Application of CGRP receptor inhibitor in preparation of medicine for relieving or treating vitiligo

Abstract

The invention relates to application of a CGRP receptor inhibitor in preparation of a medicine for relieving or treating leucoderma. According to the application disclosed by the invention, the condition that the CGRP receptor is associated with occurrence and development of the vitiligo is determined for the first time, and the CGRP receptor inhibitor can be used for effectively relieving or treating the vitiligo. The CGRP receptor inhibitor, especially Ubrogeant or Rimegeant, can be used for realizing pharmacological regulation and control on the vitiligo. The invention further provides a method for screening candidate drugs acting on the CGRP receptor so as to relieve or treat leucoderma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically, the present invention relates to the use of CGRP receptor inhibitors in preparing drugs for alleviating or treating vitiligo. Background Art

[0002] Vitiligo is a disfiguring skin disease characterized by local or large white spots due to loss of pigment. The depigmented area usually increases over time and can affect any part of the body, including the head and face. The incidence of vitiligo varies with race, geographical environment, and population, and is more common in adolescents (Ezzedine K, Eleftheriadou V, Whitton M, et al. Vitiligo [J]. Lancet, 2015, 386 (9988): 74-84). Vitiligo is not only a cosmetic problem, but also has serious consequences for patients' quality of life due to its stubbornness, long treatment time, unsuccessful treatment, frequent recurrence and aggravation.

[0003] According to the Vitiligo Global Issue Consensus Conference (Ezzedine K, Lim HW, Suzuki T, et al. Revised classification / nomenclature of vitiligo and related issues: the Vitiligo Global Issues Consensus Conference [J]. Pigment Cell Melanoma Res, 2012, 25(3): E1-E13) and the current mainstream view, Vitiligo Diagnosis and Treatment Consensus (2021 Edition), 2021, 51(4): 247-250), vitiligo is divided into four types: segmental vitiligo, non-segmental vitiligo (vulgaris), mixed vitiligo and undetermined type vitiligo. In 2012, the Vitiligo Global Issue Consensus Conference pointed out that the "vitiligo" usually refers to vitiligo vulgaris, which is the most common clinical phenotype of vitiligo. Its lesions are often symmetrically distributed on the extremities or scattered throughout the body.

[0004] The pathogenesis of vitiligo is complex, and the initiation and regulation mechanisms are still unclear. Vitiligo is characterized by depigmented spots (white spots) of varying sizes and numbers as typical skin lesions. Vitiligo histopathological examination combined with immunohistochemical staining with the melanocyte-specific antibody Melan-A showed that there were no melanocytes in the skin of the white spots. When the biopsy specimen is taken from active white spots or around inflammatory vitiligo lesions, lymphocyte infiltration can be seen, and it is most obvious at the junction of white spots and normal skin, indicating that the occurrence and progression of vitiligo mainly stems from the deliberate destruction of melanocytes. There are many theories about the causes of vitiligo melanocyte destruction, including genetic theory, autoimmune theory, oxidative stress theory, mental theory, trace element deficiency theory, etc. (Song Shuling, New Progress in the Pathogenesis and Treatment of Vitiligo [J], China Practical Medicine, 2018, 13(05): 193-194).

[0005] In recent years, with the deepening of research, researchers have basically reached a consensus that vitiligo is an autoimmune disease that kills CD8 + Melanocyte death mediated by T cells (CTL) is the direct cause of vitiligo. When immune function is disordered, the immune system will attack melanocytes, leading to cell apoptosis, decreased tyrosinase activity and reduced melanin content, resulting in white spots, but the specific mechanism is still unclear. Studies have found that CD8 + T cells can mediate the killing of melanocytes. CD8 + The content of T cells increased significantly, and the severity of the disease and the ability to kill melanocytes were significantly higher than those of CD8 + The number of T cells is positively correlated (Riding RL, Harris JE. The Role of Memory CD8 + T Cells in Vitiligo[J].J Immunol.2019Jul 1;203(1):11-19.). Therefore, CD8 + T cells are important target cells.

[0006] Cytotoxic CD8 in vitiligo + T cells and the effector factors they secrete are closely related to the development of vitiligo and have been considered as a hot target for the treatment of vitiligo. +T cells can be divided into different subtypes of cytotoxic T lymphocytes (Tc cells), including Tc1, Tc2, Tc17 and Tc22 cells. Among them, Tc1 cells can secrete IFN-γ, granzymes and perforins to kill melanocytes, leading to the occurrence and development of vitiligo (Jacquemin C, Martins C, Lucchese F, et al. NKG2D Defines aSubset of Skin Effector Memory CD8 T Cells with Proinflammatory Functions in Vitiligo[J]. J Invest Dermatol. 2020Jun; 140(6): 1143-1153.e5.). IFN-γ may play a direct role in the pathogenesis of vitiligo. IFN-γ is produced by cytotoxic CD8 + Secreted by T cells, it can cause apoptosis of melanocytes. IFN-γ can also promote keratinocytes (Rashighi M, Harris JE. Interfering with the IFN-γ / CXCL10 pathway to develop new targeted treatments for vitiligo [J]. Ann Transl Med. 2015 Dec; 3 (21): 343) and fibroblasts to secrete CXCL9 / 10 through the JAK-STAT pathway, and CXCL9 / 10 is a key chemokine for recruiting and regulating CD8+ T cells (Xu Z, Chen D, Hu Y, et al. Anatomically distinct fibroblast subsets determine skin autoimmune patterns [J]. Nature. 2022 Jan; 601 (7891): 118-124.).

[0007] The cause and pathogenesis of vitiligo have not been fully elucidated so far, and there is currently no cure, so patients often require lifelong treatment. Since vitiligo often brings serious mental and psychological burdens to patients, and the incidence rate is increasing year by year, the treatment of vitiligo is a thorny issue. Due to differences in race, skin color, age, etc., there are also differences in treatment guidelines in various countries, but after summarizing, it is found that the treatment of vitiligo is mainly based on the area of ​​white spots, the site of onset, etc. Combined with the Vitiligo Diagnosis and Treatment Consensus (2021 Edition), the current clinical treatment methods for vitiligo mainly include: hormone therapy, phototherapy, calcineurin inhibitors, vitamin D3 derivatives, traditional Chinese medicine, transplantation therapy, occlusion therapy, etc. Only in special circumstances can permanent depigmentation be used as a treatment option.

[0008] In summary, this field urgently needs to explore and develop new treatment options to alleviate vitiligo, in order to provide assistance to clinical patients and improve their quality of life. Summary of the invention

[0009] The object of the present invention is to provide the use of CGRP receptor inhibitors in preparing drugs for alleviating or treating vitiligo.

[0010] In a first aspect of the present invention, there is provided a use of a CGRP (Calcitonin Gene Related Peptide) receptor inhibitor for preparing a mixture, a pharmaceutical composition or a medicine kit for alleviating or treating vitiligo.

[0011] In another preferred embodiment, the CGRP receptor inhibitor includes: a small molecule compound that specifically inhibits / antagonizes the CGRP receptor, an agent that downregulates / loss-of-function mutation of the CGRP receptor, an antibody that specifically binds to the CGRP receptor, an interfering molecule that specifically interferes with the expression of the CGRP receptor gene, or a gene editing agent that specifically knocks out the CGRP receptor gene.

[0012] In another preferred embodiment, the CGRP receptor inhibitor is a small molecule compound that specifically inhibits / antagonizes the CGRP receptor, including: Ubrogepant and Rimegepant.

[0013] In another preferred embodiment, the pharmaceutical composition containing the CGRP receptor inhibitor is in an external dosage form, including: ointment (such as cream, paste), gel, foam, spray, powder or cream.

[0014] In another preferred embodiment, the pharmaceutical composition is an ointment preparation, comprising: a CGRP receptor inhibitor; and an adjuvant.

[0015] In another preferred embodiment, the auxiliary agent includes one or more selected from the following group: white vaseline, paraffin, lanolin, microcrystalline wax, beeswax, spermaceti, ozokerite, stearyl glyceryl, stearic acid, and cholesterol.

[0016] In another preferred embodiment, the CGRP receptor inhibitor is a CGRP receptor inhibitor that exerts a mitigating or therapeutic effect by (significantly) reducing CD8+T cells and / or (significantly) increasing melanocytes.

[0017] In another preferred embodiment, the medicine kit further comprises: instructions for use, describing methods for alleviating or treating vitiligo.

[0018] In another aspect of the present invention, a pharmaceutical composition for alleviating or treating vitiligo is provided, wherein the pharmaceutical composition comprises a CGRP receptor antagonist and a pharmaceutically acceptable carrier.

[0019] In another aspect of the present invention, a method for inhibiting CD8 + A method for increasing T cells and / or melanocytes, comprising: treating CD8 T cells with a CGRP receptor inhibitor + T cell and / or melanocyte cell system (cell culture system or cell model), thereby inhibiting CD8 + T cells and / or increase melanocytes.

[0020] In another aspect of the present invention, a method for screening a potential substance for alleviating or treating vitiligo is provided, comprising:

[0021] (1) treating a cell system expressing a CGRP receptor with a candidate substance; and

[0022] (2) detecting the expression or activity of CGRP receptors in the cell system; if the candidate substance statistically reduces the expression or activity of CGRP receptors (such as the ability to receive or transport CGRP or generate downstream signals), it indicates that the candidate substance is a potential substance for alleviating or treating vitiligo.

[0023] In another preferred embodiment, step (1) comprises: in the test group, adding the candidate substance to the expression system;

[0024] Step (2) comprises: detecting the expression or activity of CGRP receptors in the system and comparing it with a control group, wherein the control group is an expression system to which the candidate substance is not added; if the candidate substance statistically reduces the expression or activity of CGRP receptors, then the candidate substance is a potential substance for alleviating or treating vitiligo.

[0025] In another preferred embodiment, the cell system expressing CGRP receptor includes: a cell (culture) system and a subcellular (culture) system.

[0026] In another preferred embodiment, the potential substance includes a substance that has not been disclosed so far and inhibits the CGRP receptor.

[0027] In another preferred embodiment, the reduction is a statistical reduction, such as a reduction of 10% or 20% or more, preferably a reduction of 30% or 50% or more, and more preferably a reduction of 80% or 90% or more, compared with a control or substrate.

[0028] In another preferred embodiment, the candidate substances include (but are not limited to): small molecule compounds, regulatory molecules designed for CGRP receptors or their upstream or downstream proteins or genes (such as but not limited to upregulators, interfering molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands)), CRISPR constructs, and compounds from a compound library.

[0029] In another preferred embodiment, the method further comprises: conducting further cell experiments and / or animal experiments on the obtained potential substances to further select and determine substances useful for alleviating or treating vitiligo from the candidate substances.

[0030] In another aspect of the present invention, a method for alleviating or treating vitiligo is provided, comprising: administering to a subject in need of treatment an effective amount of the CGRP receptor inhibitor selected from: a small molecule compound that specifically inhibits / antagonizes the CGRP receptor, an agent that downregulates / loss-of-function mutations the CGRP receptor, an antibody that specifically binds to the CGRP receptor, an interfering molecule that specifically interferes with the expression of the CGRP receptor gene, or a gene editing agent that specifically knocks out the CGRP receptor gene; preferably, the CGRP receptor inhibitor includes: Rimegepant and Ubrogepant.

[0031] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Photographs of vitiligo mice phenotypes in the Vehicle group, Ubrogepant ointment group, and Rimegepant ointment group after 21 days of treatment.

[0033] Figure 2 Photos of the tail skin of vitiligo mice in the Vehicle group, Ubrogepant ointment group, and Rimegepant ointment group after 21 days of treatment

[0034] Figure 3 The grayscale values ​​of the tail skin of vitiligo mice in the Vehicle group, Ubrogepant ointment group and Rimegepant ointment group were counted after 21 days of treatment.

[0035] Figure 4 , CGRP receptor inhibitor ointment for the treatment of vitiligo mouse animal model, representative images of each group detected by flow cytometry.

[0036] Figure 5, CGRP receptor inhibitor ointment for the treatment of vitiligo mouse animal model, flow cytometry statistical data, Ubrogepant ointment group and Rimegepant ointment group can significantly treat vitiligo.

[0037] Figure 6 , CGRP receptor inhibitor ointment treated vitiligo mouse animal model, tail epidermal immunofluorescence staining, Ubrogepant ointment group and Rimegepant ointment group can significantly treat vitiligo.

[0038] Figure 7 , Rimegepant ointment effectively improves the disease phenotype of vitiligo patients, and the skin condition of the patients before and after taking the medicine was photographed. DETAILED DESCRIPTION

[0039] After in-depth research, the inventors have determined for the first time that the CGRP receptor is associated with the occurrence and development of vitiligo, and that CGRP receptor inhibitors can effectively alleviate or treat vitiligo. It has been verified that CGRP receptor inhibitors, especially Ubrogepant or Rimegepant, can achieve pharmacological regulation of vitiligo. Therefore, the present invention has identified a new disease target associated with vitiligo, namely the CGRP receptor. Its inhibitors, especially Ubrogepant or Rimegepant, can be used to target and inhibit CGRP receptors, alleviate or treat vitiligo. The present invention also provides a method for screening candidate drugs that act on CGRP receptors and thereby alleviate or treat vitiligo.

[0040] CGRP receptors and receptor inhibitors (antagonists)

[0041] As used herein, the "CGRP receptor inhibitor" refers to an agent that can inhibit the expression or activity of the CGRP receptor. The "CGRP receptor inhibitor" includes inhibitors of the activity or function of the CGRP receptor, and also includes nucleic acid inhibitors, antagonists, inhibitors, blockers, blocking agents, etc. of the CGRP receptor, as long as they can downregulate the expression level of the CGRP receptor, inhibit or antagonize the activity or function of the CGRP receptor. They can be compounds, small chemical molecules, and biological molecules. The biological molecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.

[0042] As used herein, the "CGRP receptor antagonist" refers to a "CGRP receptor inhibitor".

[0043] Calcitonin Gene-Related Peptide (CGRP) is a neuropeptide of the calcitonin (CT) family. It is composed of 37 amino acids and is widely expressed in the central and peripheral nervous systems. The CGRP receptor is a complex formed by the seven-transmembrane G protein-coupled receptor CALCRL (Calcitonin receptor like) and the single-transmembrane protein RAMP1 (Receptor activity modifying protein 1). It is widely distributed in neural tissues and various non-neural tissues. When cells synthesize CALCRL and RAMP1 proteins and initially localize them on the endoplasmic reticulum membrane, RAMP1 protein controls and helps them transfer to the cell membrane through glycosylation of CALCRL; after the two transfer and localize to the cell membrane, they form heterodimers through non-covalent interactions and become functional receptors that can bind to CGRP.

[0044] CGRP is an important vasodilator active substance in the body. In vascular smooth muscle cells (VSMC), CGRP binds to receptors, causing RCP to interact with Gαs protein, activating adenylate cyclase (AC), increasing intracellular cAMP levels, and leading to activation of protein kinase A (PKA) or opening of ATP-K+ channels. CGRP can also directly act on vascular endothelial cells, activating intracellular PKA and thus NOS; the increase in NO levels catalyzed by NOS further acts on VSMC, increasing its intracellular cGMP levels, and ultimately mediating vascular dilation. Based on this, CGRP plays different regulatory roles in a variety of circulatory system diseases. In the onset of migraine, the central trigeminal nerve secretes excessive CGRP under external stimulation, causing excessive dilation of meningeal blood vessels, while aggravating local inflammatory cell infiltration and inflammatory response, further worsening migraine. Monoclonal antibody drugs targeting CGRP and CGRP receptors have been shown to have good therapeutic effects on migraine through clinical trials, such as Fremanezumab, Erenumab, and Galcanezumab.

[0045] CGRP receptor antagonist small molecule compounds screened and developed for CGRP receptors have also achieved good results in the treatment of migraine. Unlike monoclonal antibodies, CGRP receptor antagonists can quickly enter the brain and take effect very quickly. However, they are metabolized by the liver, so they may cause a higher potential risk of liver damage when used internally.

[0046] In the present invention, an inhibitor that specifically acts on the CGRP receptor is provided. More particularly, the effect of Ubrogepant or Rimegepant on relieving / treating vitiligo is surprising. Vitiligo is a special disease that is clinically refractory and has significant differences from other types of diseases in terms of pathogenesis and symptoms.

[0047] Ubrogepant, molecular weight 549.54, chemical formula C29H26F3N5O3, CAS registration number 1374248-77-7. It is a white powder, insoluble in water, soluble in anhydrous ethanol, DMSO, etc.

[0048]

[0049] Ubrogepant is currently used for the acute treatment of migraine in adults.

[0050] Rimegepant, Ki = 0.027nM, IC50 = 0.14nM. Molecular weight 534.56, chemical formula C28H28F2N6O3, CAS registration number 1289023-67-1. It is a white powder, insoluble in water, soluble in anhydrous ethanol, DMSO, etc.

[0051]

[0052] Rimegepant is currently used for the acute treatment of migraine in adults and for the prevention of episodic migraine.

[0053] The CGRP receptor antagonists Ubrogepant and Rimegepant described in the present invention have been tested in the examples and shown that Ubrogepant and Rimegepant have good therapeutic effects on vitiligo animals, significantly reducing the number of epidermal CD8+T cell infiltrations, increasing the number of epidermal melanocytes, and improving the disease phenotype of vitiligo animal models. At the same time, Rimegepant ointment has a significant effect in the clinical treatment of vitiligo patients. Therefore, CGRP receptor inhibitors can be used as drugs and pharmaceutical compositions for the treatment of vitiligo.

[0054] In the present invention, the Ubrogepant or Rimegepant also includes its pharmaceutically acceptable salts, esters, isomers, solvates or prodrugs, as long as they also have the same or substantially the same functions as Ubrogepant or Rimegepant. In the present invention, "pharmaceutically acceptable" ingredients are substances that are suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit / risk ratio. The "pharmaceutically acceptable salt" can be an acid salt or a basic salt of Ubrogepant or Rimegepant.

[0055] "Pharmaceutically acceptable acid salts" refer to salts that retain the biological activity and properties of the free base without undesirable changes in biological activity or other aspects. Such salts can be formed from inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid and similar acids.

[0056] "Pharmaceutically acceptable basic salts" refer to salts that retain the biological activity and properties of the free acid without undesirable changes in biological activity or other aspects. These salts are made by adding an inorganic base or an organic base to the free acid. Salts obtained with inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like.

[0057] Those skilled in the art should understand that after knowing the structure of Ubrogepant or Rimegepant of the present invention, Ubrogepant or Rimegepant of the present invention can be obtained by various methods well known in the art, using known raw materials, such as chemical synthesis or extraction from organisms (such as microorganisms), and these methods are all included in the present invention. In addition, Ubrogepant or Rimegepant is also a commercial drug, so its finished product is easily available to those skilled in the art.

[0058] The present invention includes the use of Ubrogepant or Rimegepant or a pharmaceutically acceptable salt thereof in the preparation of a drug targeting a CGRP receptor. As used herein, the term "targeting a CGRP receptor" generally refers to specifically or selectively acting on a CGRP receptor.

[0059] Drug screening for the treatment / relief of vitiligo

[0060] The present invention also provides a method for screening drugs (including potential drugs) for relieving or treating vitiligo, comprising: (1) treating a system comprising cells expressing CGRP receptors with a candidate substance; and (2) detecting the expression or activity of cellular CGRP receptors in the system; if the candidate substance statistically down-regulates (significantly down-regulates, such as down-regulating by more than 10%, more than 20%, more than 50%, more than 80%, etc., or making it non-expressed or inactive) the expression or activity of cellular CGRP receptors, then the candidate substance is a drug (including potential drug) for relieving or treating vitiligo.

[0061] In some embodiments, step (1) includes: in a test group, adding a candidate substance to the expression system; and / or, step (2) includes: detecting the expression or activity of cellular CGRP receptors in the system, and comparing with a control group, wherein the control group is an expression system to which the candidate substance is not added; if the candidate substance statistically downregulates (significantly downregulates, such as downregulating by more than 10%, more than 20%, more than 50%, more than 80%, etc., or making it non-expressed or inactive) the expression or activity of cellular CGRP receptors, then the candidate substance is a drug for alleviating or treating vitiligo.

[0062] In some embodiments, the cell-containing system is selected from: a cell system (such as a cell or cell culture expressing a CGRP receptor), a subcellular (culture) system, a tissue system, or an organ system.

[0063] In some embodiments, the cell-containing system is an animal system (mammalian system), and the animal includes (but is not limited to): rodents and non-human primates.

[0064] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules or constructs designed for cellular CGRP receptors, fragments or variants thereof, their encoding genes or their upstream and downstream molecules or signaling pathways (such as shRNA, siRNA, gene editing reagents, expression vectors, recombinant viruses or non-viral constructs, etc.), chemical small molecules (such as specific inhibitors or antagonists), interactive molecules, etc.

[0065] In another preferred embodiment, the method further comprises: conducting further cell experiments and / or animal experiments on the obtained potential substances to further select and determine substances useful for alleviating or treating vitiligo from the candidate substances.

[0066] Herein, a "pharmaceutical composition" is a composition containing the CGRP receptor inhibitor described herein, in particular Ubrogepant or Rimegepant or a pharmaceutically acceptable salt thereof, and a physiologically / pharmaceutically acceptable carrier and excipient.

[0067] The Ubrogepant or Rimegepant in the pharmaceutical composition is present in a therapeutically effective amount or a palliative effective amount. An effective amount is an amount sufficient to improve or alleviate in some way the symptoms associated with a disease. The dosage is an amount effective to improve or eliminate one or more symptoms, and can be determined by a person of ordinary skill in the art based on the age, sex, physical condition, etc. of the subject. The dosage may cure the disease, but the administration is usually to improve the symptoms of the disease. Repeated administration is generally required to achieve the desired symptom improvement. Herein, the subject is a mammal, particularly a human.

[0068] The pharmaceutical composition of the present invention can be prepared into any conventional formulation form by conventional methods. The dosage form can be varied, as long as it is a dosage form that can effectively allow the active ingredient to reach the mammalian body. For example, it can be selected from: injections, infusions, tablets, capsules, and pills. The active ingredient (preferably Ubrogepant or Rimegepant) can be present in a suitable solid or liquid carrier or diluent. The pharmaceutical composition of the active ingredient of the present invention can also be stored in a sterile device suitable for injection or instillation. The effective dose of the active ingredient (preferably Ubrogepant or Rimegepant) in the pharmaceutical composition can vary with the mode of administration and the severity of the disease to be treated, which can be based on the experience and advice of the clinician.

[0069] In a specific embodiment of the present invention, a series of dosing regimens of Ubrogepant or Rimegepant are proposed. It also relates to a dosing regimen using an animal model such as a mouse. It is known to those skilled in the art that the dosage of a mouse can be converted to a dosage suitable for a human, for example, according to the Meeh-Rubner formula. It should be understood that the conversion of the dosage can vary depending on the drug and the clinical situation, based on the evaluation of an experienced pharmacist.

[0070] In a preferred embodiment, the Ubrogepant or Rimegepant of the present invention is combined with preferred pharmaceutically acceptable excipients to form an external dosage form with excellent effects.

[0071] The present invention also provides a drug box or a test kit targeting CGRP receptors, wherein the drug box or the test kit contains Ubrogepant or Rimegepant according to the present invention. Alternatively, the drug box or the test kit contains a pharmaceutical composition or mixture of Ubrogepant or Rimegepant according to the present invention. Alternatively, the drug box or the test kit contains: a container, and Ubrogepant or Rimegepant according to the present invention placed in the container.

[0072] The medicine box or test kit may also contain some auxiliary medication materials, such as measuring tools and containers such as syringes required for using or applying the composition in various dosage forms. The medicine box or test kit may also contain instructions for use.

[0073] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0074] Example 1. Preparation of ointment

[0075] The production process of Ubrogepant and Rimegepant ointment includes: ① API dissolution process; ② excipient melting process; ③ homogenization mixing process; ④ cooling process; ⑤ filling process. The detailed description is as follows:

[0076] ①Raw material dissolution process

[0077] Turn on the temperature control device in advance and set the temperature to 70°C. Take the prescribed amount of anhydrous ethanol, add Ubrogepant or Rimegepant API, protect from light, stir and dissolve at 70°C, stir for no less than 30 minutes, until the API is completely dissolved by visual observation and set aside.

[0078] ②Auxiliary material melting process

[0079] Weigh the prescribed amount of white vaseline, place it in a beaker / stainless steel barrel and maintain the temperature at 70°C.

[0080] ③Homogeneous mixing process

[0081] The homogenizing speed was 8000 rpm and the mixing temperature was 70°C. Under continuous stirring, the API solution was slowly added to the matrix excipients and the homogenizing stirring was continued for 5 min.

[0082] ④Material cooling process

[0083] After stopping stirring, adjust the temperature of the temperature control instrument to 40℃±5℃, wait for the material to cool down to 40℃±5℃, and keep it warm for later use.

[0084] ⑤Filling process (small preparation amount, can be canned manually)

[0085] Add the ointment to the feeding tank, manually debug the equipment for 2-3 turns to ensure that the installation and debugging are in place before formal filling. The target filling speed is 30-40 pieces / min. If the filling volume is unstable, set the hourly output according to the filling weight stability. During the filling process, ensure that the amount of ointment in the feeding barrel can maintain a stable filling weight. During the filling process, check the filling volume every 20±5min.

[0086] The tail sealing machine seals and labels.

[0087] Table 1: Unit dose formulation composition

[0088]

[0089] Example 2: Experiment on the treatment of vitiligo mouse model with Ubrogepant and Rimegepant ointments

[0090] Take 15 C57BL / 6 mice of 9 weeks old, inject skin melanoma cells B16F10 on the lower right side of the back, and remove the melanocytoma surgically 12 days later. Continue to observe for 15 days after surgery, select animals with whitening hair in the surgery area, and successfully construct a vitiligo animal model. On the 15th day after surgery, select 12 vitiligo mice with successful modeling and divide them into 3 groups. They are blank ointment (prepared according to Table 1 but without Ubrogepant / Rimegepant ointment) Vehicle group, 0.1% Ubrogepant ointment treatment group, 0.1% Rimegepant ointment treatment group, 4 experimental mice in each group.

[0091] Starting from the 16th day after surgery, control ointment or Ubrogepant / Rimegepant ointment was applied to the tails of mice, 60 mg / tail, once a day for 3 consecutive weeks. On the last day, the tail phenotypes of mice in each group were recorded, and then the mice were killed by cervical stenosis.

[0092] Remove the tail, peel off the coccyx, unfold it, and take photos.

[0093] Take 2cm of the middle 1 / 3 2 Tail skin was divided into 6-8 small sections. It was placed in 2mg / ml Dispase II and treated at 37°C for 30 minutes. It was washed 3 times with HBSS solution and the true epidermis was separated. The epidermis was digested with 0.05% TE at 37°C for 10 minutes. It was neutralized with an equal amount of HBSS containing 5% FBS, passed through a 70μm cell sieve, and centrifuged at 400g for 5 minutes. Staining: AF700-CD45, PE-CY7-CD117, PE-CF594-CD3e, APC-CY7-CD8a. Flow cytometry antibodies 1:200. Staining was performed at 4°C for 30 minutes, protected from light. It was washed twice with FACS buffer. It was resuspended with FACS containing DAPI (1:1000) and loaded onto the flow cytometer.

[0094] Immunofluorescence staining was performed on the tail epidermis, and the primary antibodies were incubated overnight with anti-mouse CD8a (1:100, host = Rat) and anti-mouse DCT (1:300, host = Rabbit). The secondary antibodies were incubated at room temperature for 1 hour with anti-Rat-488 (1:500), anti-Rabbit-555 (1:500), and anti DAPI (1:1000). The slides were sealed and the images were taken with confocal microscope.

[0095] Observe the disease phenotype (the extent of whitening of the back hair and tail) in the animal model, such as Figure 1 , Figure 2 and Figure 3 , after 21 days of treatment, the white area of ​​the back hair increased, white spots appeared on the tail skin, and melanocytes were lost in the Vehicle group, showing a worsening of the disease. After 21 days of treatment, the degree of whitening of the back hair and tail of the animals in the 0.1% Ubrogepant ointment group and the 0.1% Rimegepant ointment group was significantly reduced. The back hair of the animals in both groups remained black, and the pigmentation of the tail skin increased (****P<0.0001).

[0096] Melanocytes and CD8+T cells in the epidermal skin of the tails of mice in each group were detected by flow cytometry. The results showed that ( Figure 4 and Figure 5 ): The melanocytes in the tail epidermis of the Vehicle group decreased significantly, and the CD8+T cells increased significantly. The disease of the animals in the 0.1% Ubrogepant ointment group was significantly improved, the melanocytes increased significantly (*P<0.05), and the CD8+T cells decreased significantly (****P<0.0001), indicating that 0.1% Ubrogepant ointment has a good therapeutic effect. The disease of the animals in the 0.1% Rimegepant ointment group was significantly improved, the melanocytes increased significantly (**P<0.01), and the CD8+T cells decreased significantly (****P<0.0001), indicating that 0.1% Rimegepant ointment has an excellent therapeutic effect.

[0097] Although the CD8+T cells in both the Ubrogepant ointment group and the Rimegepant ointment group were significantly reduced and there was no difference between the two groups, the number of melanocytes in the Rimegepant ointment group was significantly greater (*P<0.05), so the therapeutic effect of Rimegepant ointment was significantly better than that of Ubrogepant ointment.

[0098] The results of immunofluorescence staining of the tail epidermis of each group were consistent with the results of flow cytometry ( Figure 6 ).

[0099] Example 3: Analysis of the effect of CGRP receptor inhibitor Rimegepant ointment in treating local vitiligo

[0100] In this embodiment, an intervention study was conducted on patients with vitiligo.

[0101] A total of 10 patients with vitiligo were recruited in this trial, and their basic information is shown in Table 2.

[0102] Patient's medication: Apply the drug once a day to the test area, 30 mg / cm 2 , take the medicine continuously for 30 days.

[0103] After 30 days of medication, the patient's skin was observed for re-pigmentation (white spot re-pigmentation: white spots were restored to skin color). The re-pigmentation rate was calculated as shown in Tables 2 and 3.

[0104] Table 2. Related questions and evaluation criteria of the Vitiligo Visualization Scale

[0105]

[0106] Table 3. Rimegepant ointment in the treatment of vitiligo patients

[0107]

[0108]

[0109] Among the 10 patients with vitiligo mentioned above, 8 were revisited, and 7 of them improved. From the clinical status of the disease, the total treatment efficiency is very high. Under the treatment condition without phototherapy, the condition of 2 patients was effectively improved; in particular, No. 1 was completely improved, with a re-pigmentation rate of 100%. No. 2 was slightly improved, with a re-pigmentation rate of 25-50%. Under the treatment condition of combined phototherapy, the condition of 5 patients was effectively improved. In particular, No. 3 was significantly improved, with a re-pigmentation rate of 50-75%. Nos. 6, 8, 9 and 10 were slightly improved, with a re-pigmentation rate of <25%.

[0110] Representative pictures of patients' skin recovery Figure 7 , Rimegepant ointment effectively improves the disease phenotype of vitiligo patients.

[0111] In summary, the above results show that CGRP receptor antagonist ointment can very effectively improve the phenotype of vitiligo mouse model, effectively treat vitiligo patients, and has a very significant clinical treatment effect on vitiligo.

[0112] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. Use of CGRP receptor inhibitors for preparing mixtures, pharmaceutical compositions or kits for alleviating or treating vitiligo.

2. The use according to claim 1, characterized in that The CGRP receptor inhibitors include: small molecule compounds that specifically inhibit / antagonize CGRP receptors, reagents that downregulate / loss-of-function mutations of CGRP receptors, antibodies that specifically bind to CGRP receptors, interfering molecules that specifically interfere with CGRP receptor gene expression, or gene editing reagents that specifically knock out CGRP receptor genes.

3. The use according to claim 2, characterized in that The CGRP receptor inhibitor is a small molecule compound that specifically inhibits / antagonizes the CGRP receptor, including: ubegepam and remegipam.

4. The use according to claim 1, characterized in that The pharmaceutical composition containing the CGRP receptor inhibitor is an external dosage form; Included is selected from: ointment, gel, foam, spray, powder or cream; Preferably, the pharmaceutical composition is an ointment preparation, comprising: a CGRP receptor inhibitor; and an adjuvant; Preferably, the auxiliary agent includes one or more selected from the following group: white vaseline, paraffin, lanolin, microcrystalline wax, beeswax, spermaceti, ozokerite, stearyl glyceryl, stearic acid, and cholesterol.

5. The use according to claim 1, characterized in that The CGRP receptor inhibitor is a CGRP receptor inhibitor that exerts a mitigating or therapeutic effect by reducing CD8+T cells and / or increasing melanocytes.

6. A pharmaceutical composition for alleviating or treating vitiligo, comprising a CGRP receptor antagonist and a pharmaceutically acceptable carrier.

7. A CD8 inhibitor + T cells and / or a method for increasing melanocytes, characterized in that The method comprises: treating CD8 + T cell and / or melanocyte cell system, thereby inhibiting CD8 + T cells and / or increase melanocytes.

8. A method for screening a potential substance for alleviating or treating vitiligo, the method comprising: (1) treating a cell system expressing a CGRP receptor with a candidate substance; and (2) detecting the expression or activity of CGRP receptors in the cell system; if the candidate substance statistically reduces the expression or activity of CGRP receptors, it indicates that the candidate substance is a potential substance for alleviating or treating vitiligo.

9. The method according to claim 8, characterized in that Step (1) comprises: in a test group, adding a candidate substance to the expression system; Step (2) comprises: detecting the expression or activity of CGRP receptors in the system and comparing it with a control group, wherein the control group is an expression system to which the candidate substance is not added; if the candidate substance statistically reduces the expression or activity of CGRP receptors, then the candidate substance is a potential substance for alleviating or treating vitiligo.

10. A method for alleviating or treating vitiligo, characterized in that: The method comprises: administering to a subject in need of treatment an effective amount of the CGRP receptor inhibitor selected from: a small molecule compound that specifically inhibits / antagonizes the CGRP receptor, an agent that downregulates / loss-of-function mutations the CGRP receptor, an antibody that specifically binds to the CGRP receptor, an interfering molecule that specifically interferes with the expression of the CGRP receptor gene, or a gene editing agent that specifically knocks out the CGRP receptor gene; preferably, the CGRP receptor inhibitor comprises: remeditam and ubegepam.

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