Application of hair follicle immunohomeostatic regulation strategy in treatment of alopecia

Abstract

The application provides an application of a hair follicle immune homeostasis regulation strategy in treating alopecia diseases. A group of pathological LAMP3 + CCR7 + IL4I1 + DC3 dendritic cell subpopulation. A DC3 dendritic cell subpopulation marker gene Il4i1 Drive conditionally knockout one of the CGRP receptor complexes Calcrl The constructed spontaneous alopecia areata animal model can well simulate the progressive and active hair loss of alopecia areata patients, form stable hair loss patches, and combine with pathological changes such as hair follicle immune cell infiltration, which indicates that the dendritic cell CGRP signal loss is an important inducement for mediating systemic alopecia areata. The application also discloses a pharmaceutical composition or kit for preparing a drug for relieving / treating hair loss, improving hair follicle homeostasis, or enhancing hair growth / black coloration (reducing white hair), which comprises: (a) a JAK kinase inhibitor, and (b) a neuropeptide CGRP or a receptor agonist thereof.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine; more specifically, this invention relates to the application of hair follicle immune homeostasis regulation strategies in the treatment of hair loss diseases. Background Technology

[0002] Alopecia areata is a common chronic autoimmune skin disease with a global prevalence of approximately 2%. Its clinical manifestations include sudden, non-scarring, well-defined patches of hair loss that are persistent, prone to recurrence, and continuously progressive, affecting the entire scalp and even the entire body. Treatment response rates are low, seriously harming the physical and mental health of patients. The breakdown of follicular immune immunity mediated by genetic and environmental disturbances is a significant contributing factor to alopecia areata. Specifically, exposure and abnormal presentation of autoantigens derived from hair follicle epithelial cells during the anagen phase can lead to the activation of cytotoxic T cells, inducing follicular damage and premature regression, thereby triggering hair loss.

[0003] JAK-STAT is a key downstream pathway of IFN-γ and IL-15, important pathogenic factors in alopecia areata. JAK kinase inhibitor therapy, based on drugs such as baricitinib and litexitinib, is currently an important treatment for moderate to severe alopecia areata. While JAK kinase inhibitor use has brought hair regrowth and improved quality of life to some patients with moderate to severe alopecia areata, many patients still experience poor responses, or their disease worsens or even deteriorates during tapering or discontinuation of medication, suggesting that the collapse of the hair follicle immune immunity has not been effectively repaired. Furthermore, JAK kinase inhibitors have broad-spectrum immunosuppressive effects, and their administration can cause infections, hematological adverse reactions, increased risk of malignancy, and gastrointestinal adverse reactions. Therefore, given the unmet treatment needs of many patients, treatment strategies targeting the specific pathogenic mechanisms of alopecia areata urgently need to be developed.

[0004] Currently, mouse models used for research on the pathogenesis and treatment of alopecia areata include spontaneous models, transplantation models, and induced models. Spontaneous models include C3H / HeJ, DEBR, and C3H / HeJBir, but the success rate of spontaneous models is extremely low, <10% or even less than 1%, and the disease cycle is long. The success rate of C3H / HeJ mice is 0.25% for males and 0.035% for females, and female mice over 18 months of age have a chance to reach a 20% spontaneous rate. Transplantation models include: transplanting spontaneous alopecia areata lesions from C3H / HeJ mice into normal mice, or transplanting normal human scalp into immunodeficient mice and injecting in vitro cultured donor peripheral blood-derived cytotoxic cells. However, due to factors such as the low spontaneous rate of the C3H / HeJ strain and the difficulty in obtaining human samples, the internationally recognized model is currently the induction model: that is, inducing C3H / HeJ mice by intravenous injection of IFN-γ for more than 6 months. The success rate of reproduction is low. Other literature reports that IFN-γ combined with subcutaneous injection of poly(I:C) can achieve an incidence rate of 75% within 8 weeks. Overall, the time, labor and economic costs are high. At the same time, it bypasses the initial stage of alopecia areata and directly enters the stage of immune cell activation and killing. As an animal model of the disease, it has inherent limitations.

[0005] In summary, there is an urgent need in this field to explore and develop new animal models of alopecia areata, and to screen for more effective therapeutic drugs or treatment strategies in order to find more solutions for alopecia areata. Summary of the Invention

[0006] The purpose of this invention is to provide the application of targeted regulation of dendritic cell immune function in the treatment of hair loss diseases.

[0007] In a first aspect of the invention, there is provided the use of a mixture for preparing a pharmaceutical composition or kit for relieving / treating hair loss, improving hair follicle homeostasis, or enhancing hair growth / black pigmentation (reducing gray hair); said mixture comprising: (a) a JAK kinase inhibitor, and (b) a neuropeptide CGRP or a receptor agonist thereof.

[0008] In one or more embodiments, the hair loss includes alopecia areata or anagen baldness.

[0009] In one or more embodiments, the JAK kinase inhibitor comprises: a small molecule compound that specifically inhibits JAK kinase; an interfering molecule that specifically interferes with the expression of the JAK kinase gene; a gene editing agent that specifically knocks out the JAK kinase gene; or an antibody or ligand that specifically binds to the protein encoded by the JAK kinase gene; preferably, the JAK kinase inhibitor is a small molecule compound that specifically inhibits JAK kinase, such as Ruxolitinib (e.g., a cream).

[0010] In one or more embodiments, the neuropeptide CGRP receptor agonist comprises: a CGRP peptide or a modification thereof, a small molecule compound that specifically agonizes the CGRP receptor, an agent that upregulates / enhances the function of the CGRP receptor, an interfering molecule that specifically promotes the release of CGRP or the expression of its receptor gene, or a gene editing agent that specifically overexpresses the CGRP peptide or a modification thereof, or the CGRP receptor.

[0011] In another aspect of the invention, a pharmaceutical composition or kit is provided for relieving / treating hair loss or enhancing hair growth / blackening (reducing gray hair), the pharmaceutical composition or kit containing: (a) a JAK kinase inhibitor and (b) the neuropeptide CGRP or its receptor agonist; preferably, the pharmaceutical composition further includes a pharmaceutically or physiologically acceptable carrier or excipient.

[0012] In one or more embodiments, the mass-molar ratio of (a) to (b) is: 10–80 mg : 0.12–1 nmol; preferably 20–70 mg : 0.2–0.8 nmol; more preferably 30–60 mg : 0.3–0.7 nmol; even more preferably 35–45 mg : 0.4–0.6 nmol.

[0013] In one or more embodiments, according to the unit dose, (a) the amount is 10-80 mg, and (b) the amount is 0.12-1 nmol; preferably, (a) the amount is 20-70 mg, and (b) the amount is 0.2-0.8 nmol; more preferably, (a) the amount is 30-60 mg, and (b) the amount is 0.3-0.7 nmol; even more preferably, (a) the amount is 35-45 mg, and (b) the amount is 0.4-0.6 nmol.

[0014] In one or more embodiments, the medicine box further includes: instructions for use, describing methods for relieving / treating alopecia areata or enhancing hair growth / coloration.

[0015] In one or more embodiments, the " / " sign means "and" or "or".

[0016] In one or more embodiments, the JAK kinase inhibitor and the CGRP are placed separately in different containers (such as syringes) within the kit.

[0017] In another aspect of the invention, a pharmaceutical composition for relieving / treating alopecia areata or enhancing hair growth / pigmentation is provided, the pharmaceutical composition comprising a JAK kinase inhibitor and CGRP, and a pharmaceutically acceptable carrier.

[0018] In another aspect of the invention, a mixture for relieving / treating alopecia areata or enhancing hair growth / pigmentation is provided, the mixture comprising a JAK kinase inhibitor and CGRP.

[0019] In one or more embodiments, the dosage form of the pharmaceutical composition is: injection, infusion, tablet, capsule, pill; preferably an injection.

[0020] In another aspect of the present invention, a method for preparing a spontaneous alopecia areata animal model is provided, comprising: in a mixture of... Il4i1 Drivers of Cre recombinase expression Il4i1 Cre Knockout in animals Calcrl Genes were used to obtain animal models with spontaneous alopecia areata phenotype.

[0021] In one or more embodiments, conditional expression is performed using the Cre / loxP recombination system to achieve [the desired result]. Il4i1 Drivers of Cre recombinase expression Il4i1 Cre Knockout in animals Calcrl Gene.

[0022] In one or more embodiments, the method includes: (1) establishing a system composed of... Il4i1 Drivers of Cre recombinase expression Il4i1 Cre Animals; (2) Establishing a Cre / loxP-based recombination system Calcrl (2) Conditional knockout animals; (3) Cross the animals in (1) and (2) to obtain offspring that are animal models with spontaneous alopecia areata phenotype.

[0023] In one or more embodiments, the method includes: (1) providing an animal a, in which Il4i1 Positive( Il4i1 + ) cells ( Il4i1 + Introducing Cre recombinase expression elements into the genome of dendritic cells, which can interact with the genome... Il4i1 The Cre recombinase expression element contains a Cre recombinase-encoded nucleic acid; (2) includes: providing animal b, in which conditional knockout is introduced into its genome. Calcrl Gene elements; preferably, the elements target Calcrl The gene's exon 6 can be specifically knocked out; when Cre recombinase is expressed, it recognizes LoxP and undergoes cleavage (cleavage between two LoxPs), thus... Il4i1 Continuous knockout of positive cells and their progeny cells Calcrl Gene.

[0024] In one or more embodiments, (1) the Cre recombinase expression element further includes: a 2A peptide-encoded nucleic acid located upstream of the Cre recombinase-encoded nucleic acid, and a termination sequence located downstream of the Cre recombinase expression element located downstream of the Cre recombinase-encoded nucleic acid.

[0025] In one or more embodiments, the Cre recombinase expression element is introduced into the animal genome. Il4i1 The position of the stop codon (after exon 9 and before 3'UTR).

[0026] In one or more embodiments, the Cre recombinase expression element is introduced by providing homologous arms on both sides. Il4i1 In the genome of positive cells.

[0027] In one or more embodiments, the 2A peptide encodes a nucleic acid such that the Cre recombinase encodes a nucleic acid that is compatible with... Il4i1 Nucleic acid ligation ( Il4i1 -2A-Cre-termining sequences (such as polyA) are used for fusion expression.

[0028] In one or more embodiments, in (2), in the Loxp-terminator-Loxp, an resistance selection gene expression element is further provided upstream of the terminator.

[0029] In one or more embodiments, the animal is a rodent; preferably, the rodent includes mice and rats.

[0030] In one or more embodiments, the animals exhibit progressive hair loss and hair discoloration during their growth process (e.g., at 3–5 months of age), and subsequently (e.g., at 6–12 months of age or 7–11 months of age (e.g., 8, 9, 10 months of age)) develop patches of hair loss, exhibiting a typical alopecia areata phenotype.

[0031] In one or more embodiments, the skin of the animal exhibits regressive characteristics such as miniaturization, vacuolation, and depigmentation of hair follicles, with visible reticular dermal expansion and inflammatory cell infiltration around the hair follicles.

[0032] In another aspect of the invention, the application of the spontaneous alopecia areata animal model obtained by any of the methods described above is provided for: as an animal model for screening candidate drugs or therapeutic agents to alleviate or treat alopecia areata; or as an animal model for studying alopecia areata.

[0033] In one or more embodiments, the method or application is a non-diagnostic or non-therapeutic method or application, and is not intended for the diagnosis or treatment of a disease.

[0034] In another aspect of the invention, a method is provided for screening candidate drugs or therapeutic agents to alleviate or treat alopecia areata, the method comprising: (1) Prepare a spontaneous alopecia areata animal model using any of the methods described above; (2) The candidate substance is given to the animal model of (1) and the candidate substance is observed to have a relieving or therapeutic effect on alopecia areata. If the alopecia areata symptoms of the animal model are relieved (significantly relieved or statistically significant relieved), the candidate substance is a substance that relieves or treats alopecia areata.

[0035] In one or more embodiments, the candidate substances include (but are not limited to): small molecule compounds, biomacromolecules such as active genes, proteins, small regulatory molecules (such as interfering molecules (such as siRNA), editing reagents (such as gRNA), nucleic acid inhibitors, binding molecules (such as antibodies or ligands)), etc.

[0036] In one or more embodiments, the test may include setting up control animals that are not given the candidate substance.

[0037] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions, and these combinations are also included in this invention. Attached Figure Description

[0038] Figure 1 This image shows the gene expression, pathway enrichment, cell communication, and localization analysis of the pathological DC3 dendritic cell subsets in patients with alopecia areata.

[0039] A: Dimensionally reduced UMAP plots of myeloid cell subsets in scalp samples from healthy individuals (HS) and patients with alopecia areata during the acute phase (AP) / chronic phase (CP).

[0040] B: Stacked area diagram of the proportion of myeloid cell subpopulations in individual samples of each HS / AP / CP group.

[0041] C: Pie chart showing the combined expression levels of pro-inflammatory / chemotactic / antigen-presenting molecules in various myeloid cell subsets.

[0042] D: Heatmap of pathogenic signaling pathways in various myeloid cell subsets.

[0043] E: Cell communication analysis diagram of DC3 dendritic cell subset and pathogenic T cell subset.

[0044] F: DC3 dendritic cell subset (LAMP3) + CCR7 +Spatial transcriptome localization analysis diagram.

[0045] G: Representative image of immunofluorescence staining of the DC3 dendritic cell subset.

[0046] Figure 2 To investigate the expression of CGRP receptor in the pathological DC3 dendritic cell subsets of patients with alopecia areata and the regulatory effect of CGRP on the immune phenotype of dendritic cells.

[0047] A: Expression diagram of the CGRP receptor complex in the DC3 dendritic cell subset.

[0048] B: Spatial transcriptomic colocalization map of DC3 dendritic cell subsets and CGRP receptor complex.

[0049] C: Representative figure of immunofluorescence staining results of sensory nerve fibers (βIII tubulin, red), CGRP (green), and cell nuclei (DAPI, gray) in hair follicles of healthy donors (HD) and patients in the acute phase (AA_AP) / chronic phase (AA_CP).

[0050] D: Histogram and flow cytometry of the mean fluorescence intensity of CGRP on the immunophenotypic regulation of human monocyte-derived dendritic cells under LPS stimulation.

[0051] Figure 3 Transgenic construction and breeding strategies for spontaneous alopecia areata model mice.

[0052] A: LAMP3 + CCR7 + IL4I1 + Schematic diagram of gene expression patterns characteristic of the DC3 dendritic cell subset.

[0053] B: Il4i1 A strategy for constructing gene-driven transgenic lines of DC3 dendritic cell subset-specific Cre enzymes.

[0054] C: Transgenic construction strategy for spontaneous alopecia areata model mice.

[0055] D: Breeding strategy for spontaneous alopecia areata model mice.

[0056] Figure 4 To assess disease-related phenotypes in a mouse model of spontaneous alopecia areata.

[0057] A: A holistic view of skin lesions at different stages of the disease in a mouse model of spontaneous alopecia areata.

[0058] B: Representative H&E staining images and CD45 of skin lesions from 45-week-old spontaneous alopecia areata model mice and dorsal skin from littermate control mice. +Representative image of immunofluorescence staining. Unlike control mice where the skin is in the resting phase, with hair follicles withdrawing from the reticular dermis and returning to the papillary dermis, the skin lesions of the spontaneous alopecia areata model mice show regression phase characteristics such as follicle miniaturization, vacuolation, and depigmentation. Simultaneously, reticular dermal expansion and CD45 staining around the hair follicles are also observed. + Cell infiltration.

[0059] C: A representative figure illustrating the efficacy of the JAK kinase inhibitor Ruxolitinib in treating a mouse model of spontaneous alopecia areata.

[0060] Figure 5 Figure showing the synergistic effect of CGRP and its receptor antagonist Rimegepant with the JAK kinase inhibitor Ruxolitinib in inducing anagen hair growth and pigmentation during the regressive phase.

[0061] A: Overall experimental design diagram; B: A representative diagram of hair growth in mice of each group throughout the entire experimental period; C: Statistical chart of response rate during the entire germination period of the experiment, hair growth induction area of ​​each group at the experimental endpoint, and hair growth index; D: Representative H&E staining images of mouse skin in each group at the experimental endpoint; E: Statistical chart of the number of hair follicles per unit area in the growth phase for each group at the experimental endpoint. Detailed Implementation

[0062] This invention discloses the application of a hair follicle immune homeostasis regulation strategy targeting dendritic cells in the treatment of alopecia areata. Based on single-cell transcriptome sequencing of clinical samples, a group of pathological LAMP3 cells for alopecia areata was identified. + CCR7 + IL4I1 + The DC3 dendritic cell subset highly expresses antigen-presenting, chemotactic, and pro-inflammatory molecules and interacts closely with cytotoxic T cells. This DC3 dendritic cell subset specifically highly expresses the calcitonin-related neuropeptide CGRP (calcitonin gene-related peptide) receptor complex CALCRL / RAMP1, while CGRP signaling around hair follicles is significantly absent in patients with alopecia areata. Marker genes from the DC3 dendritic cell subset were used to... Il4i1 One of the driving forces for conditional knockout of the CGRP receptor complex Calcrl The constructed spontaneous alopecia areata animal model can well simulate the progressive and active hair loss of patients with alopecia areata, forming stable patches of hair loss combined with pathological changes such as infiltration of immune cells in hair follicles, suggesting that the lack of CGRP signal on dendritic cells is an important inducing factor for systemic alopecia areata.

[0063] Exogenous supplementation of the CGRP pathway can negatively regulate dendritic cell immune responses, improve hair follicle homeostasis, and enhance hair growth and pigmentation. This invention also discloses pharmaceutical compositions or kits for preparing relieving / treating hair loss, improving hair follicle homeostasis, or enhancing hair growth / pigmentation (reducing gray hair), comprising: (a) a JAK kinase inhibitor, and (b) the neuropeptide CGRP or a receptor agonist thereof.

[0064] Drug combination

[0065] Based on the new findings of this invention, a method is provided to regulate the immune effect of dendritic cells and enhance hair follicle growth and pigmentation.

[0066] As used in this invention, the term "JAK kinase inhibitor" includes inhibitors of JAK kinase activity or function, as well as nucleic acid inhibitors, antagonists, suppressors, blockers, and blocking agents of JAK kinase, as long as they can downregulate the expression level of JAK kinase or inhibit its activity or function. They can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0067] The JAK kinase inhibitors described herein can be various substances that can reduce the activity and stability of JAK kinase, downregulate its expression, or shorten its effective duration. These substances can all be used in this invention as useful for downregulating JAK kinase. For example, the JAK kinase inhibitors can be: nucleic acid inhibitors, protein inhibitors, antibodies, ligands, compounds, nucleases, nucleic acid binding molecules, etc., provided that they can downregulate JAK kinase expression and inhibit its activity or function. The nucleic acid inhibitors include: shRNAs that target the encoding gene of JAK kinase or its transcripts for inhibition or silencing, antisense nucleic acids, small interfering RNAs, microRNAs, or constructs that can express or form said shRNAs, antisense nucleic acids, small interfering RNAs, or microRNAs.

[0068] As a preferred embodiment of the present invention, the JAK kinase inhibitor is a small molecule compound that specifically inhibits JAK kinase, including Ruxolitinib or its analogues. This small molecule compound is particularly effective when used in combination with a neuropeptide CGRP receptor agonist (such as CGRP).

[0069] As another optional embodiment of the present invention, the JAK kinase inhibitor is an interfering molecule that specifically interferes with the expression of the JAK kinase gene. RNA interference is a technique for silencing gene expression. The principle of RNA interference is that long double-stranded RNA is cleaved by the specific nuclease Dicer into small interfering RNAs of 21-23 nt, composed of sense and antisense strands. The small interfering RNAs then form a silencing complex (RISC) that unwinds into single strands. The antisense strand guides the silencing complex to specifically bind to the target mRNA through base pairing, causing the mRNA to degrade. Short hairpin RNA (shRNA) is an RNA sequence that forms a sharp bend and can silence genes via RNA interference. The interfering molecule that specifically interferes with the expression of the JAK kinase gene can be either an shRNA molecule or a siRNA molecule targeting JAK kinase.

[0070] As another optional embodiment of the present invention, the JAK kinase inhibitor is an inhibitor targeting mutations, gene editing, or gene recombination of the JAK kinase gene. As a more specific embodiment, the JAK kinase is converted into a mutant using any of the above methods, thereby rendering it inactive. As a more specific embodiment, gene editing is performed using a CRISPR / Cas system. Suitable sgRNA target sites result in higher gene editing efficiency; therefore, suitable target sites can be designed and identified before proceeding with gene editing.

[0071] As used in this invention, the "neuropeptide CGRP receptor agonist" includes any substance that can activate / activate the neuropeptide CGRP receptor, including agonists, expression upregulators, activity promoters, etc.

[0072] As a preferred embodiment of the present invention, the neuropeptide CGRP receptor agonist may be a CGRP peptide or its modification, a small molecule compound that specifically agonizes the CGRP receptor, an agent that upregulates / enhances the function of the CGRP receptor, an interfering molecule that specifically promotes the release of CGRP or the expression of its receptor gene, or a gene editing agent that specifically overexpresses the CGRP peptide or its modification, or the CGRP receptor.

[0073] This invention provides a method of combined drug administration, including a method of administration using an inhibitor targeting JAK kinase in combination with a neuropeptide CGRP receptor agonist.

[0074] The present invention also provides the use of JAK kinase inhibitors and neuropeptide CGRP receptor agonists for the preparation of mixtures, pharmaceutical compositions or kits for relieving / treating hair loss or enhancing hair growth / blackening (reducing gray hair).

[0075] During administration, JAK kinase expression or activity can be downregulated first using a JAK kinase inhibitor, followed by inhibition with a neuropeptide CGRP receptor agonist; alternatively, both methods can be used simultaneously. It should be understood that multiple administration methods are included in this invention.

[0076] The present invention also provides a pharmaceutical composition comprising: (a) an effective amount of a JAK kinase inhibitor; (b) an effective amount of a neuropeptide CGRP receptor agonist; and (c) a pharmaceutically acceptable carrier or excipient.

[0077] In this invention, the term "containing" indicates that various ingredients may be used together in the mixtures or compositions of this invention. Therefore, the terms "consistent with..." and "composed of..." are included in the term "containing".

[0078] In this invention, a "pharmaceuticalally acceptable" ingredient is a substance that is suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., has a reasonable benefit / risk ratio.

[0079] In this invention, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent, or excipient used to deliver the active ingredient of this invention to animals or humans. A "pharmaceutically acceptable carrier" can be a liquid or a solid.

[0080] The pharmaceutical compositions or mixtures of the present invention can be formulated into any conventional dosage form using conventional methods. Dosage forms can be diverse, as long as they enable the active ingredient to effectively reach the mammalian body.

[0081] The effective doses of the JAK kinase inhibitors and neuropeptide CGRP receptor agonists used may vary depending on the dosing regimen and the severity of the disease being treated. When necessary, the JAK kinase inhibitors and neuropeptide CGRP receptor agonists may also be administered in combination with other active ingredients or drugs.

[0082] The present invention also provides a kit for relieving / treating hair loss or enhancing hair growth / blackening (reducing gray hair), the kit containing: a container 1, and a JAK kinase inhibitor placed in the container 1; and a container 2, and a neuropeptide CGRP receptor agonist placed in the container 2. Furthermore, the kit may also contain some adjuvant medication materials.

[0083] In addition, the medicine box may also contain instructions for use, explaining how to use the combined medication method of the present invention to relieve / treat hair loss or enhance hair growth / blackening (reduce gray hair).

[0084] This invention reveals for the first time that the combined application of JAK kinase inhibitors and neuropeptide CGRP receptor agonists has excellent therapeutic effects, and the two have a synergistic effect.

[0085] Animal models and their applications

[0086] As used in this invention, "animal" refers to a mammal; preferably, "animal" includes rodents, such as mice and rats.

[0087] LAMP3 + CCR7 + IL4I1 + Dendritic cells specifically aggregate in the hair follicles of patients with alopecia areata, recruiting and activating cytotoxic T cells by expressing chemokines, pro-inflammatory factors, and antigen-presenting molecules. Based on this, the present invention provides... Il4i1 + A dendritic cell-mediated spontaneous alopecia areata mouse model effectively mimics the characteristics of systemic alopecia areata, including spontaneous and progressive hair loss mediated by abnormal immune activation during the hair growth phase, resulting in chronic patchy alopecia lesions and abnormal activation of the peripheral immune system. This dendritic cell subset specifically highly expresses the calcitonin-related neuropeptide CGRP receptor complex CALCRL / RAMP1, while CGRP signaling around hair follicles is significantly absent in alopecia areata patients. Exogenous CGRP supplementation negatively regulates the dendritic cell immune response, improves hair follicle nutrition and homeostasis, and enhances hair growth and pigmentation during the anagen phase. This invention has significant scientific and clinical translational value for studying the mechanisms of systemic alopecia areata mediated by abnormal neuro-immune interactions and for developing novel strategies for treating alopecia diseases based on the regulation of hair follicle immune homeostasis.

[0088] Based on the novel findings of this invention, firstly, this invention identifies a group of LAMP3 molecules that play a key pathogenic role in the pathological microenvironment of alopecia areata. + CCR7 + IL4I1 + Dendritic cell subset. Furthermore, this dendritic cell subset is located in the diseased hair follicle region, and characteristically expresses IL4I1 / LAMP3 / CCR7. It plays an important regulatory role in the chemotaxis, activation, and killing function of cytotoxic T cells in the disease microenvironment, and also highly expresses the neuropeptide CGRP receptor CALCRL / RAMP1.

[0089] Secondly, this invention provides a strategy for constructing a spontaneous alopecia areata animal model. Furthermore, this spontaneous alopecia areata mouse model simulates the alopecia areata microenvironment IL4I1. +Conditional loss of the neuropeptide CGRP receptor in dendritic cell subsets can well simulate the skin lesions such as active hair loss and stable hair loss patches in the progressive stage of alopecia areata patients, suggesting that abnormal neuroimmune interaction mediates the occurrence and development of systemic alopecia areata, filling the gap in the current animal models of alopecia areata mediated by neuropathological changes.

[0090] In a preferred embodiment of the present invention, a method for constructing a spontaneous alopecia areata mouse model is provided: expressing a dendritic cell subset-specific Cre recombinase... Il4i1 Cre [Tg / Tg] strains and CGRP receptor conditional knockout strains Calcrl [flox / flox] homozygous genotype parents were crossed to obtain Il4i1 Cre [Tg / +]: Calcrl [flox / +] heterozygous genotype F1 generation; then the F1 generation is self-crossed to obtain genotypes of [flox / +]. Il4i1 Cre [Tg / +]: Calcrl [flox / flox] or Il4i1 Cre [Tg / Tg]: Calcrl A spontaneous alopecia areata mouse model of [flox / flox]. In a specific embodiment of the present invention, C57BL / 6J mice were used for construction.

[0091] In a preferred embodiment of the present invention, the spontaneous alopecia areata model mice constructed by the present invention are housed in a specific pathogen-free (SPF) barrier facility with a light-to-dark ratio of 12 hours:12 hours, a temperature of 22±1℃, and a humidity of 40-70%. They are weaned after 4 weeks of age and exhibit varying degrees of progressive hair loss and hair depigmentation around 4 months of age. By around 10 months of age, irreversible alopecia patches form. Pathological sections show the presence of telogen / regression phase hair follicles in the dermis characterized by miniaturization, vacuolation, and depigmentation, with significant lymphocyte infiltration around the hair follicles. Compared to the C3H / HeJ mouse subcutaneous IFN-γ injection model, the C3H / HeJ mouse lesion flap transplantation model, and humanized animal models, this model simulates spontaneous and progressive alopecia areata, does not rely on exogenous cytokine injection or surgical transplantation, and has high similarity to clinical alopecia areata patients; it is an ideal mouse model for spontaneous alopecia areata.

[0092] The animal models constructed in this invention can be used for screening and testing specific drugs. In drug screening, candidate drugs or therapeutics refer to substances known to have certain pharmacological activities or substances currently being tested that may have certain pharmacological activities, including but not limited to nucleic acids, proteins, carbohydrates, chemically synthesized small or large molecular compounds, and cells. The administration routes for candidate drugs or therapeutics can be oral, intravenous, intraperitoneal, subcutaneous, spinal, or direct intracerebral injection.

[0093] The animal model constructed in this invention can serve as a powerful tool for scientific research and new drug evaluation.

[0094] As those skilled in the art will understand, due to the complexity of the organism's genes, the influence of multiple signaling pathways on diseases, and the existence of the organism's own compensatory or repair mechanisms, it is difficult to obtain animal models that exhibit typical disease symptoms. This invention, through optimized design, overcomes these technical challenges.

[0095] In the study of disease mechanisms, the animal model of alopecia areata stably constructed using this invention can be used to investigate the disease mechanism, explore the key factors leading to alopecia areata from common suppression, and explore the intermediate mechanisms that prevent or delay the development of this disease. The model system of this invention helps to better understand alopecia areata and to explore / identify candidate drugs / therapeutic agents that can prevent, delay, or reverse the disease process.

[0096] In preclinical drug testing, the animal model constructed using this invention, which stably exhibits alopecia areata, is expected to be used for preclinical drug metabolism, toxicity, and efficacy testing. This animal model is physiologically close to the human body and supports long-term sampling, detection, and tracking, facilitating the advancement of new drug development. In this invention, there are no particular limitations on the types of candidate drugs used for drug testing; they can be obtained from various sources, including synthetic or natural compound libraries. For example, there are various methods for the random and directed synthesis of a variety of organic compounds and biomolecules, including the expression of random oligonucleotides and oligopeptides; or, natural compound libraries in the form of bacterial, fungal, plant, and animal extracts can be obtained or readily generated. Furthermore, libraries and compounds generated by natural or synthetic methods can be readily modified by conventional chemical, physical, and biochemical methods and can be used to generate combinatorial libraries. Known pharmacological reagents can be chemically modified (e.g., acylation, alkylation, esterification, amidation, etc.) to generate structural analogs.

[0097] The method for preparing animal models in this invention is simple and controllable, and the resulting animal models are stable and can well simulate the human alopecia areata phenotype. The resulting disease symptoms are very typical, and phenotypic changes are easy to observe. The animal models of this invention provide new ideas for the study of the pathogenesis of alopecia areata, drug screening, and clinical treatment.

[0098] Based on the technical solution disclosed in this invention, this invention also provides a kit for preparing an animal model of alopecia areata, the kit comprising: a Cre recombinase expression element, including a Cre recombinase encoding nucleic acid, conditionally knocked out. Calcrl Gene elements; preferably, conditional knockout elements target Calcrl The exon 6 of the gene can be specifically knocked out.

[0099] The kit may also include an instruction manual describing the method for preparing the animal model of the present invention, so as to facilitate application by those skilled in the art.

[0100] After obtaining the animal model of the present invention, substances of interest can be screened based on this model, which can (or potentially can) alleviate or treat alopecia areata. Following screening, truly useful drugs can be identified from the substances of interest.

[0101] Therefore, the present invention also provides a method for screening potential substances, the method comprising: (1) preparing an animal model of alopecia areata using the method or kit described above; (2) administering a candidate substance to the animal model of (1) and observing whether the candidate substance has an alleviating or therapeutic effect on alopecia areata; if the alopecia areata symptoms of the animal model are observed to be relieved, then the candidate substance is a substance for alleviating or treating alopecia areata.

[0102] In a preferred embodiment of the present invention, during screening, a control group may be set up to make it easier to observe changes in the symptoms of alopecia areata. The control group may be an animal model that does not contain the candidate substance.

[0103] As a preferred embodiment of the invention, the method further includes: conducting further cell experiments and / or animal experiments and / or clinical trials on the obtained potential substances to further select and identify truly useful substances.

[0104] On the other hand, the present invention also provides potential substances of interest obtained using the aforementioned screening method. These initially screened substances can form a screening library, from which relatively ideal, safe substances that can ultimately be screened out can be identified as having a real effect on alleviating or treating alopecia areata.

[0105] This invention provides important experimental evidence for the study of the mechanism of systemic alopecia areata mediated by abnormal neuro-immune interactions, fills the current gap in animal models of alopecia areata mediated by neuropathological changes, and has important scientific and clinical translational significance for developing new strategies for treating alopecia areata based on regulatory neuropeptides.

[0106] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0107] Example 1: Transcriptional characteristics and localization of pathogenic DC3 dendritic cell subsets in the scalp of patients with alopecia areata

[0108] 1. Acquisition and single-cell transcriptome sequencing of scalp samples from healthy donors and patients with alopecia areata.

[0109] After obtaining informed consent from the donor, a dermatologist will obtain healthy scalp and scalp samples from the acute / chronic phases of alopecia areata via biopsy. Inclusion criteria: ① Age group of 20 to 30 years old; ② Patients with alopecia areata whose SALT level is ≥85 and who have not used systemic medication within the past 2 months.

[0110] Scalp samples were stored in physiological saline at 4°C. The dermis and epidermis were separated using Dispase II, and single-cell suspensions of individual samples were prepared by digesting the dermis with a collagenase P / DNase I mixture and the epidermis with 0.05% trypsin. Dead cells were stained with DAPI, and non-adhesive single viable cells were sorted by flow cytometry and reselected into PBS containing 0.04% BSA.

[0111] 10 x Genomics single-cell transcriptome sequencing libraries were prepared by Shanghai Xuran Biotechnology Co., Ltd. according to the manufacturer's instructions, and sequencing was performed using an Illumina HiSeq 4000. Post-assembly data were processed using CellRanger, and further filtering, processing, and analysis were performed using R and the Seurat package. In the initial processing, cells with fewer than 200 but more than 5000 genes, mitochondrial content exceeding 5%, and cells of mixed origin (doublets) were removed.

[0112] 2. Dimensionality reduction, quantification, gene expression, signaling pathways, and cell communication analysis of scalp myeloid cells from healthy donors and alopecia areata patients.

[0113] (1) Analysis of the total dataset revealed that myeloid cells are the main source of expression for the pathogenic factor IL-15. Myeloid cell subpopulations were extracted from the total transcriptome dataset of single cells from the scalp of healthy donors / alopecia areata patients using LYZ gene expression. The top 2000 most variable genes were selected for principal component analysis (PCA), with a PCA ratio of 1:30 used for dimensionality reduction clustering, and a resolution of 0.5. The analysis results showed that myeloid-derived cells were divided into 10 main subpopulations, such as... Figure 1 As shown in Figure A.

[0114] (2) The proportion of each myeloid cell subpopulation in individual samples of each group was analyzed and stacked area plotted using the ggplot function. The results showed that the DC3 subpopulation is a dendritic cell subpopulation specific to alopecia areata, with an increased proportion of lesions in the acute phase (AP) and still present in the chronic phase (CP), such as... Figure 1 As shown in B.

[0115] (3) Quantitative analysis of the expression of genes related to cytokines, chemokines, and antigen-presenting molecules in myeloid cells was performed using the FlexDotPlot function. The results showed that the DC3 subset exhibited a significant advantage in both the proportion and level of various pro-inflammatory factors, such as... Figure 1 As shown in C.

[0116] (4) Enrichment analysis and heatmap creation of pro-inflammatory signaling pathways in major myeloid cell subsets were performed using the GSVA package. The DC3 subset was highly enriched for disease-related pro-inflammatory pathways such as antigen presentation, T cell chemotaxis, and cytotoxic effector function. Figure 1 As shown in D.

[0117] (5) The communication network between DC3 and various effector T cells was analyzed using CellChat. The DC3 subset plays a dominant role in communication within the alopecia areata microenvironment, extensively interacting with cytotoxic effector T cells such as CCL / CXCL / TNF / LGALS9, for example... Figure 1 As shown in E.

[0118] 3. Spatial transcriptomic localization and immunofluorescence validation of DC3 dendritic cell subsets in scalp samples from patients with alopecia areata.

[0119] (1) Spatial Transcriptome Localization

[0120] After obtaining informed consent from the donor, fresh skin biopsy tissue was obtained from alopecia areata patients via biopsy by a dermatologist and embedded in an OCT complex for quick freezing on dry ice. 10 μm frozen sections of skin were prepared using a cryostat and placed on 10xGenomics Visium slides. Bright-field images were obtained after hematoxylin and eosin (H&E) staining. Optimized permeability and tissue dissection were performed, and barcode-tagged cDNA was collected via reverse transcription followed by enzymatic digestion. The cDNA library was then sequenced on an Illumina NovaSeq 6000 platform. Data were processed using 10x Genomics SpaceRanger, mapped to the GRCh38v93 genome, and visualized using the 10x Genomics Loupe Browser 8 software package.

[0121] The results showed that CCR7 + LAMP3+ DC3 dendritic cell subsets, characterized by the gene, are concentrated in the hair follicle region of the skin lesion, such as... Figure 1 As shown in F.

[0122] (2) Immunofluorescence verification

[0123] 10 μm frozen sections of alopecia areata lesions were fixed with acetone pre-chilled at -20°C for 5 minutes, washed with PBS, blocked with PBS containing 5% BSA at room temperature for 1 hour, incubated overnight at 4°C with anti-LAMP3 (Cell Signaling Technology, #47778, dilution 1:500) monoclonal antibody, washed with PBS, incubated with fluorescein-labeled secondary antibody (Thermo Fisher Scientific, dilution 1:500) at room temperature in the dark for 1 hour, washed with PBS, stained with DAPI at room temperature for 5 minutes to show cell nuclear morphology, washed with PBS, mounted with anti-fluorescence quencher (Beyotime, #P0126) and photographed under a confocal microscope (Leica, Stellaris 8 Diva).

[0124] The results showed that, consistent with the spatial transcriptome, LAMP3 + DC3 is located at the hair follicles of alopecia areata lesions, such as Figure 1 As shown in G.

[0125] Example 2: Negative Regulation of Immune Effector Function of DC3 Dendritic Cell Subpopulation by CGRP Signaling Pathway

[0126] 1. Colocation Analysis

[0127] Analysis using the Plot_Density_Joint_Only function in the scCustomize package revealed that the CGRP receptor complex CALCRL / RAMP1 is related to LAMP3. + Colocalization exists in the DC3 subgroup.

[0128] Depend on Figure 2 As can be seen, all three are co-expressed in the DC3 subset, suggesting that the DC3 subset is regulated by the neuropeptide CGRP.

[0129] 2. Lesion localization analysis

[0130] Visualize CCR7 using 10x Genomics Loupe Browser 8 + LAMP3 + The spatial colocalization (green) of the DC3 dendritic cell subset (yellow) and the CGRP receptor complex CALCRL / RAMP1 (blue) in alopecia areata lesions.

[0131] Depend on Figure 2 B shows that CCR7 expresses the CGRP receptor complex. + LAMP3 + DC3 dendritic cell subsets aggregate in diseased hair follicles.

[0132] 3. Evaluation of the expression of nerves and neuropeptide CGRP around hair follicles in healthy donor hair follicles (HD) and in the acute phase (AA_AP) / chronic phase (AA_CP) of alopecia areata patients using tissue transparency imaging.

[0133] (1) Obtaining hair follicles from healthy donors and patients with alopecia areata

[0134] Informed consent was obtained from both healthy donors and patients with alopecia areata before sample acquisition. Hair follicles from healthy donors were obtained from scalp samples obtained through routine surgery, while scalp samples from patients with alopecia areata were obtained from scalp biopsies. Individual hair follicles were separated by dermatologists using the FUT (Follicular Unit Transplantation) technique.

[0135] (2) Full follicle embedding and transparent staining

[0136] Hair follicle samples were fixed overnight at room temperature with 4% paraformaldehyde solution. After washing with PBS at room temperature, the samples were permeabilized for 1 day on a shaker in 2% PBST solution (2% Triton X-100, 0.05% sodium azide in PBS). After washing with PBS, the samples were incubated at 4°C for 2 days in freshly prepared blocking buffer (10% ordinary goat serum, 1% Triton-X 100, 2.5% DMSO, 0.2% sodium azide in PBS). The samples were then incubated at 4°C for 3 days in antibody dilution buffer (containing 1% ordinary goat serum, 0.2% Triton-X 100, 2.5% DMSO, 0.2% sodium azide in PBS) containing mouse anti-human CGRP (abcam, ab18207, dilution ratio 1:100) and rabbit anti-human βIII tubulin (abcam, ab81887, dilution ratio 1:100). Finally, the samples were incubated in 1x Wash Buffer (containing 3% NaCl, 0.2% Triton-X) for 3 days. Wash overnight at 4°C with 100% PBS; incubate for 2 days at 4°C with antibody dilution buffer containing Alexa Fluor™ 488 conjugated with goat anti-rabbit fluorescent secondary antibody (Invitrogen, A-11008, dilution 1:500) and Alexa Fluor™ 555 conjugated with goat anti-mouse fluorescent secondary antibody (Invitrogen, A-21422, dilution 1:500); wash overnight at 4°C with 1x Wash Buffer; stain cell nuclei with instant DAPI solution and incubate at room temperature for 1.5 hours; after washing with PBS, transfer the sample to 5 times its volume of RapiClear® clearing agent and incubate at room temperature in the dark for 2–3 days; transfer the cleared sample to an iSpacer® microchamber and embed the sample in freshly preheated RapiClear® solution at 37°C; photograph the sample using a Leica® Stellaris 8 confocal microscope and process the images using Bitplane Imaris.

[0137] Depend on Figure 2 As can be seen in C, the interfollicular epithelium, follicular bulge, and follicular base of HD hair follicles all have abundant peripheral nerve innervation (βIII tubulin, red) and CGRP expression (green); in the acute lesion area, there are still residual hair shafts, but the nerve innervation and CGRP expression in each area are significantly reduced; in the chronic lesion area, there is further reduction in nerve innervation, significant loss of CGRP signal, and the hair follicle miniaturization feature.

[0138] 4. Evaluate the regulatory effect of CGRP on dendritic cell immunophenotype using human monocyte-derived dendritic cells.

[0139] (1) Obtaining dendritic cells derived from peripheral blood mononuclear cells

[0140] Peripheral blood samples were collected from three healthy individuals from the Shanghai First People's Hospital Health Examination Center and from age-matched healthy volunteers in the experimental group. After venous collection, the blood was deposited into anticoagulant tubes containing 3.2% sodium citrate. Mononuclear cells from PBMCs were enriched with CD16 magnetic beads and seeded in six-well plates (2.5 x 10⁻⁶ mcg / m²). 6 Dendritic cells derived from peripheral blood mononuclear cells were cultured in RPMI 1640 (containing 2 mM L-glutamine, 50 mM 2-ME, 10 mM HEPES, penicillin-streptomycin antibiotics, and 1% human heparinized plasma) at 100 ng / mL on day 0 of culture. On days 2, 4, and 6, 0.3 mL of medium was removed and 0.5 mL of fresh medium containing cytokines was added to replenish the medium. Peripheral blood mononuclear cells were obtained on day 7 of culture.

[0141] (2) Regulation of the immunophenotype of peripheral blood mononuclear cell-derived dendritic cells by CGRP

[0142] To avoid differences in differentiation and stimulus response due to individual variations, dendritic cells from a single sample were divided into two groups, with three replicates in each group. The control group cells were cultured in complete medium containing only LPS (0.5 μg / ml), while the CGRP-treated group cells were cultured in medium containing recombinant CGRP (10 μg / ml). -7 M) and LPS (0.5 μg / ml) complete culture medium were incubated in a 37°C incubator with 5% CO2 for 4 hours. The expression of co-stimulatory molecule CD86, antigen-presenting molecule HLA-DR, chemokine CCR7 and co-inhibitory molecule PD-L1 on the surface of dendritic cells in different groups was identified by flow cytometry, and statistical analysis was performed using paired t-tests.

[0143] Depend on Figure 2 As can be seen from D, the neuropeptide CGRP can significantly downregulate the expression of dendritic cell costimulatory molecule CD86 and antigen-presenting molecule HLA-DR, mildly downregulate chemokine CCR7, and have little effect on co-inhibitory molecule PD-L1.

[0144] Example 3: Construction and breeding of a spontaneous alopecia areata mouse model based on DC3 hyperfunction.

[0145] 1. Selection of DC3 marker genes

[0146] The expression characteristics of the DC3 characteristic genes IL4I1 / LAMP3 / CCR7 are displayed using the Plot_Density_Custom function in the scCustomize package.

[0147] like Figure 3 As shown in Figure A. LAMP3It is not expressed in the mouse DC3 subset. CCR7 It can be simultaneously expressed on naïve T cells, and selected IL4I1 As a characteristic driver gene of the DC3 subgroup.

[0148] 2. Construction and breeding of a spontaneous alopecia areata mouse model

[0149] In this embodiment, the C57BL / 6J mouse model was established. Figure 3 As shown in BC, the construction method includes the following steps: (1) Establish by Il4i1 Drivers of Cre recombinase expression Il4i1 Cre [Tg / Tg] strain A homologous recombination vector was constructed, which includes a 5' homologous arm (5'am) (SEQ ID NO: 1), a 2A-Cre-WPRE-polyA (SEQ ID NO: 2), and a 3' homologous arm (SEQ ID NO: 3).

[0150] 5' Homologous arm (SEQ ID NO: 1):

[0151] 2A-Cre-WPRE-polyA (SEQ ID NO: 2):

[0152] 3' Homologous arm (SEQ ID NO: 3):

[0153] Using CRISPR / Cas9 technology, homologous recombination is employed to... Il4i1 The gene stop codon site was introduced into the 2A-Cre-WPRE-polyA expression cassette, located after exon 9 and before the 3'UTR. Cas9 mRNA, sgRNA, and the homologous recombination vector were microinjected into C57BL / 6J zygotes to obtain F0 generation mice. Positive F0 generation mice were then mated with C57BL / 6J mice to obtain the F1 generation. Il4i1 -Cre-positive mice, that is, mice formed by... Il4i1 Drivers of Cre recombinase expression Il4i1 Cre [Tg / Tg] strain.

[0154] (2) Calcrl Conditional knockout mice

[0155] Calcrl Conditional knockout mice: C57BL / 6JCya- Calcrl em1flox / Cya; Product Number: S-CKO-11816 (Cyagen Group website: https: / / www.cyagen.cn / mice-bank / S-CKO-11816).

[0156] Will be IL4I1 Drivers of Cre recombinase expression IL4I1 Cre [Tg / Tg] strains and Calcrl The spontaneous alopecia areata mouse strain was obtained by crossbreeding conditional knockout mice.

[0157] The strategy for preparing spontaneous alopecia areata mouse strains is as follows: Figure 3 C, IL4I1 Cre [Tg / Tg] strains and Calcrl [flox / flox] homozygous genotype parents were crossed to obtain IL4I1 Cre [Tg / +]: Calcrl [flox / +] heterozygous genotype F1 generation. Then, the F1 generation is self-crossed to obtain the genotype [flox / +]. IL4I1 Cre [Tg / +]: Calcrl [flox / flox] or IL4I1 Cre [Tg / Tg]: Calcrl A mouse model of spontaneous alopecia areata with [flox / flox], such as Figure 3 D.

[0158] Example 3: Disease phenotype assessment of a spontaneous alopecia areata mouse model

[0159] In Example 2, a spontaneous alopecia areata mouse model was established. In this example, phenotypic evaluation was performed.

[0160] (1) Observation of the feeding process

[0161] The spontaneous alopecia areata model mice were housed in a specific pathogen-free (SPF) barrier facility with a light-to-dark ratio of 12 hours:12 hours, a temperature of 22±1℃, and a humidity of 40-70%. They were weaned at 4 weeks of age and continuously observed.

[0162] Observations show that spontaneous alopecia areata mice exhibit progressive hair loss and discoloration around 4 months of age, and by around 10 months of age, they form non-reversible patches of hair loss, exhibiting typical alopecia areata phenotypes, such as... Figure 4 As shown in Figure A.

[0163] (2) Skin analysis after euthanasia

[0164] After euthanasia at the observation endpoint, skin lesions and H&E pathology and CD45 levels were collected from the skin of spontaneous alopecia areata model mice and littermate control mice. + Evaluation of immunofluorescence staining results.

[0165] Analysis showed that, unlike the telogen effluvium pattern in control mice, the spontaneous alopecia areata model mice exhibited regression characteristics such as follicle miniaturization, vacuolation, and depigmentation. Simultaneously, reticular dermal dilatation and inflammatory cell infiltration (CD45, red) around the hair follicles were observed. Figure 4 As shown in B.

[0166] (3) Treatment of spontaneous alopecia areata mouse model

[0167] Two 4-month-old spontaneous alopecia areata model mice were selected and treated with JAK kinase inhibitors for 2 weeks in stable skin lesion areas. Specifically, commercially available Ruxolitinib cream (Opzelura®) was applied topically to the affected area once daily. After two weeks of treatment, the treatment was stopped and observed for one week, with photos taken weekly.

[0168] Observations showed that the JAK kinase inhibitor ruxolitinib exhibited a certain therapeutic effect on promoting hair growth in two mice with spontaneous alopecia areata during the treatment period. Figure 4 As shown in C.

[0169] Therefore, this mouse model exhibits typical symptoms of refractory alopecia areata, and its sensitivity to alopecia areata treatment drugs is evident in experiments. This mouse model can be used for screening treatment drugs related to refractory alopecia areata.

[0170] Example 4: CGRP maintains hair follicle homeostasis and synergistically enhances hair growth and pigmentation with JAK kinase inhibitors.

[0171] 1. The effect of CGRP or its receptor antagonist Rimegepant in synergy with the JAK kinase inhibitor Ruxolitinib on hair growth.

[0172] For each experiment, 30 eight-week-old male C57BL / 6J rats in the synchronized follicular resting phase were selected. After removing the hair from their backs, they were randomly divided into 6 groups. The specific treatments for each group are as follows: Figure 5 As shown in A, photos were taken and recorded daily, as shown in Table 1.

[0173] Table 1

[0174] 2. Evaluation of the effects of CGRP and its receptor antagonist Rimegepant on hair follicles inducing anagen phase.

[0175] Figure 5B is a representative image of mice in each experimental period, from... Figure 5 As shown in Figure B, both the UT and PBS groups maintained the resting phase throughout the experimental period; CGRP alone induced hair growth at rarely used injection sites; JAK kinase inhibitor (JAKi) cream effectively induced telogen effluvium in anagen follicles, but with a delayed effect. The combination of JAKi and CGRP was significantly superior to the solvent control group JAKi and PBS, exhibiting a faster induction of the anagen phase response and a larger germination area at the experimental endpoint. Furthermore, the germination effect extended to the back skin on the non-JAKi-treated side; the CGRP receptor antagonist Rimegepant inhibited the JAKi-induced telogen phase, manifested as a decrease in the overall response rate and a reduction in the telogen area at the experimental endpoint.

[0176] Melanin deposition in the back skin occurs concurrently with the progression of the anagen phase, and therefore can be used to assess the anagen follicle induction index. Image J was used to invert the images taken in each group during the experimental period and perform grayscale value statistics. The anagen follicle induction index = grayscale value of the treated side / grayscale value of the untreated side. A ratio ≥1.5 was considered as entering the anagen phase. Figure 5 C statistically analyzed the follicle induction response rate during the entire experimental cycle of the anagen phase, the percentage of the anagen phase area on the treated side at the experimental endpoint, and the follicle induction index on the treated side at the endpoint of the second experiment for each group in the two repeated experiments. Figure 5 As can be seen from C, the overall success rate of JAKi in inducing telogen effluvium hairs to enter the anagen phase in two repeated experiments was approximately 50%. The JAKi+CGRP combination group showed a significantly improved response speed and response rate compared to other groups in inducing telogen effluvium. Figure 5 C, above), and only the JAKi+CGRP combination group showed a significant difference in the percentage of growing area on the treatment side at the experimental endpoint compared to the UT / PBS / CGRP group ( Figure 5 C, Middle). Furthermore, the JAKi+CGRP combination group showed a significant difference in the hair follicle induction index during the anagen phase compared to the UT / PBS / CGRP group at the experimental endpoint. Figure 5 C, below), the combined effect is better than the JAKi+PBS solvent control group.

[0177] Figure 5 D represents the H&E staining results of skin samples taken from the treated side after euthanasia of mice, representing the experimental endpoint. Figure 5 E represents the number of hair follicles in the anagen phase per unit area in the H&E staining results image. Telogen effluvium follicles are generally located in the papillary dermis, while anagen follicles extend downwards into the reticular dermis. Figure 5As shown in Figure D, hair follicles in the UT / PBS group exhibited characteristics of the regression phase; the positive effect of CGRP alone was limited; hair follicles in the anagen phase of the JAKi+PBS and JAKi+RIME groups showed morphological fragmentation and increased inflammatory cell infiltration; while the combination of JAKi+CGRP effectively protected the integrity of the hair follicle structure without significant inflammatory cell infiltration, indicating that CGRP plays an important role in maintaining hair follicle homeostasis and the immunosuppressive environment during the anagen phase.

[0178] In summary, JAKi+CGRP has a synergistic effect, and when used together, it has the characteristics of rapid response during the hair growth phase, high response rate, large hair growth area, and good hair follicle stability, which is significantly superior to using it alone.

[0179] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. Use of the mixture for preparing pharmaceutical compositions or kits for relieving / treating hair loss, improving hair follicle homeostasis, or enhancing hair growth / pigmentation; said mixture comprising: (a) JAK kinase inhibitors, and (b) neuropeptide CGRP or its receptor agonists.

2. The use as described in claim 1, characterized in that: The hair loss mentioned includes alopecia areata or anagen baldness.

3. The use as described in claim 1, characterized in that, The JAK kinase inhibitor comprises: a small molecule compound that specifically inhibits JAK kinase; an interfering molecule that specifically interferes with the expression of the JAK kinase gene; a gene editing reagent that specifically knocks out the JAK kinase gene; or an antibody or ligand that specifically binds to the protein encoded by the JAK kinase gene; preferably, the JAK kinase inhibitor is a small molecule compound that specifically inhibits JAK kinase, such as Ruxolitinib; or The neuropeptide CGRP receptor agonist includes: CGRP peptide or its modifications, small molecule compounds that specifically agonize CGRP receptors, agents that upregulate / enhance the function of CGRP receptors, interfering molecules that specifically promote CGRP release or the expression of its receptor gene, or gene editing agents that specifically overexpress CGRP peptide or its modifications, or CGRP receptors.

4. A pharmaceutical composition or kit for relieving / treating hair loss or enhancing hair growth / blackening, said pharmaceutical composition or kit containing: (a) a JAK kinase inhibitor and (b) a neuropeptide CGRP or its receptor agonist; preferably, said pharmaceutical composition further comprising a pharmaceutically or physiologically acceptable carrier or excipient.

5. The use as described in any one of claims 1-3, or the pharmaceutical composition or kit as described in claim 4, wherein, The mass-molar ratio of (a) to (b) is: 10–80 mg : 0.12–1 nmol; preferably 20–70 mg : 0.2–0.8 nmol; more preferably 30–60 mg : 0.3–0.7 nmol; even more preferably 35–45 mg : 0.4–0.6 nmol; or According to the unit dosage, (a) the amount is 10-80 mg, and (b) the amount is 0.12-1 nmol; preferably, (a) the amount is 20-70 mg, and (b) the amount is 0.2-0.8 nmol; more preferably, (a) the amount is 30-60 mg, and (b) the amount is 0.3-0.7 nmol; even more preferably, (a) the amount is 35-45 mg, and (b) the amount is 0.4-0.6 nmol.

6. A method for preparing a spontaneous alopecia areata animal model, comprising: In the Il4i1 Drivers of Cre recombinase expression Il4i1 Cre Knockout in animals Calcrl Genes were used to obtain animal models with spontaneous alopecia areata phenotype.

7. The method as described in claim 6, characterized in that, Conditional expression is achieved using the Cre / loxP recombination system to enable... Il4i1 Drivers of Cre recombinase expression Il4i1 Cre Knockout in animals Calcrl Gene; Preferably, the method includes: (1) Establish by Il4i1 Drivers of Cre recombinase expression Il4i1 Cre animal; (2) Establish a Cre / loxP-based recombination system Calcrl Conditional knockout animals; (3) Cross the animals in (1) and (2) to obtain offspring that are animal models with spontaneous alopecia areata phenotype. More preferably, in the method: (1) includes: providing animal a, in which Il4i1 Introducing Cre recombinase expression elements into the genome of positive cells allows them to interact with the genome. Il4i1 The Cre recombinase expression element contains a Cre recombinase-encoded nucleic acid; (2) includes: providing animal b, and introducing conditional knockout into its genome. Calcrl Gene elements; preferably, the elements target Calcrl Exon 6 of the gene can be specifically knocked out; When Cre recombinase is expressed, it recognizes LoxP and undergoes cleavage, thus... Il4i1 Continuous knockout of positive cells and their progeny cells Calcrl Gene.

8. The method as described in any one of claims 6 to 7, characterized in that, The animal is a rodent; preferably, the rodent includes: mouse and rat; The animals exhibit progressive hair loss and discoloration during their growth, subsequently forming patches of hair loss, exhibiting a typical alopecia areata phenotype; and / or, The skin of the animals exhibited regressive characteristics such as miniaturization, vacuolation, and depigmentation of hair follicles, with visible reticular dermal expansion and inflammatory cell infiltration around the hair follicles.

9. The application of the spontaneous alopecia areata animal model obtained by the method of any one of claims 6 to 8, for: serving as an animal model for screening candidate drugs or therapeutic agents to alleviate or treat alopecia areata; or serving as an animal model for studying alopecia areata.

10. A method for screening candidate drugs or therapeutic agents to alleviate or treat alopecia areata, characterized in that, The method includes: (1) A spontaneous alopecia areata animal model is prepared using the method described in any one of claims 6 to 8; (2) The candidate substance is given to the animal model of (1) and the candidate substance is observed to have a relieving or therapeutic effect on alopecia areata. If the alopecia areata symptoms of the animal model are relieved, the candidate substance is a substance that relieves or treats alopecia areata.

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