair loss, acne, and dermatitis.

[0031] Furthermore, the above hair loss includes stress-induced follicular aging and / or hair loss in males and females, androgenic follicular aging and / or hair loss. Furthermore, the prevention and / or treatment of the above-mentioned hair loss involves promoting hair growth. Furthermore, the above-mentioned acne includes stress-induced acne in males and females, and androgenic acne. Furthermore, the above-mentioned dermatitis includes stress-induced dermatitis and androgenic dermatitis in males and females.

[0032] Another aspect of the present invention provides the use of the polypeptides and their derivatives, or substances capable of binding to and functioning with the GPR158 receptor, in the preparation of detection reagents targeting brain, adrenal gland, female gonad (ovary and uterus), and male gonad (testis and epididymis) tissues.

[0033] Another aspect of the present invention provides the use of the polypeptides and their derivatives, or substances that can bind to and function with the GPR158 receptor, in the preparation of drugs that inhibit the elevation of steroidocorticoids or glucocorticoids.

[0034] Another aspect of the present invention provides the use of the polypeptides and their derivatives, or substances that can bind to and function with the GPR158 receptor, in the preparation of drugs that inhibit inflammatory responses caused by elevated steroidocorticoids or glucocorticoids or elevated cortisol.

[0035] Another aspect of the present invention provides the use of a substance that can bind to and inhibit the function of the GPR158 receptor in the preparation of a product for a hair loss-related research model.

[0036] A further aspect of the present invention is a drug for counteracting, preventing and / or treating related symptoms such as hair follicle aging, hair loss, acne, and dermatitis, wherein the drug is a complex of a polypeptide, polypeptide-derived peptide or derivatives of polypeptides and polypeptide-derived peptides, and its agonist and antagonist that bind to the GPR158 receptor with an active ingredient. The above-mentioned drug may contain one or more pharmaceutically acceptable carriers. The above-mentioned carrier is preferably a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorbent carrier, surfactant, or lubricant. The above-mentioned drugs can be further prepared in various forms such as drops, tablets, granules, capsules, oral solutions, inhalation solutions, topical solutions, sprays, microneedles, or injections, and drugs in various dosage forms can be prepared according to conventional methods in the pharmaceutical field. The above-mentioned drugs are administered by oral administration, topical contact, suppository administration, intravenous, intraperitoneal, intramuscular, intrafocal, intrathecal, intranasal, or subcutaneous injection. The routes of administration include various methods such as parenteral administration (intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, ventricular, and intracranial), and transmucosal administration (e.g., transbuccal, sublingual, oral lid, transgingival, transnasal, transvaginal, transrectal, or transdermal). Other modes of administration include, but are not limited to, liposomal formulations, intravenous injection, and transdermal patches.

[0037] Another aspect of the present invention is a method for treating or preventing stress-induced hair follicle aging and / or hair loss, seborrheic hair follicle aging and / or hair loss, stress-induced acne and / or dermatitis, seborrheic acne and / or dermatitis, the method comprising the step of administering a polypeptide, polypeptide-derived peptide or derivatives of polypeptides and polypeptide-derived peptides, or a substance that can bind to and function with the GPR158 receptor, The polypeptides described above were selected from osteocalcin-inducible peptides and homologous polypeptides from different species that use them as a backbone, and their sequences are as follows: YLYQWLGAPVPYPDPLEPR SEQ ID No.1 YLGASVPSPDPLEPT SEQ ID No.2 LDPGLGAPAPYPDPLEPR SEQ ID No.3 NYNRFYNARAAPTPDPLEPL SEQ ID No.4 SNLRNAVFGTPVRDPLESK SEQ ID No.5 AGAVTAADLSLTQLESL SEQ ID No.6 YAQDSGVAGAPPNPLEAQ SEQ ID No.7 SDFYER YFMHYKTPMEQM SEQ ID No.8 The polypeptide-derived peptide described above is a conserved fragment in osteocalcin derived from a different species of organism, and the polypeptide-derived peptide is at the same position as the conserved fragment where the sequence described in any one of SEQ ID NOs: 1-8 is located, and the different species of organism described above are selected from mammals, reptiles, amphibians, bony fish, birds and birds, and the polypeptide-derived peptide is the sequence X1-X2- ...... X n Furthermore, the immediate upstream and downstream sequences of the polypeptide-derived peptide in osteocalcin are both conversion enzyme cleavage sites, where X represents an amino acid and n is any integer between 8 and 25. The derivatives of the polypeptide described above are products obtained after modification of the terminal or side chain of the polypeptide or polypeptide-derived peptide, or products obtained by attaching a tag used for the detection or purification of the polypeptide or protein, or products obtained by isotopic labeling modification. Furthermore, the product obtained after the above modifications is selected from products obtained by fluorescent group modification, or products obtained by phosphorylation modification, or products obtained by cyclization modification based on disulfide bonds, or products obtained by biotin labeling modification, or products obtained by photosensitizer modification, or products obtained by azide modification, or products obtained by PEG modification, or products obtained by methylation modification, or products obtained by fluorescent quenching group modification, or products obtained by protein coupling modification, or products obtained by small molecule compound modification, or products obtained by amination modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, esterification modification, or glycosylation modification. Furthermore, the above terminal modifications are selected from acetylation modifications at the N-terminus and amination modifications at the C-terminus of the polypeptide. Furthermore, the above side chain modifications are selected from modifications at the R group of the amino acid side chain in the polypeptide. Furthermore, the fluorescent dye used for modifying the above-mentioned fluorescent group is selected from AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, and ICG, and this modification can be used for fluorescence detection. Furthermore, the above phosphorylation modification is selected from one or more combinations of p-Ser, p-Thr, and p-Tyr. Furthermore, the glycosylation modification described above is selected from one or more combinations of Ser, Asn, Thr, and Tyr. Furthermore, the above nitration modification is selected from one or more combinations of Tyr. Furthermore, regarding the labeling of biotin, the biotin is selected from D-biotin, biotin hydrazide, photosensitive biotin, and biotin-dUTP. Furthermore, the above photosensitizer modification can be used in the preparation of photosensitive formulations. Furthermore, the above azide modification can be used in secondary bonding reactions. Furthermore, the above PEG modification can be used in the preparation of drug carriers. Furthermore, the present invention provides a method in which the isotopes used for the above-mentioned isotope labeling are selected from one or more combinations of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, and 131I.

[0038] Furthermore, the step of administering the polypeptide or its derivatives to the target population includes administering the polypeptide or its derivatives orally, intravenously, intramuscularly, transdermally, or via the mucous membrane.

[0039] The beneficial effects of this invention are as follows:

[0040] (1) The polypeptides provided by the present invention and derived polypeptides based on the same backbone can combat problems such as hair follicle aging due to stress, and / or androgens, and / or seborrheic dermatitis, and / or hair loss, and / or acne, and / or dermatitis.

[0041] (2) The candidate polypeptides provided by the present invention and derived polypeptides based on them are easy to synthesize and modify, and are low-cost, thus offering a wide range of potential applications.

[0042] (3) The candidate polypeptides provided by the present invention and derived polypeptides based on them provide a wide range of strategies for preparing therapeutic compounds or drugs for their practical application.

[0043] (4) The candidate polypeptides provided by the present invention and derived polypeptides based thereon are applicable to the treatment of hair loss and related symptoms.

[0044] (5) The present invention demonstrates the relationship between GPR158 and hair loss and hair follicles, and can be used to establish an animal model of hair loss, as well as a research and observation tool in experimental animals for hair loss disease modeling.

[0045] (6) Polypeptides from different species were found to have relatively high binding ability when docking with the human receptor GPR158, which expands the types of active polypeptides that can be used to treat conditions such as alopecia and dermatitis. [Brief explanation of the drawing]

[0046] [Figure 1] This figure illustrates the effect of mouse polypeptide (mO15-OCG) bound to the dye indole green-ICG, targeting the brain, ovaries, and uterus of female mice in vivo. The figure shows the fluorescence signal distribution and results in the brain, ovaries, and uterus of three mouse groups: the PBS group, the ICG group, and the ICG-bound polypeptide group. [Figure 2] This figure illustrates the effect of Cy5-labeled mouse polypeptide (Cy5-mO15) on targeting the adrenal glands of female mice in vivo. The figure shows the fluorescence signal distribution and results in the adrenal glands of the mO15 group and the control group mice. [Figure 3]This figure illustrates the effect of Cy5-labeled mouse polypeptide (Cy5-mO15) on targeting the brain, gonads, and adrenal glands of male mice in vivo. The figure shows the fluorescence signal distribution and results in the adrenal glands, brain, testes, and epididymis of three mouse groups: the PBS group, the control polypeptide group, and the mO15 group. [Figure 4] This shows the expression and localization results of the polypeptide receptor GPR158 in human brain, ovary, testis, and skin tissue, compiled from single-cell sequencing results of various human tissues in the human Protein Atlas database. [Figure 5] This figure shows the results of GPR158 immunofluorescence staining in the hypothalamus, pituitary gland, and ovary of mice. In the figure, GPR158 shows specific expression in the subventricular region of the hypothalamus. [Figure 6] This shows the results of molecular docking simulations between homologous polypeptide sequences from different species and the receptor GPR158. The homologous polypeptide sequences from different species have more than 60% sequence identity, and the molecular docking simulations show that all of them can bind to the extracellular domain of human GPR158 (Figure 6A). The reciprocity score for all of them is 20 or higher (Figure 6B), indicating that the different polypeptides all have relatively strong binding ability to the human GPR158 receptor. [Figure 7] This figure shows the endocytosis reaction between osteocalcin and its derived peptides and HeLa cell lines that highly express the GPR158 receptor. The figure shows that human osteocalcin (Ocn49) and its derived peptide (Ocn19) bind to and are internalized in HeLa cells that highly express the GPR158 receptor. Red represents the polypeptides and their derived peptides, located in the cytoplasm, while blue represents the cell nucleus stained with DAPI. [Figure 8] These are the results of endocytosis and signal modification experiments with different homologous polypeptides of osteocalcin and HeLa cell lines that highly express the GPR158 receptor. [Figure 8A]This refers to the binding rate between different homologous polypeptides and HeLa cells that highly express the GPR158 receptor. [Figure 8B] This represents the calcium signal intensity produced after different homologous polypeptides bound to HeLa cells that highly express the GPR158 receptor. [Figure 9] This describes the therapeutic effect of polypeptides in a mouse stress-induced alopecia model. [Figure 9A] This shows the hair growth status of the neck and back of mice in models treated with the steroid corticosteroid dexamethasone and the glucocorticoid receptor agonist prednisone, as well as in the polypeptide treatment group. [Figure 9B] This is the scoring criteria for mouse skin color. [Figure 9C] The results in Figure 9A are the results quantified according to the criteria in Figure 9B. [Figure 10] This shows the morphological and genetic detection results of the cervical and dorsal skin of a stress-induced alopecia model mouse. [Figure 10A] These are the HE staining results for the neck and back of mice treated with dexamethasone and polypeptides. [Figure 10B] Figure 10A shows the count results of hair follicles in the skin after morphological observation. [Figure 10C] This shows the expression results of the cortisol-related enzyme genes 3betaHSD, Cyp21, Cyp11B1, the testosterone-related enzyme gene 17betaHSD3, the estrogen-related enzyme gene Cyp19, and the glucocorticoid receptor gene GR in the skin. [Figure 11] This describes the therapeutic effect of polypeptides in a mouse androgen (seborrheic) alopecia model. [Figure 11A] This shows the hair growth status of the neck and back of mice in the androgen group and the androgen + polypeptide treatment group. [Figure 11B] This is the scoring criteria for mouse skin color. [Figure 11C] The results in Figure 11A are the results quantified according to the criteria in Figure 11B. [Figure 12]This shows the morphological and genetic detection results of the cervical dorsal skin of androgen (seborrheic) alopecia model mice. [Figure 12A] These are the HE staining results for the dorsal neck of mice in the control group, the DHT-treated group, and the polypeptide-treated group. [Figure 12B] Figure 12A shows the count of hair follicles in the skin after morphological observation. [Figure 12C] This is the result of the expression of the skin Wnt signaling pathway regulatory protein β-catenin, the Wnt signaling pathway antagonist DKK1, the inflammatory factors TGF-β and IL-6, and the androgen receptor AR. [Figure 13] This image shows the expression of androgen receptor (AR) protein in the dorsal neck skin of an androgen-induced (seborrheic) alopecia model mouse (immunofluorescence). Green represents immunofluorescence staining of AR, and blue represents cell nuclei stained with DAPI. [Figure 14] This shows the gene expression of sebaceous gland-related molecular markers in the cervicoid skin of androgen (seborrheic) alopecia model mice (fluorescence quantitative PCR). LPL is a lipoprotein lipase, PPARγ promotes adipogenic differentiation, and FABP3 / 4 are fatty acid-binding proteins. [Figure 15] This is a hair loss phenotype in GPR158KO female mice (10 weeks old). [Figure 15A] This shows the hair growth status on the neck and back of wild-type mice and GPR158KO mice of the same age. [Figure 15B] This is the scoring criteria for mouse skin color. [Figure 15C] The results in Figure 15A are the results quantified according to the criteria in Figure 15B. [Figure 16] This shows the morphological and genetic detection results of the dorsal neck skin of GPR158KO female mice (10 weeks old). [Figure 16A] These are HE staining results from the dorsal neck skin of wild-type mice and GPR158KO mice of the same age. [Figure 16B] Figure 16A shows the count of hair follicles in the skin after morphological observation. [Figure 16C]This shows the expression results for cutaneous 5α-reductase (an enzyme that converts testosterone to dihydrotestosterone DHT), cortisol production-related enzyme genes 3betaHSD, Cyp21, Cyp11B1, testosterone production-related enzyme gene 17betaHSD3, estrogen production-related enzyme gene Cyp19, and glucocorticoid receptor gene GR. [Figure 17] This is the identification result of primary human dermal hair papilla cells. The green color represents fibronectin. [Figure 18] This study involved inducing a stress model in vitro using human primary dermal dermal papilla cells via cortisol treatment, followed by the mitigating effects of homologous polypeptide sequences from different species on changes in cell-related molecular markers. [Figure 18A] This is the action of homologous polypeptides from humans, mice, and livestock. [Figure 18B] This is the action of homologous polypeptides of humans, reptiles, amphibians, and fish. [Figure 18C] This involves the action of homologous polypeptides in humans, birds, and avian species. GR is a glucocorticoid receptor, AR is an androgen receptor, 5α-reductase promotes the conversion of testosterone to dihydrotestosterone (DHT), STIP1 is a stress-induced phosphorylated protein, GAS6 is a hair growth marker, TGF-β1 and IL-6 are inflammatory factors, Caspase-3, Bax, and Bcl-2 are hair follicle cell apoptosis markers, CD200 and CD133 are hair follicle stem cell markers, and MMP-9 is a hair follicle degeneration marker. [Figure 19] This study involved inducing an androgen / seborrheic model in vitro using human primary dermal dermal papilla cells via DHT treatment, followed by the mitigating effects of homologous polypeptide sequences from different species on changes in cell-related molecular markers. [Figure 19A] This is the action of homologous polypeptides from humans, mice, and livestock. [Figure 19B] This is the action of homologous polypeptides of humans, reptiles, amphibians, and fish. [Figure 19C]These are the effects of homologous polypeptides in humans, birds, and avian species. AR is the androgen receptor, 5α-reductase promotes the conversion of testosterone to dihydrotestosterone (DHT), 3βHSD and 17βHSD are hormone synthesis-related enzymes, Caspase-3, Bax, and Bcl-2 are markers of hair follicle cell apoptosis, CD200 is a hair follicle stem cell marker, MMP-9 is a hair follicle degeneration marker, TGF-β, TNFα, and IL-6 are inflammatory factors, FABP3 is a fatty acid-binding protein, and PPARγ promotes adipogenic differentiation. [Modes for carrying out the invention]

[0047] To better understand the content of the present invention, the content of the present invention will be further described below with reference to specific examples, however, the scope of protection of the present invention is not limited to the following examples.

[0048] term Stress-induced hair follicle aging and / or hair loss refers to the release of corticoids, stress hormones from the adrenal glands due to chronic stress, which prolongs the resting period of hair follicles, affects the activation of hair follicle stem cells, and causes impaired hair regeneration.

[0049] Seborrheic follicular aging and / or hair loss refers to a condition in which androgens stimulate excessive secretion of sebum from the sebaceous glands, which accumulates around the hair follicles, causing them to shrink and ultimately leading to hair loss.

[0050] Stress-induced acne and / or dermatitis refers to the condition in which stress acts on the hypothalamus-pituitary-adrenal axis, promoting the release of stress hormones and pro-inflammatory mediators, which stimulate sebaceous glands and hair follicles, leading to increased skin inflammation.

[0051] Seborrheic acne and / or dermatitis refers to acne and / or dermatitis caused by an excessive accumulation of hair follicle oils, which triggers an inflammatory response.

[0052] In the present invention, both polypeptides and polypeptide-derived peptides are derived from different species of organisms and are conserved sequence polypeptides in osteocalcin. The origins of SEQ ID NOs: 1 to 8 are as follows: SEQ ID NO: 1 is human osteocalcin-derived peptide, SEQ ID NO: 2 is mouse osteocalcin-derived peptide, SEQ ID NO: 3 is domesticated sheep osteocalcin-derived peptide (the sequence of this part is the same in otters and badgers), SEQ ID NO: 4 is reptile lizard osteocalcin-derived peptide, SEQ ID NO: 5 is amphibian aquatic frog osteocalcin-derived peptide, SEQ ID NO: 6 is bony fish Wuchang fish osteocalcin-derived peptide, SEQ ID NO: 7 is avian chicken osteocalcin-derived peptide, and SEQ ID NO: 8 is avian osidiosteocalcin-derived peptide (the sequence of this part is the same in swan goose).

[0053] The polypeptide-derived peptide described above is a conserved fragment in osteocalcin derived from a different species of organism other than those described in SEQ ID NOs: 1-8, and the polypeptide-derived peptide is located at the same position as the conserved fragment where the sequence described in any one of SEQ ID NOs: 1-8 is located, and the different species of organism described above are selected from mammals, reptiles, amphibians, bony fish, birds and birds, and the polypeptide-derived peptide is the sequence X1-X2- ...... X n Furthermore, the immediate upstream and downstream sequences of the polypeptide-derived peptide in osteocalcin are both conversion enzyme cleavage sites, where X represents an amino acid and n is any integer between 8 and 25.

[0054] Osteocalcin has two conserved sequences, and both the polypeptide and polypeptide-derived peptide in this invention are conserved sequences located close to the N-vote.

[0055] Example 1. Preparation of polypeptide sequence We synthesized the polypeptide sequence using an artificial synthesis method. The polypeptide sequence is as follows: YLYQWLGAPVPYPDPLEPR SEQ ID No.1 (Human-derived, Ocn19) YLGASVPSPDPLEPT SEQ ID No.2 (Mouse-derived, mO15 / OC15 / mOC15 / Ocn15) LDPGLGAPAPYPDPLEPR SEQ ID No.3 (Livestock - Polypeptide) NYNRFYNARAAPTPDPLEPL SEQ ID No.4 (Amphibian - Polypeptide) SNLRNAVFGTPVRDPLESK SEQ ID No.5 (Reptile - Polypeptide) AGAVTAADLSLTQLESL SEQ ID No.6 (Bone fish - Polypeptide) YAQDSGVAGAPPNPLEAQ SEQ ID No.7 (Poultry - Polypeptide) SDFYER YFMHYKTPMEQM SEQ ID No.8 (Avian Polypeptide)

[0056] The above polypeptides were synthesized using conventional solid-phase or liquid-phase synthesis methods. In this case, using the solid-phase synthesis method for polypeptides, the reaction proceeded from the C-terminal amino acid to the N-terminus, and through steps such as resin activation, amino acid linking, elution protection, and detection, the amino acid linkage was completed one by one. Then, the peptide was precipitated using excess ether, centrifuged, and the crude peptide was purified by HPLC. After mass spectrometry, it was rapidly frozen and dried with liquid nitrogen in preparation for use.

[0057] Here, SEQ ID NO: 1 is human osteocalcin-inducing peptide, SEQ ID NO: 2 is mouse osteocalcin-inducing peptide, SEQ ID NO: 3 is domesticated sheep osteocalcin-inducing peptide (the sequence of this part is the same for otters and badgers), SEQ ID NO: 4 is reptile lizard osteocalcin-inducing peptide, SEQ ID NO: 5 is amphibian aquatic frog osteocalcin-inducing peptide, SEQ ID NO: 6 is bony fish Wuchang fish osteocalcin-inducing peptide, SEQ ID NO: 7 is avian chicken osteocalcin-inducing peptide, and SEQ ID NO: 8 is avian osidiosteocalcin-inducing peptide (the sequence of this part is the same for swan goose).

[0058] Many proteins and hormones exhibit relatively high homology between humans and animals; for example, bovine and porcine insulin can be used to treat diabetic patients. Pregnant horse serum gonadotropins (PMSG) have also been used for superovulation in humans and animals. The chemical structure of oxytocin is the same in humans and most mammals. Since its isolation from pig and sheep brains in 1971, the GnRH family has grown to at least 28 species, 15 of which originate from vertebrates and 13 from invertebrates. With the exception of octo-GnRH, all GnRH peptides consist of 10 amino acids, and their molecular length and some amino acid sequences are highly conserved.

[0059] Given the homology and functional similarity between polypeptides and hormones in different species, sequences of the same species or with relatively high homology as the above polypeptides may perform similar functions.

[0060] Example 2. Preparation of polypeptide probes This example provides a polypeptide probe that utilizes the polypeptide prepared in Example 1 and is bound to ICG or Cy5 by an organic chemical reaction.

[0061] The specific method is either contract synthesis or autosynthesis. In this example, the polypeptide was bonded to ICG by click chemistry to complete the preparation of the probe, where the linker is a general-purpose DBCO. Regarding the ICG having an activating functional group, the activating functional group includes an amino group NH2, a carboxyl group COOH, an activated lipid NHS, a maleimide MAL, a mercapto group SH, an azide N3, and an alkyne ALK. This was then coupled with the polypeptide prepared in Example 1 to obtain a polypeptide ICG probe.

[0062] Example 3. Detection of the ability of polypeptide ICG / Cy5 probe to target the brain, ovaries, and uterus of female mice. The tail veins of adult female mice were injected with the polypeptide ICG probe solution / Cy5-labeled polypeptide prepared in Example 2, ICG solution (2 mM) / control polypeptide (disordered, unrelated polypeptide), and PBS. 100 μL was injected into each mouse, and after 24 hours, the samples were detected using a small animal imaging device.

[0063] The experimental results are shown in Figures 1 and 2. By comparing the results, it was found that brain tissue exhibited a clearer fluorescence signal compared to the control group, suggesting that mO15 enters the brain. Brain tissue, ovaries and uterus, and adrenal glands also exhibited a clearer fluorescence signal compared to the control group, indicating that mO15 (SEQ ID NO: 2) has specific localization binding ability to the hypothalamus, ovaries and uterus, and adrenal gland tissue.

[0064] Example 4. Detection of the ability of Cy5-labeled polypeptides to target the brain, gonads, and fat in male mice. The Cy5-labeled polypeptide from Example 2, a control polypeptide (disordered, unrelated polypeptide), and PBS were injected into the tail veins of adult male mice. 100 μL was injected into each mouse, and after 24 hours, the samples were detected using a small animal imaging device.

[0065] The experimental results are shown in Figure 3. By comparing the results of the three groups, it was found that brain tissue exhibited a clearer fluorescence signal compared to the control group, suggesting that mO15 enters the brain. Adrenal gland, testicular, and epididymal tissues all exhibited clearer fluorescence signals compared to the control group, indicating that the polypeptide has specific localization and binding ability to male brain, gonadal tissue, and adrenal gland tissue.

[0066] In summary, the results of Examples 3 and 4 demonstrate that mouse polypeptides can target the brain, adrenal gland, and gonadal tissue of female and male mice in vivo. Furthermore, in stress-induced hair loss models and androgen-induced hair loss models, the brain (cerebral cortical neurons) and adrenal gland (cortical hormones) are involved in stress regulation, the male and female gonads are involved in hormone regulation, and polypeptides can regulate the hair loss process by acting on stress- and hormone-regulated target tissues.

[0067] Example 5. Expression and distribution of polypeptide receptor GPR158 in the human brain Using the Human Protein Atlas, we investigated the expression and localization of the polypeptide receptor GPR158 in the human brain, ovaries, testes, and skin.

[0068] The search results are shown in Figure 4. Single-cell sequencing results from various human tissues revealed that the receptor GPR158 is expressed in the human brain, ovaries, testes, and skin.

[0069] Example 6. Expression and localization of the receptor GPR158 in the mouse HPO (hypothalamus-pituitary-ovary) axis. Adult C57 female mice underwent dissection and isolation of hypothalamic, pituitary, and ovarian tissue, followed by frozen sectioning and immunofluorescence staining with GPR158 antibody.

[0070] The experimental results are shown in Figure 5, revealing that GPR158 is specifically expressed in the subventricular region of the hypothalamus.

[0071] Similarly, the results of Examples 5 and 6 demonstrate that the GPR158 receptor is expressed in the brain, male and female gonads, and skin, and that polypeptides can modulate the hair loss process by acting on the GPR158 receptor in the target organs.

[0072] Example 7. Molecular docking simulation between homologous polypeptide sequences of different species and the receptor GPR158. For each species, one representative homologous polypeptide sequence was selected, and molecular docking with the receptor GPR158 was simulated using GalaxyPepDock software.

[0073] The simulation results are shown in Figure 6. Homologous polypeptides from different species exhibited more than 60% sequence identity and were all able to bind to the human GPR158 extracellular domain (Figure 6A). The reciprocity score for all was 20 or higher (Figure 6B), indicating that the different polypeptides all possessed relatively strong binding ability to the human GPR158 receptor.

[0074] The selection of polypeptide sequences is as follows: YLYQWLGAPVPYPDPLEPR SEQ ID No.1(Ocn19) YLGASVPSPDPLEPT SEQ ID No.2(mO15 / OC15 / mOC15 / Ocn15) LDPGLGAPAPYPDPLEPR SEQ ID No.3 (Livestock - Polypeptide) NYNRFYNARAAPTPDPLEPL SEQ ID No.4 (Amphibian - Polypeptide) SNLRNAVFGTPVRDPLESK SEQ ID No.5 (Reptile - Polypeptide) AGAVTAADLSLTQLESL SEQ ID No.6 (Bone fish - Polypeptide) YAQDSGVAGAPPNPLEAQ SEQ ID No.7 (Poultry - Polypeptide) SDFYER YFMHYKTPMEQM SEQ ID No.8 (Avian Polypeptide) DHTLVFSNERWPGVS SEQ ID No.9 (Unrelated polypeptide)

[0075] Example 8. Binding of human osteocalcin Ocn49 (full-length human osteocalcin) and cleaved human osteocalcin Ocn19 (SEQ ID NO: 1) to the receptor GPR158. In this example, the polypeptide prepared in Example 1 was used to observe its binding status to a HeLa cell line that highly expresses the receptor GPR158 by in vitro culture.

[0076] The specific method involved incubating Cy5-labeled full-length human osteocalcin Ocn49 and cleaved human osteocalcin Ocn19 (SEQ ID NO: 1) with HeLa cells highly expressing the receptor GPR158 at 4°C. The cells were then washed with PBS, and polypeptides that could not bind to the cells were designated as nonspecific ligands. Polypeptides that could bind to the cells were designated as specific binding ligands, and the interaction between the polypeptides and the receptor was then investigated.

[0077] The experimental results are shown in Figure 7, which demonstrate that incubation of polypeptides human Ocn46, human Ocn19, and GPR158-HeLa cells can induce internalization of the GPR158 receptor (red represents polypeptides, located in the cytoplasm). All are specific binding ligands, demonstrating that polypeptides human Ocn46 and human Ocn19 can bind to the GPR158 receptor.

[0078] Example 9. Binding of homologous polypeptides of different species to the receptor GPR158. In this example, the polypeptide from Example 7 was selected, and its internalization status and calcium signal intensity were observed in in vitro culture with a HeLa cell line that highly expresses the receptor GPR158.

[0079] The experimental results are shown in Figure 8, demonstrating that homologous polypeptides from different species possess relatively strong binding ability to human GPR158 receptor cells and can mediate calcium signaling produced by HeLa cells that highly express the GPR158 receptor. While the sequences of interspecies osteocalcin-inducing peptides show some variation, this does not affect binding to GPR158 or triggering of downstream signaling pathways, allowing the use of polypeptide sequences from different species across species.

[0080] Example 10. Mouse stress-induced alopecia model Seven-week-old male C57BL / 6 mice were coated with the steroidocorticoid dexamethasone and the glucocorticoid receptor agonist prednisone (modeling cycle was 23 days).

[0081] Hair in the resting phase was removed from the backs of the mice, and hair removal was performed using a depilatory cream. Model mice were coated once daily with dexamethasone solution (100 nM, 0.1 mL, dissolved in propylene glycol) in the depilatory area, while another group of mice were coated once daily with prednisone solution (100 nM, 0.1 mL, dissolved in propylene glycol) in the depilatory area, for 23 consecutive days. The treatment group mice received drug coating daily, along with intragastric administration of polypeptide at a concentration of 0.5 mg / kg / day.

[0082] The experimental results are shown in Figure 9. By comparing the results of the four groups, it was found that polypeptides can treat alopecia in mice induced by steroidocorticoids and glucocorticoid receptor agonists.

[0083] Furthermore, staining of mouse neck and dorsal skin sections and detection of related genes (Figure 10) revealed a significant increase in hair follicle number after polypeptide treatment, increased expression of cortisol-related enzyme genes 3betaHSD, Cyp21, and Cyp11B1 after dexamethasone treatment, a significant increase in glucocorticoid receptor gene GR, increased expression of testosterone-related enzyme gene 17betaHSD3, and decreased expression of estrogen-related enzyme gene Cyp11. Treatment with mO15 mitigated the effects of dexamethasone on the above gene expression levels.

[0084] Example 11. Mouse androgen (seborrheic) hair loss model Seven-week-old male C57BL / 6 mice were coated with DHT (the modeling and experimental cycle totaled 28 days). A 2x2cm area of ​​resting hair on the back was removed and depilated with depilatory cream. A DHT solution (0.2%, w / v) was prepared in an ethanol solution (50%, v / v). The DHT solution (0.1 mL / cm³) was applied topically to the depilation area once a day. 2 One group of mice was coated with DHT and kept in that state for 28 consecutive days. In another group, mice were coated with DHT daily and simultaneously administered polypeptides intragastricly at a concentration of 0.5 mg / kg / day.

[0085] The experimental results are shown in Figure 11. By comparing the results of the two groups, it was found that polypeptides can treat androgen-induced alopecia in mice.

[0086] Furthermore, staining of mouse neck and dorsal skin sections and detection of related genes (Figure 12) revealed that the number of hair follicles significantly increased after polypeptide treatment, the DHT-induced β-catenin gene decreased, and the expression of TGF-β, DKK1, IL-6, and AR genes increased. These gene expression levels were then alleviated after mO15 treatment. TGF-β and IL-6 belong to the category of inflammatory factors, and the experimental results demonstrate that androgens trigger inflammatory responses in the skin or hair follicles, but the polypeptide of the present invention can significantly reduce inflammatory factor levels and can be used for seborrheic acne or dermatitis.

[0087] Furthermore, immunofluorescence staining of frozen sections of mouse neck and dorsal skin (Figure 13) revealed a significant increase in androgen receptor (AR) expression in mouse neck and dorsal skin after DHT treatment, and a significant reduction in the high expression of cutaneous AR induced by DHT after intragastric administration of polypeptides.

[0088] Furthermore, the detection results of sebaceous gland-related molecular markers in the dorsal neck skin of mice (Figure 14) showed that genes such as lipase, adipocyte differentiation, and lipoproteins in the dorsal neck skin of mice increased significantly after DHT treatment, and that the expression levels of these genes were reduced after mO15 treatment.

[0089] Example 12. Alopecia phenotype in 10-week-old GPR158KO female mice Observations revealed that female GPR158KO mice, i.e., mice in which the GPR158 gene was knocked out, developed hair loss at 10 weeks of age (Figure 15). Staining of neck and dorsal skin sections of wild-type mice and GPR158KO mice of the same age and detection of related genes (Figure 16) showed that the number of hair follicles in 10-week-old GPR158KO mice was significantly reduced, and the expression of 5α-reductase (which promotes the conversion of testosterone to dihydrotestosterone DHT), cortisol-related enzyme genes 3βHSD, Cyp21, Cyp11B1, testosterone-related enzyme gene 17βHSD3, and glucocorticoid receptor gene GR was significantly increased, while the expression of estrogen-related enzyme gene Cyp19 was significantly decreased.

[0090] The inventors of this invention were the first to discover that the GPR158 receptor is associated with hair follicles and further established a correlation between the GPR158 receptor and hair loss phenotypes. This may influence the number of hair follicles and hair growth status by regulating the expression of 5α-reductase, cortisol-related enzyme genes 3βHSD, Cyp21, Cyp11B1, testosterone-related enzyme gene 17βHSD3, glucocorticoid receptor gene GR, and estrogen-related enzyme gene Cyp19.

[0091] Example 13. Purchase and identification of primary human dermal hair papilla cells Human primary dermal hair papilla cells were purchased from Shanghai Xuanke Biotechnology Co., Ltd., derived from normal scalp tissue surgically excised, and were positive for fibronectin immunofluorescence staining (Figure 17). They were identified as having a cell purity of over 90%. They were free of HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi. Fibroblast medium was used as the culture medium.

[0092] Example 14. Cortisol-induced stress hair loss cell model Human primary dermal dermal papilla cells were cultured for 24 hours after serum-starved cells were removed, using a culture medium containing 1 μM cortisol. The expression status of cell molecular markers (5α-dehydrogenase, GR, AR, CD200, IL-6, BCL2, BAX, etc.) was detected after 24 hours. Simultaneously, homologous polypeptides from different species (polypeptides from Example 7) were added and co-cultured, and the expression status of cell molecular markers was detected after 24 hours.

[0093] The results in Figure 18 show that after cortisol treatment, the expression of cellular glucocorticoid receptor GR, androgen receptor AR, and 5α-reductase increased significantly, while hair follicle cell apoptosis (Caspase-3, BCL2, BAX) increased, the expression of stress-induced phosphorylated protein STIP1 and inflammatory factors (TGF-β1, IL-6) increased, the expression of hair follicle degeneration marker MMP-9 increased, the expression of hair growth marker GAS6 decreased, and the expression of hair follicle stem cell markers CD200 and CD133 decreased. It was found that homologous sequence polypeptides from different species can mitigate these changes to a certain extent.

[0094] The polypeptide of the present invention has been shown to be usable for stress-induced hair loss, particularly hair follicle cell apoptosis, abnormally increased expression of phosphorylated protein STIP1, abnormally increased expression of inflammatory factors, stress-induced hair follicle aging or hair loss due to reduced hair follicle growth, and stress-induced acne or dermatitis.

[0095] Example 15. DHT-induced androgen / seborrheic alopecia cell model After removing serum-starved cells from human primary dermal dermal papilla cells, high androgenic treatment was performed by adding 10 nM DHT to the culture medium 24 hours later, and the expression status of cell molecular markers (5α-dehydrogenase, AR, CD200, IL-6, BCL2, BAX, FABP3, etc.) was detected after 24 hours. Simultaneously, homologous sequence polypeptides from different species (polypeptides from Example 7) were added and co-cultured, and the expression status of cell molecular markers was detected after 24 hours.

[0096] The results in Figure 19 show that after DHT treatment, the expression of cellular androgen receptor AR, 5α-reductase, and androgen synthesis-related enzymes (3βHSD, 17βHSD3) increased significantly, along with an increase in hair follicle cell apoptosis (Caspase-3, BCL2, BAX), an increase in the expression of inflammatory factors (TGF-β, TNF-α, IL-6), an increase in the expression of the hair follicle degeneration marker MMP-9, an increase in the expression of the hair follicle stem cell marker CD200, and an increase in the expression of fatty acid-binding protein FABP3 and adipogenic differentiation factor PPARγ. It was found that homologous polypeptide sequences from different species can significantly mitigate these changes.

[0097] The polypeptides of the present invention have been shown to be usable for the alleviation of androgen-induced hair loss, particularly the marked increase in the expression of androgen receptor AR5α reductase and androgen synthesis-related enzymes (3βHSD, 17βHSD3), hair follicle cell apoptosis, abnormal increase in inflammatory factor expression, androgen / seborrheic follicular aging or hair loss due to hair follicle degeneration, and androgen / seborrheic acne or dermatitis.

[0098] In summary, the polypeptides and related products provided herein can be used to prepare therapeutic agents for stress, and / or androgen, and / or seborrheic follicular aging, and / or hair loss and related diseases.

[0099] The foregoing description is merely a description of a mode for carrying out the invention of the present invention, and not all embodiments. Any equivalent transformations adopted by those skilled in the art by reading this specification are all included within the scope of the claims of the present invention.

Claims

1. The use of polypeptides, polypeptide-derived peptides, or derivatives of polypeptides and polypeptide-derived peptides in the preparation of drugs for treating stress-induced hair follicle aging and / or hair loss, seborrheic hair follicle aging and / or hair loss, stress-induced acne and / or dermatitis, and seborrheic acne and / or dermatitis, The polypeptide is selected from osteocalcin-inducible peptides and homologous polypeptides derived from different species that use it as a backbone, and its sequence is as follows: YLYQWLGAPVPYPDPLEPR SEQ ID No.1 YLGASVPSPDPLEPT SEQ ID No.2 LDPGLGAPAPYPDPLEPR SEQ ID No.3 NYNRFYNARAAPTPDPLEPL SEQ ID No.4 SNLRNAVFGTPVRDPLESK SEQ ID No.5 AGAVTAADLSLTQLESL SEQ ID No.6 YAQDSGVAGAPPNPLEAQ SEQ ID No.7 SDFYER YFMHYKTPMEQM SEQ ID No.8 The polypeptide-derived peptide is a conserved fragment in osteocalcin derived from a different species of organism, the polypeptide-derived peptide is at the same position as the conserved fragment where the sequence described in any one of SEQ ID NOs: 1 to 8 is located, the different species of organism is selected from mammals, reptiles, amphibians, bony fish, birds and birds, and the polypeptide-derived peptide is sequence X １ -X ２ - ...... X ｎ Furthermore, the immediate upstream and downstream sequences of the polypeptide-derived peptide in osteocalcin are both conversion enzyme cleavage sites, where X represents an amino acid and n is any integer between 8 and 25. The derivative is a product obtained after modification of the terminal or side chain of the polypeptide or polypeptide-derived peptide, or a product obtained by attaching a tag used for the detection or purification of the polypeptide or protein, or a product obtained by isotopic labeling modification. Preferably, the product obtained after the modification is selected from products obtained by fluorescent group modification, or products obtained by phosphorylation modification, or products obtained by cyclization modification based on disulfide bonds, or products obtained by biotin labeling modification, or products obtained by photosensitizer modification, or products obtained by azide modification, or products obtained by PEG modification, or products obtained by methylation modification, or products obtained by fluorescent quenching group modification, or products obtained by protein coupling modification, or products obtained by small molecule compound modification, or products obtained by amination modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, esterification modification, glycosylation modification, Preferably, the terminal modification is selected from acetylation modification at the N-terminus and amination modification at the C-terminus of the polypeptide. Preferably, the modification of the side chain is selected from modifications of the R group of the amino acid side chain in the polypeptide. Preferably, the fluorescent dye used to modify the fluorescent group is selected from AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, and ICG, and this modification can be used for fluorescence detection. Preferably, the phosphorylation modification is selected from one or more combinations of p-Ser, p-Thr, and p-Tyr. Preferably, the glycosylation modification is selected from one or more combinations of Ser, Asn, Thr, and Tyr. Preferably, the nitration modification is selected from one or more combinations of Tyr. Preferably, with respect to the labeling of biotin, the biotin is selected from D-biotin, biotin hydrazide, photosensitive biotin, and biotin-dUTP. Preferably, the photosensitizer modification can be used in the preparation of a photosensitive formulation. Preferably, the azide modification can be used in a secondary bonding reaction. Preferably, the PEG modification can be used in the preparation of the drug carrier. Preferably, the isotopes used for isotope labeling are selected from one or more combinations of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, and 131I. use.

2. The stress-induced hair follicle aging and / or hair loss refers to stress-induced hair follicle aging and / or hair loss in male or female subjects. Seborrheic follicular aging and / or alopecia is seborrheic follicular aging and / or alopecia in males. Stress-induced acne and / or dermatitis is stress-induced acne and / or dermatitis in females or males. Seborrheic acne and / or dermatitis is seborrheic acne and / or dermatitis in males. The use described in claim 1.

3. The use of substances that can bind to and function with the GPR158 receptor in the preparation of drugs for treating or preventing stress-induced hair follicle aging and / or hair loss, seborrheic hair follicle aging and / or hair loss, stress-induced acne and / or dermatitis, and seborrheic acne and / or dermatitis.

4. The substance that can bind to and function with the GPR158 receptor is a substance that activates the downstream pathway of the GPR158 receptor. Preferably, the substance that activates the GPR158 receptor downstream pathway is a polypeptide, a polypeptide-derived peptide, or a derivative of a polypeptide and / or polypeptide-derived peptide. The polypeptide is selected from osteocalcin-inducible peptides and homologous polypeptides derived from different species that use it as a backbone, and its sequence is as follows: YLYQWLGAPVPYPDPLEPR SEQ ID No.1 YLGASVPSPDPLEPT SEQ ID No.2 LDPGLGAPAPYPDPLEPR SEQ ID No.3 NYNRFYNARAAPTPDPLEPL SEQ ID No.4 SNLRNAVFGTPVRDPLESK SEQ ID No.5 AGAVTAADLSLTQLESL SEQ ID No.6 YAQDSGVAGAPPNPLEAQ SEQ ID No.7 SDFYER YFMHYKTPMEQM SEQ ID No.8 The polypeptide-derived peptide is a conserved fragment in osteocalcin derived from a different species of organism, the polypeptide-derived peptide is at the same position as the conserved fragment where the sequence described in any one of SEQ ID NOs: 1 to 8 is located, the different species of organism is selected from mammals, reptiles, amphibians, bony fish, birds and birds, and the polypeptide-derived peptide is sequence X １ -X ２ - ...... X ｎ Furthermore, the immediate upstream and downstream sequences of the polypeptide-derived peptide in osteocalcin are both conversion enzyme cleavage sites, where X represents an amino acid and n is any integer between 8 and 25. The derivative is a product obtained after modification of the terminal or side chain of the polypeptide or polypeptide-derived peptide, or a product obtained by attaching a tag used for the detection or purification of the polypeptide or protein, or a product obtained by isotopic labeling modification. More preferably, the product obtained after the modification is selected from products obtained by fluorescent group modification, or products obtained by phosphorylation modification, or products obtained by cyclization modification based on disulfide bonds, or products obtained by biotin labeling modification, or products obtained by photosensitizer modification, or products obtained by azide modification, or products obtained by PEG modification, or products obtained by methylation modification, or products obtained by fluorescent quenching group modification, or products obtained by protein coupling modification, or products obtained by small molecule compound modification, or products obtained by amination modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, esterification modification, glycosylation modification, More preferably, the terminal modification is selected from acetylation modification at the N-terminus and amination modification at the C-terminus of the polypeptide. More preferably, the modification of the side chain is selected from modifications of the R group of the amino acid side chain in the polypeptide. More preferably, the fluorescent dye used for modifying 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. More preferably, the phosphorylation modification is selected from one or more combinations of p-Ser, p-Thr, and p-Tyr. More preferably, the glycosylation modification is selected from one or more combinations of Ser, Asn, Thr, and Tyr. More preferably, the nitration modification is selected from one or more combinations of Tyr, More preferably, with respect to the labeling of biotin, the biotin is selected from D-biotin, biotin hydrazide, photosensitive biotin and biotin-dUTP. More preferably, the photosensitizer modification can be used in the preparation of a photosensitive formulation. More preferably, the azide modification can be used in a secondary bonding reaction. More preferably, the PEG modification can be used in the preparation of drug carriers. More preferably, the isotopes used for isotope labeling are selected from one or more combinations of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, and 131I. The use described in claim 3.

5. A method for preparing an animal model of hair loss or dermatitis, the preparation method comprising knocking out the GPR158 receptor gene in an animal, or treating the animal with a substance that can bind to the GPR158 receptor and inhibit the downstream pathway, Preferably, the substance capable of binding to the GPR158 receptor and inhibiting the downstream pathway is selected from inhibitory enzymes, peptides that bind to the screened receptor, antibodies, siRNAs, shRNAs, antisense RNAs and DNA enzymes or combinations thereof, and expression vectors containing siRNAs, shRNAs and antisense molecules. Preparation method.

6. The use of polypeptides, polypeptide-derived peptides, or derivatives of polypeptides and polypeptide-derived peptides, or substances that can bind to and function with the GPR158 receptor, in the preparation of medical and cosmetic products, cosmetics, health foods, foods, and additives for the countermeasures, prevention and / or treatment of hair follicle aging, hair loss, acne, and dermatitis, The polypeptide is selected from osteocalcin-inducible peptides and homologous polypeptides derived from different species that use it as a backbone, and its sequence is as follows: YLYQWLGAPVPYPDPLEPR SEQ ID No.1 YLGASVPSPDPLEPT SEQ ID No.2 LDPGLGAPAPYPDPLEPR SEQ ID No.3 NYNRFYNARAAPTPDPLEPL SEQ ID No.4 SNLRNAVFGTPVRDPLESK SEQ ID No.5 AGAVTAADLSLTQLESL SEQ ID No.6 YAQDSGVAGAPPNPLEAQ SEQ ID No.7 SDFYER YFMHYKTPMEQM SEQ ID No.8 The polypeptide-derived peptide is a conserved fragment in osteocalcin from organisms of different species, the polypeptide-derived peptide is in the same position as the conserved fragment where the sequence described in any one of SEQ ID NOs: 1 to 8 is located, the organisms of different species are selected from organisms of mammalian, reptilian, amphibian, teleost, avian and avian species, and the polypeptide-derived peptide is sequence X １ -X ２ - ...... X ｎ and, both the immediate upstream sequence and the downstream sequence in osteocalcin of the polypeptide-derived peptide are convertase cleavage sites, X represents an amino acid, and n is an arbitrary integer from 8 to 25 The polypeptide derivative is a product obtained after modification of the polypeptide or polypeptide-derived peptide terminal or side chain, or a product obtained by attaching a tag used for detection or purification of polypeptides or proteins, or a product obtained by isotopic labeling modification. Preferably, the product obtained after the modification is selected from products obtained by fluorescent group modification, or products obtained by phosphorylation modification, or products obtained by cyclization modification based on disulfide bonds, or products obtained by biotin labeling modification, or products obtained by photosensitizer modification, or products obtained by azide modification, or products obtained by PEG modification, or products obtained by methylation modification, or products obtained by fluorescent quenching group modification, or products obtained by protein coupling modification, or products obtained by small molecule compound modification, or products obtained by amination modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, esterification modification, glycosylation modification, Preferably, the terminal modification is selected from acetylation modification at the N-terminus and amination modification at the C-terminus of the polypeptide. Preferably, the modification of the side chain is selected from modifications of the R group of the amino acid side chain in the polypeptide. Preferably, the fluorescent dye used to modify the fluorescent group is selected from AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, and ICG, and this modification can be used for fluorescence detection. Preferably, the phosphorylation modification is selected from one or more combinations of p-Ser, p-Thr, and p-Tyr. Preferably, the glycosylation modification is selected from one or more combinations of Ser, Asn, Thr, and Tyr. Preferably, the nitration modification is selected from one or more combinations of Tyr. Preferably, with respect to the labeling of biotin, the biotin is selected from D-biotin, biotin hydrazide, photosensitive biotin, and biotin-dUTP. Preferably, the photosensitizer modification can be used in the preparation of a photosensitive formulation. Preferably, the azide modification can be used in a secondary bonding reaction. Preferably, the PEG modification can be used in the preparation of the drug carrier. Preferably, the isotopes used for isotope labeling are selected from one or more combinations of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, and 131I. Preferably, the hair loss includes stress-induced hair follicle aging and / or hair loss in males and females, androgenic hair follicle aging and / or hair loss. Preferably, the prevention and / or treatment of the hair loss is to promote hair growth. Preferably, the acne includes stress-induced acne in males and females, and androgenic acne. Preferably, the dermatitis includes stress-induced dermatitis in males and females, androgenic dermatitis. use.

7. The use of a substance capable of binding to the GPR158 receptor in the preparation of detection reagents targeting the brain, adrenal glands, and gonadal system, Preferably, the brain is a female brain, a male brain, Preferably, the adrenal glands are female and male adrenal glands. Preferably, the male gonad system consists of the testes and epididymis. Preferably, the female gonadal system consists of ovaries and a uterus. use.

8. Substances that can bind to the GPR158 receptor are polypeptides, polypeptide-derived peptides, or derivatives of polypeptides and polypeptide-derived peptides. Preferably, the polypeptide is selected from osteocalcin-inducible peptides and homologous polypeptides derived from different species that use it as a backbone, and the sequence is as follows: YLYQWLGAPVPYPDPLEPR SEQ ID No.1 YLGASVPSPDPLEPT SEQ ID No.2 LDPGLGAPAPYPDPLEPR SEQ ID No.3 NYNRFYNARAAPTPDPLEPL SEQ ID No.4 SNLRNAVFGTPVRDPLESK SEQ ID No.5 AGAVTAADLSLTQLESL SEQ ID No.6 YAQDSGVAGAPPNPLEAQ SEQ ID No.7 SDFYER YFMHYKTPMEQM SEQ ID No.8 The polypeptide-derived peptide is a conserved fragment in osteocalcin derived from a different species of organism, the polypeptide-derived peptide is at the same position as the conserved fragment where the sequence described in any one of SEQ ID NOs: 1 to 8 is located, the different species of organism is selected from mammals, reptiles, amphibians, bony fish, birds and birds, and the polypeptide-derived peptide is sequence X １ -X ２ - ...... X ｎ Furthermore, the immediate upstream and downstream sequences of the polypeptide-derived peptide in osteocalcin are both conversion enzyme cleavage sites, where X represents an amino acid and n is any integer between 8 and 25. The polypeptide derivative is a product obtained after modification of the polypeptide or polypeptide-derived peptide terminal or side chain, or a product obtained by attaching a tag used for the detection or purification of polypeptides or proteins, or a product obtained by isotopic labeling modification. More preferably, the product obtained after the modification is selected from products obtained by fluorescent group modification, or products obtained by phosphorylation modification, or products obtained by cyclization modification based on disulfide bonds, or products obtained by biotin labeling modification, or products obtained by photosensitizer modification, or products obtained by azide modification, or products obtained by PEG modification, or products obtained by methylation modification, or products obtained by fluorescent quenching group modification, or products obtained by protein coupling modification, or products obtained by small molecule compound modification, or products obtained by amination modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, esterification modification, glycosylation modification, More preferably, the terminal modification is selected from acetylation modification at the N-terminus and amination modification at the C-terminus of the polypeptide. More preferably, the modification of the side chain is selected from modifications of the R group of the amino acid side chain in the polypeptide. More preferably, the fluorescent dye used for modifying 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. More preferably, the phosphorylation modification is selected from one or more combinations of p-Ser, p-Thr, and p-Tyr. More preferably, the glycosylation modification is selected from one or more combinations of Ser, Asn, Thr, and Tyr. More preferably, the nitration modification is selected from one or more combinations of Tyr, More preferably, with respect to the labeling of biotin, the biotin is selected from D-biotin, biotin hydrazide, photosensitive biotin and biotin-dUTP. More preferably, the photosensitizer modification can be used in the preparation of a photosensitive formulation. More preferably, the azide modification can be used in a secondary bonding reaction. More preferably, the PEG modification can be used in the preparation of drug carriers. More preferably, the isotopes used for isotope labeling are selected from one or more combinations of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, and 131I. The use described in claim 7.

9. The use of substances that can bind to and function with the GPR158 receptor in the preparation of drugs, medical and cosmetic products, cosmetics, health foods, foods, and additives that inhibit the increase of steroidocorticoids or glucocorticoids, or inhibit inflammatory responses caused by the increase of steroidocorticoids or glucocorticoids or cortisol.

10. The use of a substance that can bind to and inhibit the function of the GPR158 receptor in the preparation of a product for a hair loss-related research model, Substances capable of binding to and inhibiting the function of the GPR158 receptor are selected from inhibitory enzymes, peptides that bind to the screened receptor, antibodies, siRNAs, shRNAs, antisense RNAs and DNA enzymes or combinations thereof, and expression vectors containing siRNAs, shRNAs and antisense molecules. use.

11. A method for treating or preventing stress-induced hair follicle aging and / or hair loss, seborrheic hair follicle aging and / or hair loss, stress-induced acne and / or dermatitis, seborrheic acne and / or dermatitis, the method comprising the step of administering a polypeptide, polypeptide-derived peptide, or derivatives of polypeptides and polypeptide-derived peptides, or a substance that can bind to and function with the GPR158 receptor, The polypeptide is selected from osteocalcin-inducible peptides and homologous polypeptides derived from different species that use it as a backbone, and its sequence is as follows: YLYQWLGAPVPYPDPLEPR SEQ ID No.1 YLGASVPSPDPLEPT SEQ ID No.2 LDPGLGAPAPYPDPLEPR SEQ ID No.3 NYNRFYNARAAPTPDPLEPL SEQ ID No.4 SNLRNAVFGTPVRDPLESK SEQ ID No.5 AGAVTAADLSLTQLESL SEQ ID No.6 YAQDSGVAGAPPNPLEAQ SEQ ID No.7 SDFYER YFMHYKTPMEQM SEQ ID No.8 The polypeptide-derived peptide is a conserved fragment in osteocalcin derived from a different species of organism, the polypeptide-derived peptide is at the same position as the conserved fragment where the sequence described in any one of SEQ ID NOs: 1 to 8 is located, the different species of organism is selected from mammals, reptiles, amphibians, bony fish, birds and birds, and the polypeptide-derived peptide is sequence X １ -X ２ - ...... X ｎ Furthermore, the immediate upstream and downstream sequences of the polypeptide-derived peptide in osteocalcin are both conversion enzyme cleavage sites, where X represents an amino acid and n is any integer between 8 and 25. The polypeptide derivative is a product obtained after modification of the polypeptide or polypeptide-derived peptide terminal or side chain, or a product obtained by attaching a tag used for the detection or purification of polypeptides or proteins, or a product obtained by isotopic labeling modification. Preferably, the product obtained after the modification is selected from products obtained by fluorescent group modification, or products obtained by phosphorylation modification, or products obtained by cyclization modification based on disulfide bonds, or products obtained by biotin labeling modification, or products obtained by photosensitizer modification, or products obtained by azide modification, or products obtained by PEG modification, or products obtained by methylation modification, or products obtained by fluorescent quenching group modification, or products obtained by protein coupling modification, or products obtained by small molecule compound modification, or products obtained by amination modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, esterification modification, glycosylation modification, Preferably, the terminal modification is selected from acetylation modification at the N-terminus and amination modification at the C-terminus of the polypeptide. Preferably, the modification of the side chain is selected from modifications of the R group of the amino acid side chain in the polypeptide. Preferably, the fluorescent dye used to modify the fluorescent group is selected from AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, and ICG, and this modification can be used for fluorescence detection. Preferably, the phosphorylation modification is selected from one or more combinations of p-Ser, p-Thr, and p-Tyr. Preferably, the glycosylation modification is selected from one or more combinations of Ser, Asn, Thr, and Tyr. Preferably, the nitration modification is selected from one or more combinations of Tyr. Preferably, with respect to the labeling of biotin, the biotin is selected from D-biotin, biotin hydrazide, photosensitive biotin, and biotin-dUTP. Preferably, the photosensitizer modification can be used in the preparation of a photosensitive formulation. Preferably, the azide modification can be used in a secondary bonding reaction. Preferably, the PEG modification can be used in the preparation of the drug carrier. Preferably, the isotopes used for isotope labeling are selected from one or more combinations of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, and 131I. method.

12. The step of administering the polypeptide or its derivatives to the target includes administering the polypeptide or its derivatives orally, intravenously, intramuscularly, transdermally, or via the mucous membrane. The method according to claim 11.

13. A drug for the countermeasure, prevention and / or treatment of hair follicle aging, hair loss, acne, dermatitis and related symptoms, wherein the drug is a complex of a polypeptide, polypeptide-derived peptide or derivatives of polypeptides and polypeptide-derived peptides, and its agonist and antagonist that bind to the GPR158 receptor with an active ingredient. The drug may include one or more pharmaceutically acceptable carriers. The carrier is preferably a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorbent carrier, surfactant, or lubricant. The polypeptide is selected from osteocalcin-inducible peptides and homologous polypeptides derived from different species that use it as a backbone, and its sequence is as follows: YLYQWLGAPVPYPDPLEPR SEQ ID No.1 YLGASVPSPDPLEPT SEQ ID No.2 LDPGLGAPAPYPDPLEPR SEQ ID No.3 NYNRFYNARAAPTPDPLEPL SEQ ID No.4 SNLRNAVFGTPVRDPLESK SEQ ID No.5 AGAVTAADLSLTQLESL SEQ ID No.6 YAQDSGVAGAPPNPLEAQ SEQ ID No.7 SDFYER YFMHYKTPMEQM SEQ ID No.8 The polypeptide-derived peptide is a conserved fragment in osteocalcin derived from a different species of organism, the polypeptide-derived peptide is at the same position as the conserved fragment where the sequence described in any one of SEQ ID NOs: 1 to 8 is located, the different species of organism is selected from mammals, reptiles, amphibians, bony fish, birds and birds, and the polypeptide-derived peptide is sequence X １ -X ２ - ...... X ｎ Furthermore, the immediate upstream and downstream sequences of the polypeptide-derived peptide in osteocalcin are both conversion enzyme cleavage sites, where X represents an amino acid and n is any integer between 8 and 25. The polypeptide derivative is a product obtained after modification of the polypeptide or polypeptide-derived peptide terminal or side chain, or a product obtained by attaching a tag used for the detection or purification of polypeptides or proteins, or a product obtained by isotopic labeling modification. Preferably, the product obtained after the modification is selected from products obtained by fluorescent group modification, or products obtained by phosphorylation modification, or products obtained by cyclization modification based on disulfide bonds, or products obtained by biotin labeling modification, or products obtained by photosensitizer modification, or products obtained by azide modification, or products obtained by PEG modification, or products obtained by methylation modification, or products obtained by fluorescent quenching group modification, or products obtained by protein coupling modification, or products obtained by small molecule compound modification, or products obtained by amination modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, esterification modification, glycosylation modification, Preferably, the terminal modification is selected from acetylation modification at the N-terminus and amination modification at the C-terminus of the polypeptide. Preferably, the modification of the side chain is selected from modifications of the R group of the amino acid side chain in the polypeptide. Preferably, the fluorescent dye used to modify the fluorescent group is selected from AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, and ICG, and this modification can be used for fluorescence detection. Preferably, the phosphorylation modification is selected from one or more combinations of p-Ser, p-Thr, and p-Tyr. Preferably, the glycosylation modification is selected from one or more combinations of Ser, Asn, Thr, and Tyr. Preferably, the nitration modification is selected from one or more combinations of Tyr. Preferably, with respect to the labeling of biotin, the biotin is selected from D-biotin, biotin hydrazide, photosensitive biotin, and biotin-dUTP. Preferably, the photosensitizer modification can be used in the preparation of a photosensitive formulation. Preferably, the azide modification can be used in a secondary bonding reaction. Preferably, the PEG modification can be used in the preparation of the drug carrier. Preferably, the isotopes used for isotope labeling are selected from one or more combinations of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, and 131I. Drugs.

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