Use of muscarinic receptor type 5 receptor antagonists

Abstract

The present application discloses the use of muscarinic receptor 5 antagonists. In the present application, new use of muscarinic receptor 5 antagonists for treating chronic pruritus, especially chronic pruritus caused by psoriasis is found; new target M5 receptor for treating chronic pruritus, especially chronic pruritus caused by psoriasis is found, and effective means for treating chronic pruritus, especially chronic pruritus caused by psoriasis is developed.

Description

Technical Field

[0001] This invention relates to biomedicine, and in particular to the use of a muscarinic receptor type 5 antagonist. Background Technology

[0002] The muscarinic receptor (M1-M5) is a type of G protein-coupled receptor (GPCR) with a conserved seven-helix transmembrane structure. Five different subtypes of the muscarinic receptor (M1-M5) have been identified, widely distributed in vivo and performing different biological functions. In keratinocytes, the M1 receptor is mainly expressed in the upper epidermis, while M3 and M5 are mainly expressed in the middle and lower epidermis, namely the spinous and basal layers.

[0003] M5, a newly discovered membrane receptor, has been found to possess numerous unique functions. For example, M5 regulates depressive and anxious behaviors in the VTA brain region; activation of M3 and M5 can lead to vasodilation of small arteries; activation of M5 receptors on lymphocytes can increase lymphocyte signaling, promote the release of various cytokines, and participate in the regulation of immune system activity; activation of M5 in the urinary system can lead to bladder hyperactivity; M5 also has effects different from the other four types of M-type receptors in areas such as glandular secretion, osteoporosis, gastrointestinal regulation, and drug addiction. Research on the mechanisms of itch sensation has suggested a correlation between M3 receptors and itch sensation; for example, pharmacological animal experiments have demonstrated that intradermal injection of cholinergic drugs mediates itch sensation through M3 receptors. However, there are no reports on whether M5 receptors are involved in the regulation of itch sensation.

[0004] Psoriasis is a genetically predisposed, multifactorial, erythematous scaly disease often accompanied by intense itching. Chronic itching can trigger scratching, which in turn worsens the rash, creating a vicious cycle and negatively impacting the prognosis of psoriasis patients. Currently, psoriasis can be treated with oral antihistamines to relieve itching, but these are not very effective for some patients with chronic itching. Even if the underlying disease causing the itching improves, they may still experience unbearable itching.

[0005] Therefore, there is an urgent need in this field to study the mechanisms of chronic itching, especially chronic itching caused by psoriasis, in order to develop new drugs with better efficacy. Summary of the Invention

[0006] The purpose of this invention is to provide the use of a muscarinic receptor type 5 receptor antagonist.

[0007] The purpose of this invention is to provide the use of a type IV histamine receptor antagonist.

[0008] Another object of the present invention is to provide a method for preventing and / or treating chronic itching caused by psoriasis.

[0009] To address the aforementioned technical problems, a first aspect of the present invention provides the use of a muscarinic receptor type 5 receptor antagonist for:

[0010] (i) Prevention and / or treatment of chronic pruritus;

[0011] (ii) To prepare medicines for the prevention and / or treatment of chronic pruritus; and / or

[0012] (iii) Non-therapeutic reduction of PLC, PKC and / or Gβγ expression levels in vitro.

[0013] In some preferred embodiments, the muscarinic receptor type 5 receptor antagonist is WXY-1-1, WXY-3-5, or VU0488130; preferably VU0488130.

[0014] In some preferred embodiments, the muscarinic receptor type 5 antagonist is used as a research reagent to reduce the expression levels of intracellular PLC, PKC and / or Gβγ in vitro in a non-therapeutic manner.

[0015] In some preferred embodiments, the chronic itching is chronic itching caused by psoriasis.

[0016] In some preferred embodiments, the use includes administering a therapeutically effective amount of a muscarinic receptor type 5 antagonist to a subject.

[0017] In some preferred embodiments, the effective therapeutic dose is 0.01 μg-100 mg / kg body weight / day.

[0018] In some preferred embodiments, the muscarinic receptor type 5 receptor antagonist is administered via transdermal or injectable route.

[0019] In some preferred embodiments, the administration frequency of the muscarinic receptor type 5 antagonist is once daily, twice daily, three times daily, once every two days, once every three days, or once weekly.

[0020] In some preferred embodiments, the muscarinic receptor type 5 receptor antagonist is prepared as a topical or injectable formulation for application.

[0021] In some preferred embodiments, the topical preparation is a liquid or semi-solid topical preparation.

[0022] In some preferred embodiments, the concentration of the active ingredient in the liquid or semi-solid topical formulation is from 0.1 μg / 1 μL to 10 μg / 1 μL.

[0023] In some preferred embodiments, the injectable formulation is a liquid, wherein the concentration of the active ingredient in the injectable formulation is from 0.1 μg / 1 μL to 10 μg / 1 μL.

[0024] A second aspect of the invention provides the use of a muscarinic receptor type 3 receptor antagonist for:

[0025] (i) Prevention and / or treatment of chronic itching caused by psoriasis; and / or

[0026] (ii) To prepare medicines for the prevention and / or treatment of chronic itching caused by psoriasis.

[0027] A third aspect of the present invention provides a method for preventing and / or treating chronic pruritus, the method comprising the steps of:

[0028] Administer a therapeutically effective dose of a muscarinic receptor type 5 antagonist to the test subject; or,

[0029] A therapeutically effective amount of a pharmaceutical composition containing a muscarinic receptor type 5 antagonist is administered to a test subject.

[0030] In some preferred embodiments, the chronic itching is chronic itching caused by psoriasis.

[0031] A fourth aspect of the present invention provides a method for preventing and / or treating chronic itching caused by psoriasis, the method comprising the steps of:

[0032] Administer a therapeutically effective dose of a type IV histamine receptor antagonist to the test subject; or...

[0033] Administering a therapeutically effective amount of a pharmaceutical composition containing a type IV histamine receptor antagonist to a test subject; or,

[0034] Administer a therapeutically effective dose of a muscarinic type 3 receptor antagonist to the test subject; or

[0035] A therapeutically effective amount of a pharmaceutical composition containing a type IV histamine receptor antagonist is administered to a test subject.

[0036] Compared with the prior art, the present invention has at least the following advantages:

[0037] (1) This invention develops a new use for muscarinic receptor type 5 antagonists for the treatment of chronic pruritus, especially chronic pruritus caused by psoriasis;

[0038] (2) This invention has discovered a new target for the treatment of chronic pruritus, especially chronic pruritus caused by psoriasis—the M5 receptor;

[0039] (3) The present invention has developed an effective means of treating chronic pruritus, such as administering an M5 receptor antagonist to a test subject. The present invention has also developed an effective means of treating chronic pruritus caused by psoriasis, such as administering an M5 receptor antagonist, an M3 receptor antagonist, a type IV histamine receptor antagonist, or a pharmaceutical composition containing them to a test subject.

[0040] 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

[0041] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0042] Figure 1A This is a flowchart of the IMQ psoriasis mouse animal model creation process according to an embodiment of the present invention;

[0043] Figure 1B This is a comparison image of the neck and back skin of the IMQ modeling experimental group and the control group according to an embodiment of the present invention;

[0044] Figure 1C This is a pathological comparison of the neck and back skin tissues of the experimental group and the control group according to an embodiment of the present invention;

[0045] Figure 2A The figure shows the spontaneous scratching behavior of the IMQ model group and the Vaseline control group according to the embodiment of the present invention. n = 6-7, *0.01 < P < 0.05, **0.001 < P < 0.01, ***P < 0.001, and all data are mean ± SEM.

[0046] Figure 2B This is a diagram of scratching behavior induced by CQ according to an embodiment of the present invention, n=5-7, *0.01<P<0.05, **0.001<P<0.01, ***P<0.001, all data are mean±SEM;

[0047] Figure 2C This is a diagram of histamine-induced scratching behavior according to an embodiment of the present invention, n = 6-7, *0.01 < P < 0.05, **0.001 < P < 0.01, ***P < 0.001, all data are mean ± SEM;

[0048] Figure 3A This is a graph showing the changes in scratching behavior of a cheek model mouse representing itching according to an embodiment of the present invention, n=5-6, *0.01<P<0.05. All data are mean±SEM;

[0049] Figure 3B This is a graph showing the changes in wiping behavior in two groups of mice representing pain according to an embodiment of the present invention, n=5-6, *0.01<P<0.05. All data are mean±SEM;

[0050] Figure 4A This figure shows the effect of low (0.3 μg / 10 μL) and high (0.6 μg / 10 μL) concentrations of the type I histamine receptor antagonist Chlorpheniramine injected intrathecally on pruritus behavior in mice, according to embodiments of the present invention. n = 5-7, *0.01 < P < 0.05. All data are mean ± SEM.

[0051] Figure 4B This figure shows the effect of intrathecal injection of a high concentration (10 μg / 10 μL) of the type IV histamine receptor antagonist JNJ7777120 on pruritus behavior in mice, according to an embodiment of the present invention. n = 5-6, *0.01 < P < 0.05. All data are mean ± SEM.

[0052] Figure 5A The graph shows the expression levels of M1, M3, and M5 mRNA in primary sensory neurons of mice in the control group according to the embodiments of the present invention. n=3, *0.01<P<0.05, **0.001<P<0.01, and all data are mean±SEM.

[0053] Figure 6A This is a graph showing the effect of applying low-dose (3 μg / 10 μL) and high-dose (30 μg / 10 μL) Pirenzepine, an M1 receptor-specific antagonist, on pruritus behavior in mouse models after modeling, according to an embodiment of the present invention. n = 7-8, **0.001 < P < 0.01, all data are mean ± SEM;

[0054] Figure 6B This is a graph showing the effect of intrathecal injection of a high dose (2 μg / 10 μL) of the M3 receptor-specific antagonist 4-DAMP on the pruritus behavior of mice in the model group according to an embodiment of the present invention. n=5, **0.001<P<0.01, all data are mean±SEM;

[0055] Figure 6C This is a graph showing the effect of intrathecal injection of a high dose (10 μg / 10 μL) of the M5 receptor-specific antagonist ML381 on the pruritus behavior of mice in the model group according to an embodiment of the present invention. n = 5-8, **0.001 < P < 0.01, all data are mean ± SEM.

[0056] Figure 6DThis is a graph showing the effect of intrathecal injection of low-dose (0.5 μg / 10 μL) and high-dose (1 μg / 10 μL) M2 receptor-specific antagonist Methoctramine on pruritus behavior in model mice according to embodiments of the present invention. n=5, **0.001<P<0.01, all data are mean±SEM.

[0057] Figure 6E Figure n=6, **0.001<P<0.01, all data are mean±SEM; This is based on an embodiment of the present invention. The effect of intrathecal injection of low-dose (5μg / 10μL) and high-dose (10μg / 10μL) M4 receptor-specific antagonist Tropicamide on pruritus behavior in model mice.

[0058] Figure 7A The graphs show the expression of M3 and M5 in the DRG of mice in the IMQ model group and the control group according to the embodiments of the present invention, where B and C are statistical graphs respectively.

[0059] Figure 7B This is a graph showing the positive ratio of DRG M3 in the IMQ group and the control group according to an embodiment of the present invention, with blue arrows indicating M3 positive neurons;

[0060] Figure 7C This is a graph showing the DRG M5 positive ratio of the IMQ group and the control group according to an embodiment of the present invention. The blue arrows indicate M5 positive neurons, and n=5.

[0061] Figure 7D This is a co-localization map of M3, IB4, and CGRP in the DRG of the neck and back of a normal mouse according to an embodiment of the present invention. The blue arrows represent M3 positive neurons, and the white arrows represent IB4 and M3 colocalized neurons.

[0062] Figure 7E This is a co-labeled statistical plot of M3 and IB4 in the DRG of normal mice in the neck and back according to the embodiment of the present invention. **0.001 < P < 0.01, all data are mean ± SEM;

[0063] Figure 7F According to the embodiments of the present invention, M5, IB4, and CGRP are co-labeled in the DRG of the neck and back of normal mice. The blue arrows are M5 positive neurons, and the white arrows are IB4 and M5 co-localized neurons.

[0064] Figure 7G This is a co-labeled statistical graph of M5, IB4, and CGRP in the DRG of normal mice in the neck and back according to the embodiment of the present invention. **0.001 < P < 0.01, all data are mean ± SEM;

[0065] Figure 8AThis is a graph showing the effect of intradermal injection of a high dose (10 μg / μL) of M5 agonist on pruritus behavior in normal mice according to an embodiment of the present invention. n = 5-6, *0.01 < P < 0.05, all data are mean ± SEM.

[0066] Figure 9A This is a graph showing the effect of intrathecal injection of low concentration (2.5 μg / 10 μL) and high concentration (5 μg / 10 μL) of PKA antagonist H-89 on pruritus sensation in psoriasis mice according to embodiments of the present invention. n = 5-8, *0.01 < P < 0.05, *0.001 < P < 0.01, all data are mean ± SEM;

[0067] Figure 9B This is a graph showing the effect of low (1 nM / 10 μL) and high (10 nM / 10 μL) concentrations of the intrathecal injection of PLC antagonist U73122 on the pruritus behavior of psoriasis mice according to an embodiment of the present invention. n = 5, n = 5-8, *0.01 < P < 0.05, *0.001 < P < 0.01, all data are mean ± SEM;

[0068] Figure 9C This is a graph showing the effect of intrathecal injection of low concentration (0.25 μg / 10 μL) and high concentration (0.5 μg / 10 μL) of the PKC antagonist GF109203X on pruritus behavior in psoriatic mice according to embodiments of the present invention. n = 5-12, n = 5-8, *0.01 < P < 0.05, *0.001 < P < 0.01, all data are mean ± SEM;

[0069] Figure 9D The figure shows the effect of intrathecal injection of Gβγ antagonist Gallein (0.3 μg / 10 μL, 3 μg / 10 μL) on chronic pruritus sensation in mice according to the embodiments of the present invention. n = 5-6, n = 5-8, *0.01 < P < 0.05, *0.001 < P < 0.01, all data are mean ± SEM.

[0070] Figure 10A This is a graph showing the effect of intrathecal injection of a high dose (10 nM / 10 μL) of PLC antagonist U73122 on M5 agonist-induced pruritus according to an embodiment of the present invention. n = 7, *0.01 < P < 0.05, and all data are mean ± SEM.

[0071] Figure 10B This is a graph showing the effect of intrathecal injection of a high dose (0.5 μg / 10 μL) of the PKC antagonist GF109203X on M5 agonist-induced pruritus according to an embodiment of the present invention. n = 7-8, *0.01 < P < 0.05, and all data are mean ± SEM.

[0072] Figure 11 This is a graph showing the effect of the M5 receptor antagonist VU0488130 on the pruritus behavior of mice with chronic pruritus psoriasis according to an embodiment of the present invention, where N is 5-8 mice per group. Detailed Implementation

[0073] Through extensive and in-depth research, the inventors discovered a new target for treating chronic pruritus, especially chronic pruritus caused by psoriasis—the M5 receptor. Based on this target, they screened and obtained the M5 receptor antagonist VU0488130, which is effective in treating chronic pruritus caused by psoriasis.

[0074] Uses of muscarinic receptor type 5 antagonists

[0075] This invention relates to the use of muscarinic receptor type 5 antagonists for: (i) prevention and / or treatment of chronic pruritus; (ii) preparation of medicaments for prevention and / or treatment of chronic pruritus; and / or (iii) reduction of PLC, PKC and / or Gβγ expression levels.

[0076] In a preferred embodiment of the present invention, the chronic itching is chronic itching caused by psoriasis.

[0077] In this invention, the "muscarinic receptor type 5 antagonist" can be any muscarinic receptor type 5 antagonist, such as compounds WXY-1-1, WXY-3-5, or VU0488130. For the treatment of chronic pruritus caused by psoriasis, the muscarinic receptor type 5 antagonist is preferably VU0488130, which exhibits superior itch-relieving effects.

[0078] In a preferred embodiment of the present invention, the muscarinic receptor type 5 antagonist prevents and / or treats chronic pruritus caused by psoriasis by reducing the expression levels of PLC, PKC and / or Gβγ.

[0079] In a preferred embodiment of the invention, the use includes administering a therapeutically effective amount of a muscarinic receptor type 5 antagonist to a subject.

[0080] In a preferred embodiment of the present invention, the muscarinic receptor type 5 receptor antagonist is administered via transdermal administration (e.g., local transdermal administration) or injection (e.g., intrathecal injection).

[0081] In a preferred embodiment of the present invention, the therapeutically effective dose of the muscarinic receptor type 5 antagonist is 0.01 μg-100 mg / kg body weight / day.

[0082] In a preferred embodiment of the present invention, the muscarinic receptor type 5 receptor antagonist is prepared as a topical formulation for application.

[0083] compound

[0084] The compound VU0488130 (ML381) in this invention has the structural formula shown in Formula I below. It is a commercially available mAChRM5 antagonist with CAS number 1623481-80-0 and chemical formula: C 25 H 25 N3O8; Molecular weight: 495.68. VU0488130 is soluble in DMSO, hM5IC50 = 0.45μM, hM1IC50 ≥ 10μM, hM2IC50, hM3IC50, hM4IC50 ≥ 30μM.

[0085] The VU0488130 used in this invention was purchased from Aobious, product number: AOB2555, specification: 5mg.

[0086]

[0087] The structural formula of the compound “WXY-1-1” in this invention is shown in Formula I-2 below.

[0088]

[0089] The structural formula of the compound "WXY-3-5" in this invention is shown in Formula I-3 below.

[0090]

[0091] Pharmaceutical Composition

[0092] This invention relates to pharmaceutical compositions containing muscarinic type 5 receptor antagonists. In addition to comprising at least one muscarinic type 5 receptor antagonist as an active ingredient, the pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient. In some embodiments of the invention, the pharmaceutical composition comprises at least one compound selected from WXY-1-1, WXY-3-5, and VU0488130; and a pharmaceutically acceptable carrier or excipient. In the most preferred embodiment, the pharmaceutical composition comprises VU0488130; and a pharmaceutically acceptable carrier or excipient.

[0093] The pharmaceutical compositions of the present invention are preferably used as topical formulations. Preferably, they are prepared as liquid or semi-solid topical formulations. In the liquid or semi-solid topical formulations, the concentration of the active ingredient is from 0.1 μg / 1 μL to 10 μg / 1 μL, preferably from 0.2 μg / 1 μL to 8 μg / 1 μL, preferably from 0.3 μg / 1 μL to 7 μg / 1 μL, preferably from 0.4 μg / 1 μL to 6 μg / 1 μL, preferably from 0.5 μg / 1 μL to 5 μg / 1 μL, preferably from 0.5 μg / 1 μL to 1 μg / 1 μL, or any other range constrained by these values.

[0094] The pharmaceutical compositions of the present invention are preferably used as injectable formulations. Preferably, they are prepared as liquid injectable formulations. In the liquid injectable formulation, the concentration of the active ingredient is from 0.1 μg / 1 μL to 10 μg / 1 μL, preferably from 0.2 μg / 1 μL to 8 μg / 1 μL, preferably from 0.3 μg / 1 μL to 7 μg / 1 μL, preferably from 0.4 μg / 1 μL to 6 μg / 1 μL, preferably from 0.5 μg / 1 μL to 5 μg / 1 μL, preferably from 0.5 μg / 1 μL to 1 μg / 1 μL, or any other range constrained by these values.

[0095] Treatment

[0096] This invention relates to a method for preventing and / or treating chronic pruritus caused by psoriasis, comprising the steps of: administering a therapeutically effective amount of a muscarinic receptor type 5 antagonist to a test subject; or administering a therapeutically effective amount of a pharmaceutical composition containing a muscarinic receptor type 5 antagonist to a test subject.

[0097] In a preferred embodiment of the present invention, the muscarinic receptor type 5 receptor antagonist is administered via transdermal administration, oral administration, or injection, preferably via transdermal administration or injection, and more preferably via local transdermal administration or intrathecal injection.

[0098] In a preferred embodiment of the present invention, the effective therapeutic dose of the muscarinic receptor type 5 antagonist is 0.01 μg-100 mg / kg body weight / day, for example, 0.01 μg-100 mg / kg body weight / day, 0.05 μg-100 mg / kg body weight / day, 0.1 μg-100 mg / kg body weight / day, 0.2 μg-100 mg / kg body weight / day, 0.3 μg-100 mg / kg body weight / day, 0.4 μg-100 mg / kg body weight / day, 0.5 μg-100 mg / kg body weight / day, 1 μg-100 mg / kg body weight / day, 2 μg-100 mg / kg body weight / day, 3 μg-100 mg / kg body weight / day, 4 μg-100 mg / kg body weight / day, 5 μg- 100mg / kg body weight / day, 6μg-100mg / kg body weight / day, 7μg-100mg / kg body weight / day, 8μg-100mg / kg body weight / day, 9μg-100mg / kg body weight / day, 10μg-100mg / kg body weight / day, 20μg-100mg / kg body weight / day, 30μg-100mg / kg body weight / day, 40μg-100mg / kg body weight / day, 50μg-100mg / kg body weight / day, 60μg-100mg / kg body weight / day, 70μg-100mg / kg body weight / day, 80μg-100mg / kg body weight / day, 90μg-100mg / kg body weight / day, or 100μg-100mg / kg body weight / day.

[0099] In a preferred embodiment of the present invention, the administration frequency of the muscarinic receptor type 5 antagonist is once daily, twice daily, three times daily, once every two days, once every three days, or once weekly.

[0100] In another aspect, the present invention provides the use of M3 receptor antagonists and type IV histamine receptor antagonists in the treatment of chronic pruritus caused by psoriasis.

[0101] Uses of type IV histamine receptor antagonists

[0102] This invention relates to the use of type IV histamine receptor antagonists for: (i) prevention and / or treatment of chronic pruritus caused by psoriasis; and / or (ii) preparation of medicaments for prevention and / or treatment of chronic pruritus caused by psoriasis.

[0103] As used in this invention, "histamine receptor" belongs to G protein-coupled receptors (GPCRs). Histamine receptors are divided into four subtypes: H1R, H2R, H3R and H4R. The term "type IV histamine receptor" is H4R.

[0104] In this invention, the "type IV histamine receptor antagonist" can be any type IV histamine receptor antagonist. In a preferred embodiment of this invention, the type IV histamine receptor antagonist is JNJ7777120.

[0105] compound

[0106] The compound JNJ7777120 in this invention, with the structural formula shown in Formula II, is a commercially available selective histamine H4 receptor antagonist. Its CAS number is 459168-41-3. Molecular formula: C 14 H 16 ClN3O, molecular weight: 277.75.

[0107]

[0108] Treatment

[0109] The present invention also relates to a method for preventing and / or treating chronic pruritus caused by psoriasis, comprising the steps of: administering a therapeutically effective amount of a type IV histamine receptor antagonist to a test subject; or administering a therapeutically effective amount of a pharmaceutical composition containing a type IV histamine receptor antagonist to a test subject.

[0110] the term

[0111] As used herein, the term "muscarinic receptor" refers to a G protein-coupled receptor (GPCR) with a conserved seven-helix transmembrane structure. Five different subtypes of muscarinic receptors (M1-M5) have been identified. The terms "M5 receptor" and "muscarinic receptor type 5 receptor" are used interchangeably.

[0112] As used herein, the term "antagonist" refers to an agent that does not act directly on the receptor and does not elicit a receptor response, but rather antagonizes or cancels the agonist's effect by blocking the binding of the agonist to the receptor. In embodiments of the present invention, the M5 receptor antagonist binds to the M5 receptor but does not possess intrinsic activity, thereby blocking the activation of the M5 receptor.

[0113] As used herein, the terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," "physiologically acceptable carrier," or "physiologically acceptable excipient" refer to pharmaceutically acceptable materials, compositions, or solvents, such as liquid or solid fillers, diluents, solvents, or encapsulating materials. In one embodiment, each component is "pharmaceutically acceptable," meaning it is compatible with other components in a pharmaceutical preparation and suitable for contact with human or animal tissues or organs without excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications, with a reasonable benefit / risk ratio. In some embodiments, these materials may be liposomes, animal or vegetable oils, lanolin, beeswax, petrolatum, paraffin, liquid paraffin, dimethyl silicone oil, or other oleophobic matrices; or methylcellulose, sodium carboxymethylcellulose, polyethylene glycol, or other water-in-oil matrices; or calcium soaps, lanolin, monoglycerides, fatty alcohols, or other water-in-water matrices; or sodium soaps, triethanolamine soaps, fatty alcohol sulfates, polysorbates, etc.

[0114] The term "topical preparation" refers to a dosage form in which a drug is formulated using a suitable carrier for external, non-oral administration. Non-limiting examples of topical preparations include creams, ointments, pastes, patches, rubber plasters, plasters, poultices, gels, and films.

[0115] As used herein, the term "object" is defined as including animals, such as mammals, including but not limited to primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In a particular embodiment, the object is a human.

[0116] As used herein, the term "prevention" refers to the prevention of the onset, recurrence, or spread of a disease or condition, or one or more symptoms associated with such disease or condition. In one embodiment, such symptoms are known to those skilled in the art to be associated with the disease or condition to be prevented. In a particular embodiment, the term refers to the administration of the compound provided herein, with or without other additional active agents, to a patient at risk of developing the disease or disorder described herein, prior to the onset of symptoms. The term includes the suppression and reduction of symptoms of a particular disease. In a particular embodiment, patients with a family history of a disease are specifically considered candidates. Furthermore, patients with a history of recurrent symptoms are also potential candidates for prevention. In this regard, the term "prevention" may be used interchangeably with the term "preventive treatment."

[0117] As used herein, the term "treatment" refers to the eradication or improvement of a disease or condition, or one or more symptoms associated with such disease or condition. In one embodiment, such symptoms are known to those skilled in the art to be associated with the disease or condition to be treated. In certain embodiments, the term refers to minimizing the spread or aggravation of a disease or condition by administering one or more preventative or therapeutic agents to a subject suffering from such a disease or condition. In some embodiments, the term refers to the administration of the compound of the present invention, with or without other additional active agents, after the onset of symptoms of a particular disease.

[0118] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with 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 under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0119] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0120] Unless otherwise specified, the term “or” means the term “and / or” and is used interchangeably with the term “and / or”.

[0121] As used herein, including the appended claims, unless the context clearly indicates otherwise, the singular forms of words such as “an,” “a,” and “the” include their respective plural referents.

[0122] laboratory animals

[0123] The experimental animals used in the following examples were all healthy 8-12 week old male C57BL / 6N mice (Shanghai Slack Laboratory Animal Co., Ltd.). All mice were housed in separate cages, 5-6 mice per cage. Sufficient food and water were provided to the mice during the rearing period. The ambient temperature was maintained at 25-26℃, and the humidity was kept constant at 50%-60%. The living environment was kept quiet and clean. The circadian rhythm was: 7:00 am - 7:00 pm (bright) and 7:00 pm - 7:00 am (dark). The procedures were approved by the Animal Ethics Committee of Tongji University School of Medicine (Animal Research Ethics Number: TJAA08521103), and all operations were conducted in accordance with national guidelines for animal welfare and use.

[0124] 1. Experimental reagents

[0125]

[0126] 2. Preparation of experimental reagents

[0127] 1) Preparation of immunofluorescence staining blocking solution

[0128] 10% Triton 5mL BSA 2.5g Adjust the volume to 0.01M PBS solution. 50mL

[0129] Shake well before use. Store in a refrigerator at 4°C for extended periods. If you observe flocculent precipitate or smell an odor in the sealing solution, discard it and prepare a fresh solution.

[0130] 2) Preparation of immunofluorescence antibody dilution buffer

[0131] 10% Triton 1.5mL BSA 0.5g Adjust the volume to 0.01M PBS solution. 50mL

[0132] Shake well before use. Store at 4°C for extended periods. If flocculent precipitate is observed in the sealing solution, discard and prepare a fresh solution.

[0133] 3) 0.1M PBS preparation

[0134] <![CDATA[NaH2PO4·2H2O]]> 2.8g <![CDATA[Na2HPO4·12H2O]]> 30.8g NaCl 85g <![CDATA[Make up the volume to with ddH2O]]> 1000mL

[0135] After preparation, mix well and place the 0.1M PBS at room temperature. When using, take 100mL of 0.1M PBS, add 900mL of ddH2O, mix well, and adjust the pH to 7.4.

[0136] 4) Preparation of 4% PFA solution

[0137] <![CDATA[NaH2PO4·2H2O]]> 2.8g <![CDATA[Na2HPO4·12H2O]]> 30.8g PFA 40g <![CDATA[Volume made up to with ddH2O]]> 1000mL

[0138] After the solution becomes clear, filter it with filter paper and store the filtered 4% PFA solution at -20°C. All operations are performed in a fume hood.

[0139] 5) 20% and 30% sucrose solutions

[0140] sucrose 20g Adjust the volume to 0.01M PBS solution. 100mL

[0141] sucrose 30g Adjust the volume to 0.01M PBS solution. 100mL

[0142] Once the solution becomes clear, store it in a refrigerator at 4°C and prepare it as needed.

[0143] 3. Experimental Methods

[0144] 1) Animal preparation and grouping

[0145] Allow mice to acclimatize to the environment 3-5 days in advance, and interact with them for 3-5 minutes daily (such as stroking their fur) to reduce stress and improve the stability of experimental data. Shave the fur on the back of the neck and cheeks of the mice at least one day in advance. The shaving area on the back of the neck should be 1cm × 1cm, and the area on the back of the neck should be approximately 0.6cm × 0.6cm. When using a shaver, be gentle, especially on the delicate areas near the ears, to avoid skin injury. When shaving the cheeks, avoid the whiskers. For model establishment, 8-12 week old male C57BL / 6N mice should be randomly divided into two groups: a Vaseline control group and an IMQ model group, with 5-7 mice in each group. When conducting pharmacological and behavioral experiments, at least two different concentrations should be used, referring to the recommended concentrations in the literature. For frozen tissue immunofluorescence staining, 3-5 mice should be used per group. For paraffin slide preparation and H&E staining experiments, 5 mice should be used per group. In the q-PCR experiments, three mice were used in each group. All experiments were conducted using randomized grouping.

[0146] 2) Drug injection method

[0147] 2.1) Intraperitoneal injection: Hold the mouse's neck and back skin with your left hand and tighten it. Using a 1mL syringe, draw up 100μL of physiological saline, scopolamine, anisodamine, sodium pentobarbital solution, etc., and insert the needle into the mouse's right lower abdomen, avoiding the bladder and large intestine.

[0148] 2.2) For intradermal injection, use a 1 mL insulin syringe and draw up 50 μL of physiological saline, scopolamine, and ACh solution. Inject this solution into the nape of the neck of mice that have completed acclimatization, creating a taut wheal. Leave the needle in the skin for 3 seconds to prevent leakage. If the injection site is the cheek, the injection volume is 10 μL.

[0149] 2.3) Intrathecal injection: Using a 1 mL insulin injection needle, draw up a solution of physiological saline, scopolamine, anisodamine, and ACh, insert the needle between L4 and L5 of the mouse lumbar vertebrae, and inject 10 μL of the liquid.

[0150] 3) Establishment of a mouse pruritus model

[0151] 3.1) Establishment of psoriatic mouse models on the neck, back, and cheek: IMQ cream or petroleum jelly was squeezed into a 1 mL syringe, and air was expelled. For the neck model, two small compartments (20 μL, approximately 20 mg) of IMQ were squeezed onto the back of the mouse's neck, spreading evenly over a 1 cm × 1 cm area of ​​skin. For the cheek model, one small compartment (10 μL, approximately 10 mg) of IMQ was squeezed onto the mouse's cheek, spreading evenly over a 0.6 cm × 0.6 cm area of ​​skin. Applications were repeated for 5 consecutive days, each at 10:00 AM, with 24-hour intervals. Before each application, the skin of the mouse's neck, back, and cheek was photographed, and relevant behavioral patterns were recorded. The cheek model was used to differentiate between pain and itching behaviors in mice; the right cheek of the mouse was treated unilaterally.

[0152] 4) Behavioral measurement

[0153] 4.1) Recording Spontaneous Itching Behavior: After the mice were acclimatized to their surroundings, before applying IMQ or Vaseline daily, a 50cm × 30cm white mouse cage was placed in which a thin layer of bedding was added. The mice were placed in the cages to acclimatize, ensuring they were separated into one cage per mouse. The acclimatization period was 30 minutes. During this period, a certain distance was maintained between the two cages to ensure that the mice's behavior did not interfere with each other, and the environment was kept quiet, avoiding human movement. After the acclimatization period, without any intervention, a 1-hour video of the mice's spontaneous activity was recorded. After recording, the mice were returned to their original cages, and the experimental area was cleaned.

[0154] 4.2) Counting Itching Behaviors in Mice: In the cheek model, the criterion for itching behavior was: the mouse raising its hind limb to scratch the modeled area and then lowering its hind limb, which was counted as one effective itching behavior. During the count, scratching of non-modeled areas should be excluded. The criterion for pain behavior was: the mouse rubbing the modeled area with its forelimb on the modeled side, which was counted as one effective pain behavior. During the count, rubbing of non-modeled areas and grooming behavior using both forelimbs from behind the ears to the front should be excluded. In the neck and back model, the behavior of mice scratching their neck and back with their hind limbs was recorded. During the counting process, records were taken every 5 minutes to understand the distribution of itching behavior over a 1-hour period. A double-blind method was used for behavioral counting.

[0155] 5) Paraffin slide preparation and H&E staining

[0156] Skin samples from the back of the neck of both the Vaseline and IMQ groups were immersed in 10% formalin for 8–24 hours. The tissues were then dehydrated using ethanol solutions of varying concentrations, from low to high. After clearing, the skin tissues were placed in xylene-containing solutions and finally embedded in paraffin. The prepared paraffin blocks were fixed onto a paraffin microtome, ensuring the skin epidermis was facing upwards and the sections were 3–5 μm thin. The sections were then flattened in heated water before mounting and baking.

[0157] Melt the paraffin sections using a hairdryer, then soak them in xylene for 10 minutes, repeating this process three times. Next, soak them in 100% ethanol, 95% ethanol, and 85% ethanol for 5 minutes each, followed by rinsing with tap water for 3 minutes. Add an appropriate amount of hematoxylin dye to the tissue, incubate at room temperature for 5 minutes, rinse with tap water for 1 minute, then differentiate in 1% hydrochloric acid alcohol for 3 seconds, rinse with tap water for 1 minute, and finally blue again in running tap water for 10 minutes. Add eosin dye, incubate at room temperature for 20 seconds, and rinse with tap water for 1 minute. Soak the sections in 85% ethanol and 95% ethanol for 20 seconds and 1 minute respectively, and finally soak them in 100% ethanol for 5 minutes each for dehydration. After mounting with neutral resin, observe the histopathological changes under a microscope (Olympus upright fluorescence microscope, OLYMPUS, Japan).

[0158] 6) Frozen sections and immunofluorescence staining

[0159] After successful IMQ modeling, mice were allowed to acclimatize to their environment, minimizing sudden changes in light and sound stimulation. Intraperitoneal anesthesia was administered using 1% sodium pentobarbital at a dose of 10 mL / kg. After a period of time, once the mice's pain reflexes subsided, both upper limbs were immobilized. Using ophthalmic scissors, the skin and muscle tissue were quickly incised along the xiphoid process and lower edge of the chest to open the thoracic cavity and expose the heart. The needle of the infusion pump was inserted into the apex of the heart, taking care to avoid accidental insertion into other atria or ventricles, which could lead to incomplete perfusion. Successful entry into the left ventricle is indicated by visible blood return. The right atrial appendage was then incised with ophthalmic scissors, and 0.9% NaCl solution was rapidly injected until the liver, mesenteric vessels, and skin turned pale, indicating complete perfusion. This step aims to quickly flush out blood and prevent hemoglobin and other substances from affecting molecular experiments and staining results. Then, rapidly inject 4% paraformaldehyde solution. Once stiffening of the mouse limbs, twitching, and erection of the whiskers are observed, slow the injection rate. The sample can be collected when the mouse's body stiffens and twitching stops. It is important to note that the temperature of the 0.9% NaCl solution should be maintained at room temperature; excessively cold 0.9% NaCl solution should not be used to prevent vasoconstriction and incomplete perfusion. Furthermore, the 4% paraformaldehyde solution should be aliquoted and stored at -20°C. Before use, remove it and allow it to reach room temperature. In molecular experiments, only 0.9% NaCl solution needs to be perfused. After perfusion, quickly transfer the mouse to ice for further manipulation to prevent degradation of RNA and proteins. For immunofluorescence staining, the 4% paraformaldehyde solution should be perfused after the 0.9% NaCl solution.

[0160] After perfusion, the skin and muscles of the mouse's neck and back were removed to expose the general morphology of the spinal canal. The skull and spinal cord were disconnected from the anterior end of the cervical vertebrae. Spring scissors were used to cut open the vertebrae, exposing the white cervical spinal cord tissue. After lifting the cervical spinal cord tissue, the DRG tissue near the intervertebral foramen could be seen. Using spring scissors and forceps, the DRG tissue was removed and fixed in 4% paraformaldehyde for 4 hours. The skull was cut open, and the brain tissue was separated to expose the trigeminal ganglion tissue at the base of the skull. The connection between the trigeminal nerve and the base of the skull was shortened using spring scissors, and the tissue was fixed in 4% paraformaldehyde for 4 hours. After fixation, the tissue was dehydrated using 20% ​​PBS sucrose solution. Once dehydrated, the DRG or TG tissue was gently trimmed under a microscope (stereomicroscope, Shanghai Guangmi Instruments Co., Ltd., China) to remove fibers and excess muscle tissue. After trimming, excess water was blotted with filter paper, the tissue was placed in a mold, OCT was added for embedding, and the tissue was quickly frozen at -80°C to solidify. Remove the frozen tissue and fix it onto the base of the microtome, sectioning to a thickness of 14 μm. Use strong adhesion slides for mounting. After preparation, label the tissue slides and store them at -20°C for long-term storage. Use within 3 months if possible. Remove the mounted DRG tissue and place it at room temperature. Rinse three times with 0.01M PBS for 3 minutes each time to remove OCT tissue. Draw a hydrophobic zone with nail polish, add an appropriate amount of blocking solution, incubate at room temperature for 1 hour, discard the blocking solution, and directly add a primary antibody solution containing 1% BSA and 0.3% Triton, incubating overnight at 4°C. The primary antibodies used and their dilution ratios are: rabbit anti-ChAT antibody 1:500; Anti-Muscarinic Acetylcholine Receptor M3 antibody 1:1000; Anti-Muscarinic Acetylcholine Receptor M5 antibody 1:500; DAPI 1:50000. After overnight incubation, remove the tissue and incubate at room temperature for 30 minutes. Remove the primary antibody and wash three times with 0.01M PBS for 10 minutes each time. Add the secondary antibody and incubate at room temperature for 2 hours, taking care to avoid light. Remove the secondary antibody, add DAPI, and incubate at room temperature for 5 minutes. Remove the DAPI and wash three times with 0.01M PBS for 10 minutes each time, taking care to avoid light throughout the process. The dilution ratio of the secondary antibody is: Alexa Fluor 555 donkey anti-rabbit IgG 1:1000; Alexa Fluor 488 donkey anti-mouse IgG 1:1000. Since IB4 carries its own fluorescence, it is added during the secondary antibody incubation. After the slides have dried, mount them with 75% glycerol and store at 4°C protected from light.Image data was acquired and statistically analyzed using a fluorescence microscope (Axio Imager. M2 fluorescence microscope, Carl Zeiss Jena, Germany) via computer software and a digital imaging system. The same exposure time and contrast were used for all photographs. In the statistical analysis, the total number of cells was used as the denominator, and the total number of ChAT, M3, and M5 positive cells was used as the denominator. The positive rates of ChAT, M3, and M5 in the DRG were compared between the control group and the model group. When calculating the co-labeling of M3, M5, IB4, and CGRP, the same exposure time and contrast were used for photographs. For example, the proportion of M3 in IB4 was calculated, i.e., the percentage of cells co-localized with M3 and IB4 in the DRG was calculated out of the total number of M3-positive cells.

[0161] 7) q-PCR

[0162] After successful IMQ modeling, mice were allowed to acclimatize to their environment, minimizing sudden changes in light and sound stimulation. Intraperitoneal anesthesia was administered using 1% sodium pentobarbital at a dose of 10 mL / kg. After a period of time, once the mice's pain reflexes disappeared, both upper limbs were immobilized. Using ophthalmic scissors, the skin and muscle tissue were quickly incised along the xiphoid process and lower edge of the chest to open the thoracic cavity and expose the heart. The needle of the infusion pump was inserted into the apex of the heart, carefully controlling the depth to prevent accidental insertion into other atria or ventricles, which could lead to incomplete perfusion. Successful needle entry into the left ventricle is indicated by visible blood return. The right atrial appendage was then incised with ophthalmic scissors, and 0.9% NaCl solution was rapidly injected until the liver, mesenteric vessels, and skin turned pale, indicating complete perfusion. This step aims to quickly flush out blood and prevent hemoglobin and other substances from affecting molecular experiments. Then, bilateral trigeminal ganglia were quickly harvested on ice and placed into EP tubes containing enzyme-free Trizol. Total RNA was extracted from the trigeminal ganglion and reverse transcribed into cDNA using a reverse transcription kit. This cDNA was then used as a template for amplification. The following primers were used:

[0163] M1 primer: upstream sequence: 5'-AGTCCCAACATCACCGTCTTG-3';

[0164] Downstream sequence: 5'-CAGGTTGCCTGTCACTGTAGC-3';

[0165] M2 primer: Upstream sequence: 5'-TGGTTTGGCTATTACCAGTCCT-3';

[0166] Downstream sequence: 5'-CTGAAGGTGGCGGTTGACTT-3';

[0167] M3 primer: Upstream sequence: 5'-CCTCGCCTTTGTTTCCCAAC-3';

[0168] Downstream sequence: 5’-TTGAGGAGAAATTCCCAGAGGT-3’;

[0169] M4 primer: Upstream sequence: 5’-ATGGCGAACTTCACACCTGTC-3’;

[0170] Downstream sequence: 5’-CTGTCGCAATGAACACCATCT-3’;

[0171] M5 primer: Upstream sequence: 5’-TCAACGGCACCCCAGTAAATC-3’;

[0172] Downstream sequence: 5’-GGATGTAGGTCGTGTAGAGGTTC-3’;

[0173] GAPDH primer: Upstream sequence: 5’-GAGTGTTTCCTCGTCCCGTA-3’;

[0174] Downstream sequence: 5’-TCACCCCATTTGATGTTAGT-3’.

[0175] Prepare a 10 μL reaction system, and the amplification program is as follows: 95°C, 600 s, repeat 1 time; 95°C, 10 s; 60°C, 10 s; 72°C, 10 s, repeat 45 times; extend at 72°C for 600 s. Calculate the expression level of the target gene in the sample by 2-ΔΔCT. The mice are grouped as follows: 3 mice in each group.

[0176] 8) Data analysis

[0177] The statistical data in the embodiments of the present invention are all expressed as mean ± SEM. When the number of data groups is two, the comparison between data is performed using a t-test (student’s t test), and when the number of groups is greater than or equal to 3, one-way ANOVA is used. When 0.01 < P < 0.05, it indicates a statistical difference, *; when 0.001 < P < 0.01, it indicates a significant difference, **; when P < 0.001, ***, it indicates a highly significant difference. The graphing software used in this study is GraphPad Prism 7.0.

[0178] Example 1

[0179] The psoriasis mouse model induced by topical application of IMQ is a widely used skin lesion model for psoriasis patients. In this example, a psoriasis chronic pruritus mouse model was established using the reference experimental method. The specific steps are as follows:

[0180] Use IMQ (0.05%, 20 mg) to continuously apply to the local skin of the neck and back for 5 days ( Figure 1AOn the 3rd day, significant erythema was observed on the skin of the neck and back. On the 5th day, the skin of the neck and back showed a predominantly scaly appearance. Figure 1B The clinical symptoms are similar to those of patients with psoriasis. Histopathologically, it shows typical psoriasis histological features such as hyperkeratosis with parakeratosis, epidermal hyperplasia, and acanthosis. Figure 1C ).

[0181] like Figure 1C H&E staining results showed that, compared with the Vaseline group, the IMQ group mice on day 5 showed hyperkeratosis with parakeratosis on the back of the neck. Neutrophil synthesis was observed in the intercellular spaces of the parakeratotic cells (green arrows); epidermal hyperplasia was observed, with sinuses extending into the dermis in a columnar shape (red arrows); spinous cells were enlarged, with pale cytoplasm and normal intercellular spaces (yellow arrows); proliferating, dilated and congested blood vessels were observed in the papillary layer of the dermis (purple arrows); new hair follicles and sebaceous gland structures were observed in the dermis (blue arrows); and dermal collagen was thickened and the intercellular spaces were widened.

[0182] Example 2

[0183] In this embodiment, it was verified that the psoriasis mouse model constructed in Example 1 exhibited obvious spontaneous and induced scratching behaviors.

[0184] Continuous monitoring of mouse behavior revealed a significant increase in spontaneous scratching behavior starting on day 3, which continued to increase until day 5. Figure 2A This indicated that the model mice exhibited itching behavior. On day 5 of the model, 50 μL of CQ (4 μg / μL) and histamine (10 μg / μL) solution were injected into the neck and back, and the sensitivity of the mice to the chemical stimuli was tested. The CQ-induced scratching behavior in the model group was significantly increased (…). Figure 2B Histamine-induced scratching behavior increased significantly. Figure 2C In summary, the IMQ-induced psoriasis mouse model not only exhibits enhanced sensitivity to mechanical stimuli but also to both histamine-dependent and non-histamine-dependent chemical stimuli.

[0185] Example 3

[0186] In this embodiment, it was confirmed that the mouse model of psoriasis was mainly characterized by itching.

[0187] A facial model was established by applying IMQ (0.05%, 10 mg) to the skin of the right cheek of mice for 5 consecutive days. On day 3, a significant increase in scratching behavior of the hind limbs, representing itching, was observed in the model group, and this continued until day 5. Figure 3A Similar to the neck and back mouse model, but the forelimb swiping behavior, which represents pain, did not show significant changes. Figure 3B In other words, the IMQ-induced psoriasis mouse model is mainly characterized by itching behavior.

[0188] Example 4

[0189] In this embodiment, it was confirmed that type IV histamine receptors mediate itching sensation in a mouse model of psoriasis.

[0190] After IMQ modeling was completed, intrathecal injection of type I and IV histamine receptor antagonists Chlorpheniramine and JNJ7777120 was used to preliminarily explore the role of different histamine receptors in IMQ-induced chronic pruritus in psoriatic mice. Neither low-dose (0.3 μg / 10 μL) nor high-dose (0.6 μg / 10 μL) intrathecal injection of Chlorpheniramine alleviated pruritus behavior in the model mice. Figure 4A Intrathecal injection of JNJ7777120 (10 μg / 10 μL) significantly alleviated pruritus behavior in model mice. Figure 4B This suggests that type IV histamine receptors, rather than type I histamine receptors, mediate itching in a mouse model of psoriasis.

[0191] Example 5

[0192] M5, as a novel receptor, has not yet been fully explored in terms of its function. M5 exhibits different roles from the other four types of M-type receptors in areas such as glandular secretion, osteoporosis, gastrointestinal regulation, and drug addiction; however, its application in pruritus has not yet been reported. In this embodiment, we confirmed that the increase in M1, M3, and M5 mRNA was predominant in the primary sensory neurons of model mice.

[0193] After IMQ modeling was completed, primary sensory neurons from the mouse modeling site were used for q-PCR experiments to detect changes in the mRNA content of M1-M5 in sensory neurons of the model group. At the transcriptional level, the mRNA content of M1, M3, and M5 increased ( Figure 5A This suggests that Ach may bind to M1, M3, and M5 in the primary sensory neurons of the model group mice to mediate itching.

[0194] Example 6

[0195] In this embodiment, it was demonstrated that M3 and M5 specific antagonists can significantly reduce pruritus behavior in the model mice.

[0196] The role of M-type receptors was further explored using intrathecal injection of different M-type receptor-specific antagonists. After establishing an IMQ-induced psoriasis pruritus model, changes in pruritus behavior in model mice were observed after intrathecal injection of the M1 receptor-specific antagonist Pirenzepine, the M2 receptor-specific antagonist Methoctramine, the M3 receptor-specific antagonist 4-DAMP, the M4 receptor-specific antagonist Tropicamide, and the M5 receptor-specific antagonist ML381. High-dose intrathecal injection of 4-DAMP (2 μg / 10 μL) and high-dose ML381 (10 μg / 10 μL) alleviated pruritus behavior in the model group mice. Figure 6B (C). The above results suggest that M3 and M5 mediate pruritus in the psoriasis pruritus model.

[0197] Example 7

[0198] In this embodiment, increased expression of M3 and M5 in primary sensory neurons of model mice was confirmed.

[0199] Pharmacological results suggest that M3 and M5 play important roles in pruritus in the psoriasis model, but further experiments are needed to confirm this. After modeling, mouse DRG tissue was collected for M3 and M5 immunofluorescence staining, and the proportions of M3 and M5 were calculated. The proportions of M3 and M5 in the DRG of the model group were significantly higher than those in the control group. Figure 7A M3 is mainly expressed in the neuronal cytoplasm, while M5 is mainly expressed in the neuronal cell membrane and cytoplasm. To clarify the distribution of M3 and M5 in primary sensory neurons, M3 and M5 were co-labeled with the neuronal marker calcitonin gene-related peptide (CGRP) and the non-peptidase marker IB4. The co-labeling ratio of M3 and IB4 in the neck and back DRG of normal mice was 55.0% (a total of 2937 cells); the co-labeling ratio of M3 and CGRP was 38.7% (a total of 2009 cells). Figure 7D , Figure 7E The co-labeling ratio of M5 and IB4 in the DRG of the neck and back of normal mice was 11.0% (1842 cells in total); the co-labeling ratio of M5 and CGRP was 35.0% (2561 cells in total). Figure 7F , Figure 7G M3 is mainly expressed in both CGRP and IB4, while M5 is mainly co-labeled with CGRP.

[0200] Example 8

[0201] In this embodiment, it was confirmed that the M5 agonist can induce pruritus.

[0202] M5 antagonists significantly relieved itching in psoriatic mice. To verify the necessity of the M5 receptor in pruritus in psoriatic mice, M5 receptor activation was performed to observe whether it could induce itching sensation. Intradermal injection of VU0238429 (10 μg / μL) induced itching sensation in normal mice.

[0203] Examples 5-8 above demonstrate that M3 and M5 in primary sensory neurons are involved in the regulation of chronic pruritus in psoriasis.

[0204] Example 9

[0205] In this embodiment, it was confirmed that PLC, PKC, and Gβγ are involved in mediating itching sensation in a mouse model of psoriasis.

[0206] PKA, PLC, PKC, and Gβγ are common downstream signal transduction pathways of G protein-coupled receptors. An IMQ-induced chronic pruritus model of psoriasis was established. PKA antagonist H-89, PLC antagonist U73122, PKC antagonist GF109203X, and Gβγ antagonist Gallein were administered intrathecally to observe pruritus behavior in the psoriasis mouse model and explore the downstream pathways mediated by acetylcholine (Ach) in pruritus. Behavioral results indicated that intrathecal injection of PLC antagonist (10 nM / 10 μL), PKC antagonist (0.5 μg / 10 μL), and Gβγ antagonist (3 μg / 10 μL) alleviated pruritus behavior in the model mice. In summary, PLC, PKC, and Gβγ are involved in mediating pruritus sensation in the psoriasis mouse model.

[0207] Example 10

[0208] In this embodiment, it was confirmed that M5 can mediate the sensation of itching through PLC and PKC.

[0209] M5 agonist VU0238429 (10 μg / μL) was injected into the neck and back of the mouse, followed by intrathecal injections of PLC and PKC antagonists U73122 and GF109203X, respectively. Behavioral changes in mice were observed. Behavioral results indicated that the PLC antagonist U73122 (10 nM / 10 μL) and the PKC antagonist GF109203X (0.5 μg / 10 μL) significantly alleviated M5 agonist-induced pruritus. In conclusion, M5 can mediate pruritus through PLC and PKC.

[0210] Example 11

[0211] In this embodiment, the therapeutic effect of M5 receptor antagonists was studied.

[0212] A chronic pruritus model of psoriasis was established in mice by applying imiquimod cream (3g:0.15g) for 5 consecutive days, followed by intrathecal injection of the M5 receptor antagonist VU0488130 (5-10μg / 10μL). The number of times the mice scratched the model site with their hind limbs was observed. The experimental results are shown below. Figure 11 .

[0213] The above experimental results confirm the role of M5 in chronic itching of psoriasis. The M5 antagonist VU0488130 can alleviate itching behavior in mice with chronic itching of psoriasis. VU0488130 has significant research value and application prospects in chronic itching of psoriasis.

[0214] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes can be made in form and detail without departing from the spirit and scope of the present invention.

Claims

1. The use of a muscarinic receptor type 5 receptor antagonist for: Prepare a drug for the prevention and / or treatment of chronic pruritus, wherein the drug prevents and / or treats chronic pruritus by reducing the expression levels of PLC, PKC and / or Gβγ, and wherein the chronic pruritus is chronic pruritus caused by psoriasis, and wherein the muscarinic receptor type 5 antagonist is VU0488130.

2. Use according to claim 1, characterized in that, The intended use includes administering a therapeutically effective dose of a muscarinic receptor type 5 antagonist to a subject, wherein the therapeutically effective dose is 0.01 μg to 100 mg / kg body weight / day.

3. Use according to claim 1, characterized in that, The muscarinic receptor type 5 receptor antagonist is administered via transdermal or injectable route.

4. Use according to claim 1, characterized in that, The administration frequency of the muscarinic receptor type 5 antagonist is once daily, twice daily, three times daily, once every two days, once every three days, or once weekly.

5. The use according to claim 1, characterized in that, The muscarinic receptor type 5 receptor antagonist is prepared as a topical or injectable formulation for application.

6. The use of a muscarinic receptor type 3 receptor antagonist for: Prepare a medicine for the prevention and / or treatment of chronic pruritus caused by psoriasis, wherein the antagonist is 4-DAMP.

7. The use of a type IV histamine receptor antagonist, characterized in that, The antagonist is JNJ7777120, which is used to prepare a medicine for the prevention and / or treatment of chronic pruritus caused by psoriasis.

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