Application of CCR3 antagonist as medicine for treating stress dermatitis

Abstract

The invention belongs to the field of medicine health, and relates to application of a CCR3 antagonist as a medicine for treating stress dermatitis. Specifically, the invention provides an application of the screened CCR3 antagonist SB297006 in preparation of a pharmaceutical composition for treating stress dermatitis. The CCR3 antagonist SB297006 accurately targets a pathway of psychological stress deterioration AD, inhibits chemotaxis of eosinophilic granulocytes, and can more effectively relieve skin inflammation caused by stress deterioration.

Description

Technical Field

[0001] This invention belongs to the field of medicine and health, specifically relating to the application of CCR3 antagonists as drugs for treating stress dermatitis. Background Technology

[0002] Dermatitis typically refers to skin inflammation directly caused by physical (e.g., friction, hot or cold stimulation), chemical (e.g., detergents, cosmetics), biological (e.g., bacteria, fungi), or immune factors (e.g., allergies). Common types include contact dermatitis, atopic dermatitis, and seborrheic dermatitis. Its causes are mainly external direct stimulation or internal immune imbalance. The core issue is damage to the skin barrier (e.g., destruction of the stratum corneum) + local immune activation (e.g., mast cell degranulation, cytokine release), leading to symptoms such as erythema, itching, and exudation. The mechanism is primarily a "local inflammatory response of the skin."

[0003] Stress dermatitis is a type of inflammatory skin disease closely related to psychological stress. Its occurrence, development, or aggravation is mainly driven by psychological factors such as mental stress, anxiety, and drastic emotional fluctuations. Essentially, it is a manifestation of neuro-immune-endocrine axis disorder in the skin.

[0004] Current treatments for stress dermatitis often involve basic anti-inflammatory therapy, stress intervention, and lifestyle modifications, lacking specific medications. Therefore, there is an urgent need for a drug that can target and improve stress dermatitis. Summary of the Invention

[0005] This invention provides the use of the CCR3 antagonist SB297006 in the preparation of pharmaceutical compositions for treating stress dermatitis, and provides a Transwell cell migration model and its use in screening therapeutic drugs for stress dermatitis.

[0006] In a first aspect of the invention, there is provided the use of a CCR3 antagonist for preparing a composition for treating or improving stress dermatitis.

[0007] In another preferred embodiment, the CCR3 antagonist comprises: a small molecule drug, a nucleic acid, a polypeptide, a protein, a nanoparticle, a liposome, or a combination thereof.

[0008] In another preferred embodiment, the CCR3 antagonist blocks the binding of CCR3 and CCL11.

[0009] In another preferred embodiment, the CCR3 antagonist is SB297006.

[0010] In another preferred embodiment, SB297006 is the sole active component in the composition.

[0011] In another preferred embodiment, the stress dermatitis is sympathetic nerve cell activated dermatitis.

[0012] In another preferred embodiment, the sympathetic cell-activated dermatitis includes sympathetic cell-activated atopic dermatitis, sympathetic cell-activated non-atopic dermatitis, or a combination thereof.

[0013] In another preferred embodiment, the sympathetic nerve cells are Pdyn. + Sympathetic nerve cells.

[0014] In another preferred embodiment, the composition is used to treat or improve stress dermatitis in a subject.

[0015] In another preferred embodiment, the subjects had significantly higher CCL11 levels than normal controls.

[0016] In another preferred embodiment, "significantly higher" means that the ratio of the CCL11 level X1 of the test subject to the CCL11 level X0 of the normal control is X1 / X0 ≥ 1.1, preferably ≥ 1.2, more preferably ≥ 1.3, and most preferably ≥ 1.5.

[0017] In another preferred embodiment, the composition is used to treat or improve stress dermatitis in a subject whose PSS score is ≥14 points, preferably ≥29 points.

[0018] In another preferred embodiment, the stress dermatitis refers to dermatitis that is exacerbated by stress.

[0019] In another preferred embodiment, the stress-induced dermatitis includes: stress-induced atopic dermatitis, stress-induced non-atopic dermatitis, or a combination thereof.

[0020] In another preferred embodiment, the nonatopic dermatitis includes: contact dermatitis, seborrheic dermatitis, or a combination thereof.

[0021] In another preferred embodiment, the stress dermatitis is atopic dermatitis exacerbated by stress.

[0022] In another preferred embodiment, the concentration of the CCR3 antagonist in a unit dose of the composition is 5-80 mg / unit, more preferably 6-40 mg / unit, even more preferably 8-30 mg / unit, more preferably 10-25 mg / unit, and most preferably 12-20 mg / unit.

[0023] In a second aspect of the invention, a composition is provided, the pharmaceutical composition comprising the following components:

[0024] (Z1) The first active component, wherein the first active component is SB297006; and

[0025] (Z2) The second active component is another drug for treating stress dermatitis.

[0026] In another preferred embodiment, the composition further includes: (Z3) a pharmaceutically acceptable carrier or excipient.

[0027] In another preferred embodiment, the composition is a pharmaceutical composition or a cosmetic composition.

[0028] In another preferred embodiment, the first active component in the composition accounts for 0.1%-99% of the total mass of the composition.

[0029] In a third aspect of the invention, a pharmaceutical combination is provided, the pharmaceutical combination comprising:

[0030] (a) A CCR3 antagonist, wherein the CCR antagonist is SB297006;

[0031] (b) CCL11 detection reagent.

[0032] In another preferred embodiment, the drug combination further comprises: an eosinophil detection reagent.

[0033] In another preferred embodiment, the eosinophil detection reagent refers to a reagent for detecting the level of eosinophils in the blood.

[0034] In another preferred embodiment, the CCL11 detection reagent comprises: a CCL11 detection reagent in blood; a CCL11 detection reagent in urine; or a combination thereof.

[0035] In a fourth aspect of the invention, the use of the composition described in the second aspect of the invention or the pharmaceutical composition described in the third aspect of the invention is provided for preparing a medicine box for treating or improving stress dermatitis.

[0036] In a fifth aspect of the invention, a medicine box for treating or improving stress dermatitis is provided, the medicine box containing the composition described in the second aspect of the invention or the pharmaceutical combination described in the third aspect of the invention.

[0037] In another preferred embodiment, the medicine box also includes a PSS scale.

[0038] In another preferred embodiment, the medicine box also includes an instruction manual.

[0039] In another preferred embodiment, the instructions direct the kit to be used to treat or improve stress dermatitis.

[0040] In another preferred embodiment, the specification states that the CCR antagonist is administered when the CCL11 level of the test subject is significantly higher than that of the normal control.

[0041] In another preferred embodiment, the specification states that the CCR antagonist is administered when the eosinophil level in the test subject is significantly higher than that in the normal control.

[0042] In another preferred embodiment, the eosinophil level is the eosinophil level in a complete blood count result.

[0043] In another preferred embodiment, the eosinophil level being significantly higher than that of the normal control means that the eosinophil level M1 of the test subject is ≥1.1, preferably ≥1.2, and most preferably ≥1.3 compared with the eosinophil level L0 of the normal control.

[0044] In another preferred embodiment, the instructions state that the CCR antagonist is administered when the subject's PSS score on the kit is ≥14 points, preferably ≥29 points.

[0045] In another preferred embodiment, the specification states that the dosage of the CCR3 antagonist is 0.1-8 mg / kg, more preferably 0.12-6 mg / kg, even more preferably 0.14-5 mg / kg, even more preferably 0.16-3 mg / kg, and most preferably 0.2-1 mg / kg.

[0046] In another preferred embodiment, the specification states that the dosage of the CCR3 antagonist is 0.1-1 mg / kg.

[0047] In another preferred embodiment, the specification states that the application of the CCR antagonist comprises: multiple applications and / or a single application.

[0048] In a sixth aspect of the invention, a Transwell cell migration model is provided, wherein the upper chamber of the Transwell cell migration model comprises eosinophils and the lower chamber of the Transwell cell migration model comprises sympathetic nerve cells.

[0049] Among them, the sympathetic nerve cells are Pdyn + Sympathetic nerve cells.

[0050] In another preferred embodiment, the eosinophils are eosinophils derived from an animal model of atopic dermatitis.

[0051] In another preferred embodiment, the eosinophils are eosinophils derived from peripheral blood of an animal model of atopic dermatitis.

[0052] In another preferred embodiment, the atopic dermatitis model is an animal model induced by MC903.

[0053] In another preferred embodiment, the animal model comprises: a human.

[0054] In another preferred embodiment, the animal model comprises: a non-human mammal.

[0055] In another preferred embodiment, the non-human mammals include: mice, rats, rabbits, monkeys, and dogs.

[0056] In another preferred embodiment, the Pdyn + Sympathetic nerve cells are Pdyn cells that can be specifically activated by external stimuli. + Sympathetic nerve cells.

[0057] In another preferred embodiment, the external stimulus includes: light stimulation.

[0058] In another preferred embodiment, the light comprises: blue light at 473 nm.

[0059] In another preferred embodiment, the Pdyn + Sympathetic nerve cells originate from Pdyn Net - Sympathetic nerve cells of Catch (Catch) transgenic mice.

[0060] In another preferred embodiment, the Pdyn Net -Catch (Catch) transgenic mice are produced through... Figure 1 Transgenic mice prepared by the method described above.

[0061] In a seventh aspect of the invention, the use of the Transwell cell migration model described in the sixth aspect of the invention is provided for screening a drug for treating or improving stress dermatitis.

[0062] In an eighth aspect of the invention, a method for screening a medicament for treating or improving stress dermatitis is provided, comprising the following steps:

[0063] (S1) In the experimental group, in the presence of the test substance, in Pdyn + The Transwell cell migration model of the sixth aspect of the present invention was cultured under conditions of sympathetic nerve cell activation; and in a control group, the Transwell cell migration model of the sixth aspect of the present invention was cultured under the same conditions but without the test substance; and

[0064] (S2) The number of eosinophils that migrated to the lower chamber and the number of eosinophils remaining in the upper chamber were measured in the experimental group and the control group, respectively, and the results were calculated and compared. If there was no significant change between the ratio Y1 of the number of eosinophils that migrated to the lower chamber in the experimental group to the number of eosinophils initially inoculated and the ratio Y0 of the number of eosinophils that migrated to the lower chamber in the control group to the number of eosinophils initially inoculated, then the test substance is a potential therapeutic agent for treating or improving stress dermatitis.

[0065] The initial number of eosinophils is the sum of the number of eosinophils that migrated to the lower chamber and the number of eosinophils remaining in the upper chamber.

[0066] In another preferred embodiment, the presence of the test substance means that the upper and lower chambers of the Transwell cell migration model contain the same concentration of the test substance.

[0067] In another preferred embodiment, the test substance is a single compound, a mixture, an extract, or a compound preparation.

[0068] In another preferred embodiment, "no significant change" means that the ratio of Y1 to Y0, Y1 / Y0, is ≤1.09, preferably ≤1.08, more preferably ≤1.05, and most preferably ≤1.03.

[0069] In another preferred embodiment, "no significant change" means that the ratio of Y1 to Y0, Y1 / Y0, is ≥0.9, preferably ≥0.93, more preferably ≥0.95, and most preferably ≥0.97.

[0070] In another preferred embodiment, the method further includes: (S3) further performing activity testing or functional testing on the potential therapeutic agents selected in the previous step.

[0071] In another preferred embodiment, the activity test is performed in a non-human animal model of stress dermatitis.

[0072] 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. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0073] Figure 1 Pdyn was displayed Net-CatCh (CatCh) mouse gene strategy description. Pdyn-Cre (The Jackson Laboratory, 027958), Net-Flpo: from Professor Zhan Cheng's laboratory at the University of Science and Technology of China, CatCh (The Jackson Laboratory, 025109).

[0074] Figure 2 The diagram shows a schematic of the Transwell cell migration experiment. Eosinophils and sympathetic nerve cells from Ctrl or Catch mice with an atopic dermatitis model induced by MC903 were sorted and placed in the upper and lower chambers of the Transwell apparatus, respectively. After activating the sympathetic nerve cells of the Catch mice by irradiation with 473nm blue light, the migration of eosinophils from the upper chamber to the lower chamber was observed.

[0075] Figure 3 The results showed that the proportion of eosinophil migration was increased in the Catch group compared to the Ctrl group, indicating that sympathetic nerve activation leads to eosinophil migration. Simultaneously, ELISA experiments showed that lower ventricular sympathetic neurons in the Catch group released more CCR3 ligand CCL11.

[0076] Figure 4 The study showed a positive correlation between the Perceived Stress Scale (PSS) score and blood eosinophil (Eos) levels in patients with atopic dermatitis.

[0077] Figure 5 The study showed that the CCR3 antagonist SB297006 inhibited eosinophil migration, indicating that the sympathetic nervous system chemotactically attracts eosinophils via CCR3.

[0078] Figure 6 The study showed that stress activates sympathetic neurons, which in turn chemotactically attract eosinophils via the CCL11-CCR3 axis, exacerbating atopic dermatitis.

[0079] Figure 7 The study showed that injecting non-stressed atopic dermatitis model mice with the CCR3 antagonist SB297006 or its homologous solvent resulted in no significant difference in the severity of skin inflammation between the two groups of mice.

[0080] Figure 8 The study demonstrated that injecting atopic dermatitis model mice with the CCR3 antagonist SB297006 or its homologous solvent and subjecting them to high-stress RHS (Respiratory Health Study) showed that the CCR3 antagonist SB297006 inhibited stress-induced skin inflammation, demonstrating the important role of CCR3 in stress dermatitis. Detailed Implementation

[0081] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that a CCR3 antagonist, SB297006, can effectively improve atopic dermatitis exacerbated by stress, but has no significant effect on atopic dermatitis exacerbated by non-stress. Based on this discovery, the present invention was completed.

[0082] the term

[0083] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0084] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0085] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0086] Where a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that every intermediate integer of the value 20, every tenth of every intermediate integer of the value, any other intermediate value between the upper and lower limits of the range, and any other intermediate value within the specified range are included in this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered by this invention, but are subject to any express exclusions within the specified range. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0087] As used herein, the treatment or improvement of a disease includes treating the disease, improving disease symptoms, preventing disease symptoms from worsening, slowing the rate of disease symptom worsening, improving disease-related physiological indicators, preventing disease-related physiological indicators from worsening, slowing the rate of disease-related physiological indicator worsening, preventing the disease, or combinations thereof.

[0088] Atopic dermatitis

[0089] Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease and one of the most common skin diseases. The incidence of AD is on the rise globally, affecting people of all ages.

[0090] The clinical manifestations of Alzheimer's disease (AD) include skin barrier dysfunction, severe itching, dryness, eczema, erythema, etc., which usually occur in the joint area and on the face.

[0091] The pathogenesis of Alzheimer's disease (AD) is complex and not yet fully understood. Influencing factors include family history, environmental factors, and immune dysregulation.

[0092] Currently, the main treatment for Alzheimer's disease (AD) focuses on repairing the skin barrier, reducing allergen exposure, and establishing long-term control. Treatment for moderate to severe AD still primarily relies on corticosteroids and biologics. However, systemic use of corticosteroids and immunosuppressants has significant side effects, may damage the skin barrier, and is prone to rebound phenomena.

[0093] Stress dermatitis

[0094] Stress dermatitis, as the name suggests, is a skin inflammation caused by psychological or emotional stress. Common stimuli include psychological factors such as tension, anxiety, and depression.

[0095] Unlike allergic skin reactions, stress dermatitis does not directly involve abnormalities in the immune system, but rather is a skin response to stress.

[0096] Studies have shown that when individuals are under high stress, their skin barrier function declines, making them more susceptible to inflammation.

[0097] From a symptomatic perspective, symptoms of stress dermatitis may include localized redness, swelling, stinging, and scaling, which often worsen under conditions of high psychological stress. Patients may find that their symptoms are closely related to mood swings.

[0098] Clinical observations and experimental studies have found that psychological stress plays a significant role in the occurrence and development of Alzheimer's disease (AD), often leading to a worsening of AD symptoms.

[0099] Stress dermatitis is characterized by fluctuating symptoms that can rapidly worsen after stress. It is often accompanied by psychological symptoms such as sleep disturbances, anxiety, and depression, making it prone to becoming chronic. Repeated stress can prolong the course of the disease, making it difficult to control with anti-inflammatory drugs alone.

[0100] Currently, treatment focuses on skin barrier repair, reducing allergen exposure, and establishing long-term control. Systemic therapies such as corticosteroids and immunosuppressants fail to target the pathways that exacerbate skin inflammation due to stress. Therefore, there is an urgent need to develop more safe and effective treatments for moderate to severe Alzheimer's disease exacerbated by stress.

[0101] It is important to note that stress dermatitis and atopic dermatitis are two different classification methods. In a specific embodiment, stress dermatitis is atopic dermatitis exacerbated by stress. In a specific embodiment, stress dermatitis is non-atopic dermatitis exacerbated by stress. In a specific embodiment, stress dermatitis refers to dermatitis exacerbated by stress, which includes both atopic and non-atopic dermatitis.

[0102] CCR3 antagonists

[0103] CCR3 (CC chemokine receptor 3) is a G protein-coupled receptor (GPCR) that is mainly expressed on the surface of immune cells such as eosinophils, mast cells, and Th2 T cells.

[0104] CCR3 is a key receptor that drives the migration of eosinophils to sites of inflammation. Specifically, CCR3 is a key chemokine receptor on the surface of eosinophils. Its main function is to act like a "GPS navigation system," guiding eosinophils from the bloodstream to tissues experiencing allergies, parasitic infections, or certain inflammations (such as the lungs of asthma patients or the nasal mucosa of allergic rhinitis patients).

[0105] CCR3 antagonists are a class of small molecule compounds or biological agents that can specifically bind to CCR3. Their core function is to competitively block the binding of CCR3 to ligands.

[0106] PSS scale

[0107] The Perceived Stress Scale (PSS) is a standardized tool widely used internationally to assess an individual's perceived stress level. It was developed by American psychologist Sheldon Cohen and others in 1983.

[0108] This scale focuses on an individual's subjective perception and coping ability of stressful events in life, rather than the objective stressful events themselves. The core dimensions include: the sense of uncontrollability, overload, and difficulty in coping with stressful events.

[0109] The PSS (Persistent Stress) scale was used to inquire about the participants' feelings and thoughts over the past month. Commonly used versions include the 10-question version (PSS-10), the 14-question version (PSS-14), and the 4-question abbreviated version (PSS-4). The scale uses a 5-point Likert scale (0 = never, 4 = always), with a higher total score indicating greater perceived stress.

[0110] The Stress Study Scale (PSS) is applicable to research, clinical practice, and health management. It can help assess the association between stress and mental illnesses (such as anxiety and depression) and physical health (such as cardiovascular disease), providing a basis for stress intervention. Its advantages lie in its ease of use and strong cross-cultural adaptability, but it needs to be combined with other tools to comprehensively determine the sources and effects of stress.

[0111] The composition of the present invention

[0112] The compositions of the present invention comprise a CCR antagonist as a first active component. In a specific embodiment, the CCR antagonist is the sole active component. In a specific embodiment, the CCR antagonist is SB297006.

[0113] As used herein, the composition preferably contains 0.1-99 wt% of a first active component, with the remainder being a second active component, a pharmaceutically acceptable carrier, a diluent or solution or a salt solution.

[0114] When necessary, one or more pharmaceutically acceptable carriers may be added to the composition. These carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., commonly used in the pharmaceutical field.

[0115] The compositions provided by this invention can be in various forms, such as tablets, injections, capsules, powders, syrups, solutions, suspensions, and aerosols, and can be present in suitable solid or liquid carriers or diluents and in suitable sterilization devices for injection or infusion.

[0116] Various dosage forms of the compositions of the present invention can be prepared according to conventional pharmaceutical preparation methods. The dosage form typically contains 0.05-1000 mg of the active compound of the present invention per unit volume, and preferably, the dosage form contains 1-500 mg of the active compound of the present invention per unit volume.

[0117] The compositions of this invention are suitable for clinical use in mammals, including humans and animals, and can be administered via oral, nasal, skin, lung, or gastrointestinal routes. Skin administration is most preferred. The most preferred daily dose is 0.01-400 mg / kg body weight, administered as a single dose, or 0.01-200 mg / kg body weight, administered in divided doses. Regardless of the method of administration, the optimal dose for an individual should be determined based on the specific treatment. Generally, a low dose is started, and the dose is gradually increased until the most suitable dose is found.

[0118] As used in this article, the amount of drug administered per unit body weight can be converted to the amount of drug administered per unit body surface area.

[0119] The compositions of the present invention can be administered in various ways, such as by injection, spray, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods, into the body such as muscle, intradermal, subcutaneous, vein, or mucosal tissue; or by being mixed with or encapsulated by other substances before being introduced into the body.

[0120] Typically, the active ingredient of the present invention or a composition containing it can be administered in unit dose form via enteral or non-enteric routes, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0121] Dosage forms can be liquid, solid, or semi-solid. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including O / W, W / O, and double emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can include tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, microcapsules, pellets, suppositories, films, patches, aerosols, and sprays, etc.; semi-solid dosage forms can include ointments, gels, and pastes, etc.

[0122] The active ingredients of this invention can be formulated into ordinary formulations, sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0123] In order to formulate the active ingredient of the present invention into tablets, a wide variety of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, croscarmellose, croscarmellose sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0124] Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0125] To formulate the drug delivery unit into capsules, the active ingredient of this invention can be mixed with a diluent and a flow aid, and the mixture can be placed directly into hard or soft capsules. Alternatively, the active ingredient can be first formed into granules or microspheres with a diluent, binder, and disintegrant, and then placed into hard or soft capsules. The diluents, binders, wetting agents, disintegrants, and flow aids used to prepare the tablets of this invention can also be used to prepare the capsules of this invention.

[0126] To prepare the active ingredient of this invention into an injection, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of commonly used solubilizers, co-solvents, pH adjusters, and osmotic pressure regulators can be added. Solubilizers or co-solvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure regulators can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing a lyophilized powder injection, mannitol, glucose, etc., can also be added as a support agent.

[0127] In addition, colorants, preservatives, flavorings, tasters or other additives may be added to pharmaceutical preparations if necessary.

[0128] The active ingredients or compositions of the present invention can be taken alone or in combination with other therapeutic or symptomatic drugs.

[0129] When the active ingredient of this invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0130] use

[0131] This invention provides the application of CCR3 antagonists in the treatment of stress dermatitis.

[0132] In a specific embodiment, the present invention provides the application of CCR3 antagonists in the preparation of medicaments for treating stress dermatitis.

[0133] In a specific implementation, the CCR antagonist is SB297006.

[0134] In a specific implementation, the stress dermatitis is atopic dermatitis exacerbated by stress.

[0135] This invention provides a Transwell cell migration model and its use in screening drugs for treating or improving stress dermatitis. The upper chamber of the Transwell cell migration model contains eosinophils; the lower chamber contains sympathetic nerve cells; wherein the sympathetic nerve cells are Pdyn. + Sympathetic nerve cells.

[0136] This invention provides the application of a combination of CCR3 antagonists and CCL11 detection reagents in the companion diagnosis of stress dermatitis.

[0137] Cellular and animal experiments demonstrated the important role of the CCR3 receptor in stress dermatitis and showed that the use of CCR3 antagonists can inhibit eosinophil chemotaxis and alleviate stress dermatitis, providing a basis for drug development for stress dermatitis.

[0138] In a specific implementation, the CCR3 antagonist inhibits the chemotaxis of activated sympathetic nerve cells to eosinophils.

[0139] In another preferred embodiment, the activated sympathetic nerve cells are activated sympathetic nerve cells cultured in vitro.

[0140] In specific implementations, CCR3 antagonists alleviate stress-exacerbated atopic dermatitis in mice through one or more of the following pathways: increased TEWL (transdermal water loss), increased scratching bouts (number of scratches per hour), and increased dermal thickness.

[0141] The main advantages of this invention include:

[0142] (a) This invention provides the application of the CCR3 antagonist SB297006 in the treatment of stress dermatitis; the CCR3 antagonist precisely targets the pathway that psychological stress aggravates AD, inhibits the chemotaxis of eosinophils, and can more effectively relieve skin inflammation.

[0143] (b) When CCR3 antagonists are used to treat stress dermatitis, they are fast-acting, safe, and have few side effects. They do not cause the pigmentation, skin atrophy, or other side effects that are caused by hormone drugs.

[0144] (c) The CCR3 antagonist SB297006 provided by this invention is a targeted treatment and has a significant improvement effect on atopic dermatitis exacerbated by stress (as measured by PSS score), with a good prognosis.

[0145] 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, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0146] Example 1: Chemotaxis of activated sympathetic neurons on eosinophils in an in vitro experiment.

[0147] First, such as Figure 1 As shown, this embodiment constructs a Pdyn Net-Catch transgenic mice. Calcium-translocating channelrhodopsin (CatCh) and enhanced yellow fluorescent protein (eYFP) are selectively expressed in the Pdyn receptors of these transgenic mice. Net In sympathetic neurons, this causes Pdyn in mice to... + Sympathetic nerve cells can be specifically activated by 473nm blue light.

[0148] Catch mice and littermate control mice were euthanized and dissected on ice. Fresh paravertebral sympathetic ganglia (superior cervical ganglion SCG, stellate ganglion STG, and T2-T4 thoracic ganglia) were isolated. After removing connective tissue, the isolated sympathetic ganglia were transferred to 1 mL of calcium- and magnesium-free 1×HBSS buffer (C14175500BT, Gibco), supplemented with 2 μL of saturated NaHCO3 (S5761, Sigma), 0.35 mg of L-cysteine ​​(C7352, Sigma), and 20 U of papain (A003124-0100, Diamond), and then digested at 37°C for 30 minutes. After collecting the sample by centrifugation at 100g for 1 minute, add 1 mL of calcium- and magnesium-free HBSS buffer containing 4 mg collagenase II (40508ES76, Yisheng) and 1.25 mg dispersin II (40104ES80, Yisheng), and continue digestion at 37°C for 30 minutes. Centrifuge at 100g for 10 minutes, collect the cell pellet, and resuspend it in 1 mL of calcium- and magnesium-free HBSS buffer. After centrifugation at 100g for another 10 minutes, resuspend the cell pellet in neural basal medium (21103-049, Gibco), which contains 2% B-27 additive (17504-044, Gibco), 1% L-glutamine (35050-061, Gibco), 100 U / mL penicillin, and 100 μg / mL streptomycin. Finally, the cells were seeded onto 12mm coverslips (80344-1220, Sieg Glass) coated with 10μg / mL poly-L-lysine and incubated in a humidified incubator at 37℃ and 5% CO2 for 18-24 hours before subsequent experimental treatments.

[0149] Next, eosinophils were isolated from the peripheral blood of adult C57BL / 6J mice (GemPharmatech, NO. N000013) with an MC903-induced atopic dermatitis model. First, BD Pharm Lyse... TMMouse peripheral blood erythrocytes were lysed with lysis buffer (555899, BD Pharmingen) and centrifuged at 300g for 10 minutes. The cell pellet was collected and resuspended in 80 μL M ACS buffer (containing 0.5% bovine serum albumin, 9048-46-8, Vetec; 2 mM EDTA, 60-00-4, Sigma-Aldrich). 7 Each cell was added to 20 μL of Siglec-F magnetic beads (130-118-513, Miltenyi Biotec) and incubated at 4°C for 10 minutes. Subsequently, based on the total cell count and the number of labeled cells, eosinophils were sorted using a MACS sorting column (130-042-201, Miltenyi Biotec) and a MACS sorter (130-092-108, Miltenyi Biotec). The sorted and purified eosinophils were resuspended in RPMI-1640 (C22400500BT, Gibco) medium containing 10% fetal bovine serum (A3160801, Gibco) and 0.5% bovine serum albumin (V900933-100G, Vetec).

[0150] Next, Transwell cell migration experiments were performed. Specifically, as follows: Figure 2 As shown, the eosinophils were seeded in the upper layer of a Transwell chamber (5 μm pore size, CLS3421, Corning), while the lower chamber contained sympathetic nerve cells from littermates and Catch mice. The apparatus was cultured at 37°C and 5% CO2 for 4 hours. During the culture period, the neurons were intermittently stimulated with 473 nm blue light (20 Hz, 15 mW) for 1 minute per hour. After culture, cells from the upper and lower chambers were collected and labeled with fluorescent antibodies (Lead / Dead-FVS510 (1:1000, 564406, BD Bioscience), anti-CD45-APC (1:200, 103112, Biolegend), anti-CD11b-BV421 (1:100, 562605, BD Bioscience), and anti-Siglec-F-PE (1:100, 552126, BD Bioscience). Eosinophils were quantitatively analyzed by flow cytometry; eosinophils were defined as Lead / Dead cells. - CD45 + CD11b + SiglecF + Cells. The chemotaxis index is calculated as: number of eosinophils migrating to the lower chamber / initial number of eosinophils inoculated into the upper chamber.

[0151] The results are as follows Figure 3 As shown in the figure. The results indicate that activated sympathetic neurons have a significant chemotactic effect on eosinophils, confirming that the neuro-immune axis plays an important role in the process of psychological stress exacerbating skin inflammation.

[0152] Following psychological stress, the activation and release of sympathetic nerves led to a significant increase in the concentration of the chemokine CCL11, meaning that the lower ventricular sympathetic neurons in the Catch group released more chemokine CCL11.

[0153] Example 2: CCR3 antagonists inhibited the chemotaxis of activated sympathetic neurons to eosinophils in in vitro experiments.

[0154] The experimental procedure was the same as in Example 1, except that during the 4-hour co-culture period, 20 μM of the CCR3 antagonist SB297006 (58816-69-6, MedChemExpress) was added to both the upper and lower chambers of the Transwell cell migration model. CCR3 (C-Cchemokine receptor type 3) is a G protein-coupled receptor (GPCR) belonging to the chemokine receptor family.

[0155] Perceived stress in patients with atopic dermatitis was assessed using the PSS scale (PSS-14), and eosinophil levels in their blood were measured. Results were as follows: Figure 4 As shown, the Perceived Stress Scale (PSS) score in patients with atopic dermatitis is positively correlated with the level of eosinophils (Eos) in the blood.

[0156] After adding the CCR3 antagonist SB297006, as Figure 5 As shown, eosinophil migration was significantly inhibited, indicating that the sympathetic nervous system chemotactically attracts eosinophils through CCR3.

[0157] like Figure 6 As shown, CCL11 interacts with the chemokine receptor CCR3 on the surface of eosinophils, causing a large number of eosinophils to chemotactically migrate to the site of inflammation, leading to aggravation of skin inflammation. Specifically, CCR3, as the main chemokine receptor for eosinophils, regulates the migration and activation of immune cells by binding to a specific chemokine (Eotaxin-1 / CCL11).

[0158] The CCR3 antagonist SB297006 works by blocking the binding of CCR3 to CCL11.

[0159] Example 3: Intraperitoneal injection of a CCR3 antagonist into mice alleviated stress-induced atopic dermatitis.

[0160] In this embodiment, an MC903-induced mouse atopic dermatitis model was established. Adult C57BL / 6J mice were anesthetized by inhalation of isoflurane (2-3%), and their body temperature was maintained using a heating pad. Two days before the start of modeling, hair was shaved from a 1cm × 2cm area on the right side of the mouse's nape. MC903 (100μL, 0.1mM, dissolved in 100% ethanol; Tocris, 2700) was topically applied to the shaved area on the nape of the neck and administered for 5 consecutive days.

[0161] Starting from day 5, the mice were subjected to five days of repeated high-platform stress (RHS), in which the mice were placed on a transparent circular platform with a diameter of 10 cm (1.5 m above the ground) and subjected to stress twice a day (10:00-12:00 am and 10:00-12:00 pm), each lasting 15 minutes.

[0162] The experimental group mice were injected intraperitoneally with SB297006 (5 mg / kg) 30 minutes before stress, while the control group was injected with an equal volume of the same solvent.

[0163] Mice were tested on day 10, including transepidermal water loss (TEWL), a measure of skin barrier function; the number of scratching bouts per hour; and dermal thickness.

[0164] Transdermal water loss (TEWL): The TEWL of mice was measured using a VAPOSCAN AS-VT100 RS device (Asahi TechnoLab.Ltd., Yokohama, Kanagawa, Japan). Each mouse was measured three times, and the average value was taken.

[0165] Scratching bouts in mice per hour: Mouse behavior was recorded and analyzed using an infrared imaging system (Future LifeScience FLS-37258; China). Scratching behavior was defined as the continuous scratching of the mouse's back or affected area with its hind paws, ending when the mouse licked its paws or put its hind paws back on the bottom of the cage.

[0166] After harvesting skin tissue from the lesion areas of mice, the skin tissue was fixed in 4% paraformaldehyde (PFA) solution and then embedded in paraffin for histological analysis. The sections were stained using a hematoxylin and eosin (H&E) staining kit (ab245880, Abcam) according to the manufacturer's instructions. Five random fields of view were selected from three tissue sections from each mouse, and the vertical distance from the epidermal-dermal junction to the dermal-subcutaneous junction was measured. The average value was used to calculate the dermal thickness.

[0167] Results of non-stress atopic dermatitis model mice injected with CCR3 antagonist SB297006 or isoform solvents as follows Figure 7 As shown, there was no significant difference in the severity of skin inflammation between the two groups of mice.

[0168] When mice with atopic dermatitis were injected with the CCR3 antagonist SB297006 or its homologous solvent and subjected to high-stress RHS, the results were as follows: Figure 8 As shown, the CCR3 antagonist SB297006 significantly inhibited stress-induced skin inflammation, further demonstrating the important role of CCR3 in stress dermatitis.

[0169] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of a CCR3 antagonist, characterized in that, This is used to prepare a composition for treating or improving stress dermatitis.

2. The use as described in claim 1, characterized in that, The CCR3 antagonist is SB297006.

3. The use as described in claim 1, characterized in that, The stress dermatitis mentioned is atopic dermatitis exacerbated by stress.

4. A composition, characterized in that, The pharmaceutical composition comprises the following components: (Z1) The first active component, wherein the first active component is SB297006; and (Z2) The second active component is another drug for treating stress dermatitis.

5. A drug combination, characterized in that, The drug combination comprises: (a) A CCR3 antagonist, wherein the CCR antagonist is SB297006; (b) CCL11 detection reagent.

6. The use of the composition of claim 4 or the pharmaceutical combination of claim 5, characterized in that, This is used to prepare a medicine box for treating or improving stress dermatitis.

7. A medicine box for treating or improving stress dermatitis, characterized in that, The medicine box contains the composition of claim 4 or the drug combination of claim 5.

8. A Transwell cell migration model, characterized in that, The upper chamber of the Transwell cell migration model contains eosinophils; the lower chamber of the Transwell cell migration model contains sympathetic nerve cells. Among them, the sympathetic nerve cells are Pdyn + Sympathetic nerve cells.

9. The use of the Transwell cell migration model according to claim 8, characterized in that, Used to screen a drug for the treatment or improvement of stress dermatitis.

10. A method for screening drugs for treating or improving stress dermatitis, characterized in that, Includes the following steps: (S1) In the experimental group, in the presence of the test substance, in Pdyn + The Transwell cell migration model of claim 8 is cultured under conditions of sympathetic nerve cell activation; and In the control group, the Transwell cell migration model of claim 8 was cultured under the same conditions but in the absence of the test substance; and (S2) The number of eosinophils that migrated to the lower chamber and the number of eosinophils remaining in the upper chamber were measured in the experimental group and the control group, respectively, and the results were calculated and compared. If there was no significant change between the ratio Y1 of the number of eosinophils that migrated to the lower chamber in the experimental group to the number of eosinophils initially inoculated and the ratio Y0 of the number of eosinophils that migrated to the lower chamber in the control group to the number of eosinophils initially inoculated, then the test substance is a potential therapeutic agent for treating or improving stress dermatitis. The initial number of eosinophils is the sum of the number of eosinophils that migrated to the lower chamber and the number of eosinophils remaining in the upper chamber.

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