Use of modulation of the cannabinoid system and its downstream pathways for the prevention and / or treatment of chronic pruritus

Abstract

The application discloses a kind of by adjusting cannabinoid system and its downstream passage to prevent and / or treat chronic pruritus, it is related to the field of biological medicine technology.It is specifically related to at least one of CB2R agonist, microglial cell inhibitor, p-p38 inhibitor, IL-1 beta signal blocking agent in the application of preparation of the drug for preventing and / or treating chronic pruritus.The application first proves that CB2R expressed in spinal cord microglial cell plays an indispensable role in the formation and maintenance process of chronic pruritus, and by targeting CB2R expressed in spinal cord microglial cell, the function of microglial cell becomes a new treatment way of chronic pruritus.CB2R agonist, microglial cell inhibitor, p-p38 inhibitor, IL-1 beta signal blocking agent can effectively reduce spontaneous scratching times.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to an application for preventing and / or treating chronic pruritus by regulating the cannabinoid system and downstream pathways thereof. BACKGROUND

[0002] Pruritus is an unpleasant somatic sensation that provokes the desire to scratch. According to the duration, it can be divided into acute pruritus and chronic pruritus. In acute pruritus, whether it is chemical pruritus caused by chemical substances (such as histamine) after mosquito bites or mechanical pruritus induced by mechanical stimulation, it is an early warning system of the body defense mechanism, and is usually a protective sensation. In contrast, severe and persistent chronic pruritus under pathological conditions has a serious adverse effect on people's physical and mental health. Chronic pruritus is a stubborn disease induced by skin diseases, systemic diseases, metabolic disorders, neurological diseases, etc. or idiopathic. When the pruritus under pathological conditions is continuously aggravated and leads to excessive scratching, it can cause skin damage and infection, worsen the pruritus, and lead to a further scratch-pruritus vicious cycle, causing the patient to suffer abnormally. Some skin diseases and skin damage caused by scratching can also cause patients' appearance anxiety and inferiority, which can cause anxiety and even trigger mental depression in patients, and significantly reduce the quality of life of patients.

[0003] In the related art, although the potential mechanism of pruritus signal has been deeply explored, the related signal pathways of acute pruritus are relatively clear at present. However, the potential mechanism of chronic pruritus, which is the transformation of pruritus into a pathological chronic state, is still unclear. At present, the treatment of clinical pruritus mainly depends on antihistamine drugs, but studies have shown that histamine does not play a key role in inducing chronic pruritus, and its antagonist drugs have no effect on many types of chronic pruritus. Although the newly developed monoclonal antibody targeting peripheral inflammatory mediators can relieve chronic pruritus to a certain extent, it has the risk of immune suppression or severe hypersensitivity reaction as the immunosuppressive drug (such as cyclosporine, methotrexate, etc.).

[0004] Therefore, chronic pruritus still lacks a safe therapy that is universally effective and treats both the symptoms and the root cause, and it is urgent to develop new therapeutic targets or pathways to make up for the lack of clinical drugs. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes the use of at least one of a CB2R agonist, a microglial cell inhibitor, a p-p38 inhibitor, and an IL-1β signal blocker in the preparation of a drug for preventing and / or treating chronic pruritus, which can effectively reduce the frequency of spontaneous scratching and improve chronic pruritus.

[0006] Use of at least one of CB2R agonist, microglia inhibitor, p-p38 inhibitor, IL-1β signal blocker in the preparation of a drug for preventing and / or treating chronic pruritus according to the first aspect of the embodiment of the present application.

[0007] According to the application of the embodiment of the present application, at least the following beneficial effects are achieved:

[0008] The present application first discloses the role of cannabinoid CB2R system in chronic pruritus, and proves that CB2R expressed in spinal cord microglia cells plays an indispensable role in the formation and maintenance of chronic pruritus, and targeting CB2R expressed in spinal cord microglia cells to regulate the function of microglia cells becomes a new treatment way for chronic pruritus. It can become a new effective target for treating chronic pruritus, and provide an important direction for developing a new way for treating pruritus, and can be developed into a new type of efficient antipruritic drug to alleviate the shortage of clinical drugs, better protect the life quality of patients, and open up a new way for developing safe and efficient new drugs or new methods for treating chronic pruritus in clinic.

[0009] CB2R agonists (such as AM1241 or GW405833), microglia inhibitors (such as minocycline), p-p38 inhibitors (such as SB203580), and IL-1β signal blockers (such as IL-1ra) can treat chronic pruritus related to various diseases (such as allergic contact dermatitis, psoriasis, etc.), have good antipruritic effect in the development and maintenance stages of chronic pruritus, and can effectively reduce the frequency of spontaneous scratching.

[0010] According to some embodiments of the present application, the CB2R agonist includes at least one of AM1241, GW405833, HU308, JWH-015, JWH-133, MDA7, Anandamide, GW 833972A, A-836339, Bay 59-3074, and WIN55212-2.

[0011] According to some embodiments of the present application, the microglia inhibitor includes at least one of minocycline (Minocycline) and PLX5622.

[0012] In the present application, the term "microglia inhibitor" refers to a drug capable of inhibiting the activity and function of microglia cells, or capable of killing or removing microglia cells. The drug can be a small molecule, a peptide, a protein, an antibody or an antigen-binding fragment thereof, an antibody mimetic, an aptamer, or a nucleic acid molecule.

[0013] According to some embodiments of the present application, the p-p38 inhibitor comprises at least one of SB203580, SB239063. p-38 mainly mediates the transmission of extracellular signals to the nucleus, and its activation (phosphorylation) can regulate the activity of intracellular transcription factors and further regulate the synthesis and release of pro-inflammatory factors. The p-p38 inhibitor can reduce the number of mouse scratching by inhibiting the activation of p38.

[0014] According to some embodiments of the present application, the IL-1β signal blocker comprises at least one of IL-1ra and anti-IL-1β monoclonal antibody. The anti-IL-1β monoclonal antibody includes but is not limited to Canakinumab. The IL-1β signal blocker inhibits the binding of IL-1β to its downstream receptor, which inhibits the chronic itching signal and reduces the number of mouse scratching.

[0015] According to some embodiments of the present application, the drug can further comprise other active ingredients for preventing and / or treating chronic itching. The active ingredients preferably do not affect the effects of CB2R agonists, microglia inhibitors, p-p38 inhibitors, and IL-1β signal blockers. For example, GRPR receptor antagonists (including but not limited to PD176252, RC3095) can also be included.

[0016] According to some embodiments of the present application, the drug further comprises a pharmaceutically acceptable excipient.

[0017] According to some embodiments of the present application, the pharmaceutically acceptable excipient comprises at least one of diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, cosolvent, preservative, pH adjuster, osmotic pressure adjuster, surfactant, coating material, antioxidant or buffer.

[0018] According to some embodiments of the present application, the content of CB2R agonists, microglia inhibitors, p-p38 inhibitors, and IL-1β signal blockers in the drug is 1wt% to 99wt%.

[0019] According to some embodiments of the present application, the administration route of the drug is selected from intrathecal injection, intraperitoneal injection, intravenous injection, intramuscular injection, subcutaneous injection, oral administration or nasal administration.

[0020] According to some embodiments of the present application, the dosage form of the drug comprises at least one of suspension, granules, capsules, powders, tablets, injections, suppositories, aerosols or drops.

[0021] The administration route and dosage form of the drug are preferably capable of allowing the drug to pass through the blood-brain barrier to act on the spinal cord level. Thus, the treatment effect on chronic itch can be better. For example, the drug is administered in the form of an injection by intrathecal injection or intraperitoneal injection.

[0022] According to some embodiments of the present application, the drug is used to reduce the number of spontaneous scratches.

[0023] According to some embodiments of the present application, the CB2R agonist is used to reduce the number of spontaneous scratches, inhibit the activation of p38, reduce the synthesis of IL-1β, or inhibit the downstream GRP + and GRPR + activity of itch-specific neurons. Specifically, it can be used to inhibit the activation of p38 and the synthesis of IL-1β in microglia in the spinal cord.

[0024] CB2R is mainly expressed in peripheral immune cells (such as macrophages, neutrophils, and lymphocytes) and microglia in the central nervous system.

[0025] According to some embodiments of the present application, the chronic itch includes chronic itch associated with at least one of a skin disease, a systemic disease, a metabolic disease, and a neurological disease.

[0026] According to some embodiments of the present application, the skin disease includes, but is not limited to, atopic dermatitis, psoriasis, steatosis eczema, nodular prurigo, or allergic contact dermatitis.

[0027] According to some embodiments of the present application, the systemic disease includes, but is not limited to, a liver disease, a kidney disease, or HIV / AIDS.

[0028] According to some embodiments of the present application, the metabolic disease includes, but is not limited to, diabetes.

[0029] According to some embodiments of the present application, the neurological disease includes, but is not limited to, myelitis.

[0030] According to some embodiments of the present application, the drug can be used in the development stage, the maintenance stage, and / or the recovery stage of chronic itch.

[0031] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent from the description, or can be learned by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Modeling process of an allergic contact dermatitis mouse model (A) and photographs of the nape skin at day 12 (B);

[0033] Figure 2Therapeutic effects of CB2R selective agonists AM1241 (A, C) and GW405833 (B, D) on chronic pruritus in wild-type allergic contact dermatitis mouse models of different genders;

[0034] Figure 3 Therapeutic effects of CB2R agonist AM1241 on CB2R - / - genotype allergic contact dermatitis mouse models; wherein A and B are the scratching frequency comparison results of male (wild-type, CB2R - / - genotype) models and female (wild-type, CB2R - / - genotype) models, respectively, and C and D are the therapeutic effects of AM1241 on CB2R - / - genotype male models and CB2R - / - genotype female models, respectively;

[0035] Figure 4 Effects of CB2R selective antagonist AM630 on wild-type and CB2R - / - genotype allergic contact dermatitis mouse models of different genders during the recovery stage of chronic pruritus;

[0036] Figure 5 Therapeutic effects of intraperitoneal administration of AM1241 on wild-type allergic contact dermatitis mouse models;

[0037] Figure 6 Long-term therapeutic effects of CB2R agonists on wild-type allergic contact dermatitis mouse models during the establishment stage (A, C) and maintenance stage (B, D) of chronic pruritus;

[0038] Figure 7 Modeling process (A) and skin photos (B) of psoriasis mouse models;

[0039] Figure 8 Therapeutic effects of CB2R agonists on psoriasis mouse models with chronic treatment (A) and acute treatment (B, C);

[0040] Figure 9 Detection results of CB2R at the spinal cord level in wild-type allergic contact dermatitis mouse models; wherein A is a tissue immunofluorescence photo (scale bar is 10 μm), B is the proportion of Cnr2 + microglia cells per unit area, C is the proportion of Cnr2 + microglia cell proliferation, and D is the detection results of CB2R mRNA expression level in the cervical spinal cord;

[0041] Figure 10Flow chart of CX3CR1-GFP-mice primary microglia acquisition (A) and CB2R expression in primary microglia (B, scale bar 50 μm);

[0042] Figure 11 Changes of microglia in wild type allergic contact dermatitis mouse model; wherein, A is the activation and proliferation of microglia in the dorsal horn of the spinal cord of mice in each group (scale bar 50 μm), B is the morphology of single microglia in the spinal cord of mice in each group and the morphology after skeletonization (scale bar 10 μm), C is the statistical results of the number of microglia per unit area of the spinal cord of mice, D is the statistical results of the fluorescence intensity of microglia by immunofluorescence staining of the spinal cord, E is the statistical results of the cell body area of microglia, F is the Sholl analysis results of the length and number of processes of microglia in the spinal cord;

[0043] Figure 12 Expression levels of CD11b and Cx3Cr1 of microglia in wild type allergic contact dermatitis mouse model; wherein, A and B are the representative graph and statistical results of Western blot of CD11b, respectively, C is the RT-qPCR detection results of Cx3Cr1;

[0044] Figure 13 Therapeutic effects of intrathecal injection of minocycline on wild type allergic contact dermatitis mouse model; wherein, A and B are the therapeutic effects of acute injection of minocycline on male mouse model and female mouse model, respectively, C and D are the therapeutic effects of minocycline on male mouse model and female mouse model in the establishment stage of chronic pruritus model, respectively, E and F are the therapeutic effects of minocycline on male mouse model and female mouse model in the maintenance stage of chronic pruritus, respectively;

[0045] Figure 14 Effects of intrathecal injection of AM1241 on activated microglia and phosphorylated mitogen-activated protein kinase p38 in the spinal cord of chronic pruritus model mice; wherein, A is the immunofluorescence representative graph (scale bar 50 μm), B is the statistical results of the number of Iba1 + cells per unit area, C is the statistical results of the number of p-p38 + cells per unit area;

[0046] Figure 15 Changes of microglia in wild type and CB2R - / -Changes of spinal microglia and p-p38 in allergic contact dermatitis mouse model; Wherein A, D are immunofluorescence representative graph and immunofluorescence co-representative graph of spinal microglia (red) and p-p38 (green) respectively (scale bar is 50 μm, co-labeling experiment uses Tmem119; Ai9 mice), B is the Iba1 + Cell number statistics results, C is the p-p38 per unit area + Cell number statistics results;

[0047] Figure 16 Therapeutic effect of p-p38 inhibitor on allergic contact dermatitis mouse model; A, B are the therapeutic effects of different doses of p-p38 inhibitor on male and female mouse models respectively, C, D are the therapeutic effects of p-p38 inhibitor on male mouse model in the establishment stage and maintenance stage of chronic pruritus model respectively, E, F are the therapeutic effects of p-p38 inhibitor on female mouse model in the establishment stage and maintenance stage of chronic pruritus model respectively;

[0048] Figure 17 Changes of inflammatory mediators in different treatment groups; Wherein A is the fluorescence detection result of protein chip, the red box is the detection result of IL-1β, B is the volcano plot analysis of fluorescence detection result of protein chip, C is the mRNA level detection result of IL-1β, D is the immunofluorescence co-representative graph of spinal microglia (red, Tmem119; Ai9 mice) and IL-1β (green) (scale bar is 10 μm);

[0049] Figure 18 Therapeutic effect of intrathecal injection of IL-1β signal blocker IL-1ra on allergic contact dermatitis mouse model;

[0050] Figure 19 Inhibitory effect detection result of intrathecal injection of AM1241 on chronic pruritus mouse spinal cord activated pruritus specific neurons; Wherein A is a representative graph of RNAscope labeling c-Fos (purple), Grp (red) and Grpr (green) in spinal cord tissue of mice in different treatment groups, B is the immunofluorescence statistical result of c-Fos positive cells, C is the immunofluorescence statistical result of c-Fos and Grp double positive cells, D is the immunofluorescence statistical result of c-Fos and Grpr double positive cells, E is a pie chart of the proportion of c-Fos positive cells in the dorsal horn of the spinal cord;

[0051] Figure 20 Therapeutic effect of intrathecal injection of GRPR receptor antagonist PD176252 on allergic contact dermatitis mouse model. DETAILED DESCRIPTION

[0052] The concept and the technical effects of the present application will be described clearly and completely in combination with the embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0053] The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not marked with the manufacturer, which are all conventional products that can be obtained by market purchase.

[0054] In the description of the present application, the terms "comprising" and "having" and any variations thereof are intended to cover the non-exclusive inclusion, for example, the process, method, system, product or equipment comprising a series of steps or units do not have to be limited to the clearly listed steps or units, but can include other steps or units that are not clearly listed or inherent to these processes, methods, products or equipment.

[0055] The term "intrathecal administration" or "intrathecal injection" or "i.t." refers to the administration mode of directly delivering the drug to the subarachnoid space of the spinal cord of the conscious mouse.

[0056] In the embodiments of the present application, the wild-type mouse is C57BL / 6J mouse (8-10 weeks old, female mouse 23±1g, male mouse 25±1g) from the Experimental Animal Center of South University of Science and Technology (Shenzhen, China), in addition, Cnr2 gene knockout mouse (CB2R - / - , #005786, B6.129P2-Cnr2 tm1Dgen / J), CX3CR1 GFP / GFP mouse (#005582), Tmem119 CreER / + mouse (#031820), Ai9 mouse (R26 tdTomato , #0079050) are purchased from Jackson Company of USA, Tmem119 CreER / + mouse and Ai9 mouse (R26 tdTomatoTmem119; Ai9 mice for labeling microglia. Feeding conditions: All experimental animals before the experiment were fed in the SPF level animal room of the experimental animal center, and the light and dark cycle was maintained every 12 hours, the room temperature was maintained at 22-24℃, and the animals could freely eat and drink water. All behavior tests were completed between 8am and 6pm. The mice were transported from the experimental animal center to the behavior room 24 hours before the formal experiment to allow the mice to adapt to the test environment for more than 45 minutes, the ambient temperature was 23℃, and the animals were transported back to the experimental animal center after adaptation. The animal experiments were carried out in accordance with the European Union regulations (2010 / 63 / EU), and all animal models involved in the study were approved by the Ethics Committee of South University of Science and Technology.

[0057] The allergic contact dermatitis mouse model and the psoriasis mouse model used in the embodiments of the present application both belong to chronic itching models.

[0058] In the embodiments of the present application, AM1241 is purchased from Selleck, item number S1544;

[0059] GW405833 is purchased from Sigma, item number G1421

[0060] AM630 is purchased from Sigma, item number SML0327;

[0061] SB203580 is purchased from Selleck, item number S1076;

[0062] IL-1ra is purchased from R&D Systems, item number 480-RM-010;

[0063] PD176252 is purchased from MCE, item number HY-103286;

[0064] Minocycline is purchased from Sigma, item number M9511.

[0065] In the embodiments of the present application, the solvent injected by the solvent group and the solvents of AM1241, GW405833, minocycline, AM630, SB203580 and PD176252 are all obtained by mixing dimethyl sulfoxide, castor oil and physiological saline at a volume ratio of 1:1:18.

[0066] In the embodiments of the present application, the basal scratching level (BL) is the statistical result of the spontaneous scratching times of the constructed mouse model before grouping and administration. Mice with no significant difference in spontaneous scratching times are used for subsequent grouping and administration to reduce the error of drug antipruritic effect evaluation.

[0067] The recovery phase of chronic itching in mice is defined as the gradual decrease in the number of scratches over time, approaching that of normal mice. In an embodiment of the present invention, on day 30 (day 0 of abdominal sensitization treatment), the number of spontaneous scratches in the allergic contact dermatitis mouse model was significantly reduced, and the mice were in the recovery phase of chronic itching at this time point.

[0068] The therapeutic effect of CB2R agonists on chronic pruritus in a mouse model of allergic contact dermatitis.

[0069] By administering a CB2R agonist to a mouse model of allergic contact dermatitis, we aimed to evaluate the role of spinal cord-expressed CB2R in chronic pruritus and whether spinal cord CB2R activation mediates an antipruritic effect.

[0070] The modeling process of a mouse model of allergic contact dermatitis (DNFB-induced chronic pruritus model) is as follows: Figure 1 As shown in Figure A. Specifically:

[0071] On day 0, 50 μL of 0.5% DNFB solution (a mixture of acetone and olive oil (V / V = 4:1)) was applied to the hairless skin on the abdomen of the mice to significantly increase immunoglobulin E in the mice. Subsequently, on days 5, 7, 9, and 11, 30 μL of 0.25% DNFB solution was applied to the hairless area on the nape of the neck of the mice to stimulate an inflammatory skin response, resulting in obvious erythema, thickening, and epidermal peeling in the hairless area (e.g., Figure 1 As shown in Figure B, the skin of the control group mice showed no obvious changes.

[0072] (1) On day 12 of establishing the mouse model, the allergic contact dermatitis mouse models established from wild-type mice of each sex were randomly divided into groups. The number of mice in each group is detailed in [link to relevant documentation]. Figure 2 The solvent group received an intrathecal injection of 10 μL of solvent; the treatment group received an intrathecal administration of 10 μL of CB2R agonist (15 μg AM1241 or 30 μg GW405833). The number of spontaneous scratches in mice was counted at 1–2 h, 4–5 h, and 23–24 h after administration to assess the degree of itching.

[0073] The results are as follows Figure 2 As shown.

[0074] Compared with the solvent group, in the absence of significant differences in baseline scratching levels (BL) during chronic pruritus, acute intrathecal injection of exogenous CB2R selective agonists of different types (AM1241 or GW405833) into mouse models of allergic contact dermatitis of both sexes produced significant antipruritic effects. In particular, it significantly reduced the number of spontaneous scratches in mice 1-2 hours after administration.

[0075] (2) Construction of wild type and CB2R - / - genotype allergic contact dermatitis mouse model, during the process of mouse model construction, the mice's scratching behavior video (1 hour / day) was taken on the 6th, 8th, 10th, 12th day, and the number of spontaneous scratching of mice was counted to evaluate the degree of itching.

[0076] and the CB2R - / - genotype allergic contact dermatitis mouse model was given intrathecally 15 μg AM1241 (10 μL in volume) as the treatment group, and 10 μL solvent as the solvent group; the number of mice in each group is shown in Table 1. Figure 3 The number of spontaneous scratching of mice was counted at 1-2h, 4-5h and 23-24h after administration to evaluate the degree of itching.

[0077] The results are shown in Table 2. Figure 3

[0078] During the process of chronic pruritus model establishment, the CB2R - / - The number of scratching of mice was significantly higher than that of wild type mice, the degree of itching was more serious, and the recovery of chronic pruritus was later. Moreover, AM1241 did not produce significant antipruritic effect in CB2R - / - mice of both sexes. This indicates that the antipruritic effect of intrathecal AM1241 in wild type mice is mediated by CB2R specificity, which again verifies the important role of CB2R at the spinal cord level in chronic pruritus.

[0079] (3) Construction of wild type and CB2R - / - genotype allergic contact dermatitis mouse model, the number of mice in each group is shown in Table 1. Figure 4 On the 30th day of the chronic pruritus recovery stage, CB2R selective antagonist (5 μg AM630) was given intrathecally, and the number of spontaneous scratching of mice was counted before and 0-1h after administration to evaluate the degree of itching.

[0080] The results are shown in Table 3. Figure 4

[0081] Compared with CB2R - / - mice, wild type mice showed a significant increase in the number of scratching 0-1h after administration of AM630 due to the blocking of the antipruritic effect mediated by endogenous CB2R at the spinal cord level. However, in CB2R - / - mice, no significant increase in the number of scratching was found because AM630 lacked a target.

[0082] ​​This further demonstrates that CB2R and its endogenous ligands may also play an important role in the recovery phase of chronic pruritus in wild-type mice. Furthermore, the Gi-type G protein-coupled receptor CB2R expressed at the spinal cord level plays a crucial role in chronic pruritus; its activation leads to downstream inhibitory effects that mediate significant antipruritic effects.

[0083] (4) On day 5 of establishing the mouse model, the allergic contact dermatitis mouse models established from wild-type male mice were randomly divided into groups. The number of mice in each group is detailed in [link to relevant documentation]. Figure 5 Mice were administered medication continuously from days 5 to 11. The treatment group received an intraperitoneal injection of 5 mg / kg of the CB2R agonist AM1241 at a body weight / volume ratio of 0.1 kg / mL; the solvent group received an intraperitoneal (ip) injection of the corresponding volume of solvent. Scratching behavior videos of mice were recorded on days 6, 8, 10, and 12 (1 hour / day), and the number of spontaneous scratches was counted to assess the degree of itching.

[0084] The results are as follows Figure 5 As shown.

[0085] Compared to the solvent group, intraperitoneal injection of 5 mg / kg AM1241 on days 5-11 during the development of chronic pruritus in mice also reduced the frequency of scratching and alleviated chronic pruritus to some extent. This indicates that systemic administration of CB2R agonists has good therapeutic potential.

[0086] (5) To evaluate the long-term therapeutic effect of continuous intrathecal injection of CB2R agonists in relieving chronic pruritus, wild-type and CB2R agonists of each sex were constructed. - / - A mouse model of genotype-related allergic contact dermatitis; the number of mice in each group is detailed in [link to relevant documentation]. Figure 6 Mice were administered 10 μg AM1241 intrathecally once daily during both the chronic pruritus model establishment phase (days 5-11) and the chronic pruritus maintenance phase (days 12-23); the solvent group received 10 μL of solvent intrathecally. The number of spontaneous scratches per hour was recorded every two days to assess the degree of pruritus.

[0087] The results are as follows Figure 6 As shown.

[0088] Compared with the solvent group, continuous intrathecal AM1241 administration significantly reduced the number of scratches in both male and female mice, promoting their rapid recovery.

[0089] In summary, CB2R can inhibit the development of chronic pruritus signals in wild-type mice, but this inhibitory effect disappears when CB2R is knocked out; compared to wild-type mice, CB2R... - / -The chronic itch in mice is more severe. CB2R at the spinal cord level plays an indispensable role in the development and maintenance of chronic itch signals, and agonist drugs targeting CB2R in the spinal cord can significantly relieve itching in each stage of chronic itch.

[0090] Therapeutic effect of CB2R agonist on chronic itch symptoms in psoriasis mouse model

[0091] This example further evaluates the therapeutic effect of CB2R agonist on chronic itch symptoms in psoriasis mouse model.

[0092] The modeling process of the psoriasis mouse model is as shown in Figure 7 . Specifically:

[0093] The mouse psoriasis model was established by applying imiquimod ointment on the depilated area of the mouse neck and back for 7 consecutive days (days 0-6, 1 time / day, 62.5 mg per mouse), so that the psoriasis-like changes such as infiltrative erythema and thick layer of silver-white scales appeared in the depilated area (as shown in Figure 7 B of the accompanying drawings, the skin of the control group mice showed no obvious changes). At the same time, the behavior video of the mice was recorded for 1 hour every day in the morning of days 1-7 to count the number of scratching times.

[0094] The test method for the antipruritic effect of chronic treatment is as follows:

[0095] The wild-type male mouse psoriasis model was constructed and randomly grouped, and 10 μg of AM1241 was injected intrathecally continuously once a day during the establishment of the chronic itch model (days 0-6); the solvent group was injected intrathecally with 10 μL of solvent. The number of mice in each group is shown in Figure 8 . The number of spontaneous scratching times of the mice within 1 hour was counted once a day before intrathecal injection of the drug to assess the degree of itching.

[0096] The test method for the antipruritic effect of acute treatment is as follows:

[0097] The constructed wild-type male mouse psoriasis model was randomly grouped, and the treatment groups were injected intrathecally with 15 μg of AM1241 or 30 μg of GW405833, respectively; the solvent group was injected intrathecally with 10 μL of solvent. The number of mice in each group is shown in Figure 8 . The number of spontaneous scratching times of the mice was counted at 1-2 h, 4-5 h, and 23-24 h after administration to assess the degree of itching.

[0098] The results are shown in Figure 8 .

[0099] Compared with the solvent group, intrathecal injection of AM1241 significantly reduced the scratching times of the chronic itch mice during the establishment of the psoriasis model. After the chronic itch model was established, compared with the solvent group, intrathecal injection of 15 μg AM1241 and 30 μg GW405833 also significantly reduced the scratching times of the mice, without significant difference in the basic scratching times. This indicates that CB2R expressed at the spinal cord level also plays an indispensable role in the psoriasis model, and agonist drugs targeting CB2R at the spinal cord level can also effectively treat the chronic itch of psoriasis.

[0100] CB2R is mainly expressed in microglial cells in the mouse spinal cord and significantly increased in the chronic itch model

[0101] (1) On the 12th day of DNFB-induced chronic itch, after the mice were deeply anesthetized with avertin, the mice were perfused with pre-cooled PBS and 4% paraformaldehyde successively, the cervical spinal cord of the mice was fixed again in 4% paraformaldehyde, dehydrated with 20% and 30% sucrose solutions, and embedded after dehydration. Normal mice were used as a control group. The mRNA (Cnr2) expression and distribution of CB2R at the spinal cord level of the chronic itch model mice (DNFB group) were detected by high-sensitivity RNAscope in situ hybridization technology and tissue immunofluorescence technology; and the mRNA expression level of CB2R in the cervical spinal cord was detected by qRT-PCR.

[0102] The results are shown in Figure 9 .

[0103] The staining results of the cervical spinal cord tissue of the mice showed that Cnr2 labeled with green fluorescence was obviously co-labeled with IBA1 (Ionized calcium binding adapter molecule 1, a marker for microglial cells in the central nervous system) labeled with red fluorescence. This indicates that Cnr2 is specifically expressed in microglial cells at the spinal cord level. As shown in Figure 9 A of the figure.

[0104] The statistical results showed that although the proportion of Cnr2 expression in microglial cells did not differ significantly between the control group and the DNFB group, the expression of Cnr2 in the DNFB group was significantly increased with the proliferation of microglial cells, and the microglial cells expressing Cnr2 were significantly increased in the DNFB group. As shown in Figure 9 B and C of the figure.

[0105] Consistent with the above results, the mRNA expression level of CB2R in the cervical spinal cord was significantly increased in the chronic itch model. As shown in Figure 9 D of the figure. This further indicates that CB2R expressed at the spinal cord level is closely related to chronic itch in mice. As mentioned above, CB2R is mainly expressed in microglial cells in the mouse spinal cord and significantly increased in the chronic itch model.- / - The related results of mice are linked, and the increased expression of CB2R under chronic itching conditions can help the body to inhibit the development and maintenance of chronic itching signals.

[0106] (2) The spinal cord tissue of the 2-day-old postnatal mouse pups of CX3CR1-GFP fluorescently labeled microglia was extracted, digested and dissociated, and then cultured to purify the primary microglia from mixed glial cells. The process is as follows: Figure 10 as shown in FIG. A. And the primary microglia cells were subjected to cell fluorescence staining using CB2R antibody.

[0107] The results are shown in Figure 10 .

[0108] The red fluorescently labeled CB2R is completely co-labeled with the green fluorescently labeled CX3CR1-GFP primary microglia. The results are consistent with the immunofluorescence staining of the spinal cord tissue of the chronic itching model mice. Combined with the immunofluorescence staining of the spinal cord tissue and the staining of the primary cultured microglia in vitro, we further confirmed that the CB2R (Cnr2) at the spinal cord level is specifically expressed in the microglia.

[0109] Inhibition of microglial activation for the treatment of chronic itching

[0110] (1) The cervical spinal cord tissue of the DNFB-induced chronic itching model (DNFB group) on the 12th day was obtained and subjected to immunofluorescence staining; the process of Sholl analysis was performed on the spinal cord microglia of the DNFB group mice; and the mRNA level of the other marker Cx3cr1 and the protein level of CD11b of the microglia were detected by RT-qPCR and Western blot. Wild-type normal mice were used as controls (control group).

[0111] The results are shown in Figure 11 and 12 .

[0112] Compared with the control group mice, the spinal cord microglia of the DNFB group mice were obviously activated and proliferated, and the microglia cell bodies were significantly enlarged, the processes were retracted and shortened, and the complexity of the processes was significantly reduced; the number of microglia per unit area of the spinal cord of the DNFB group mice was significantly increased, the fluorescence intensity of the microglia in the immunofluorescence staining of the spinal cord was significantly enhanced, and the cell body area of the microglia was significantly increased. Through Sholl analysis, it was found that the process length and process number of the spinal cord microglia of the DNFB group mice were significantly reduced compared with the control group mice. This indicates that the morphology of the microglia in the chronic itching model mice is changed to an obvious activated state.

[0113] In addition, the expression levels of the other markers Cx3cr1 and CD11b of the microglia were also significantly increased.

[0114] In summary, spinal microglia showed significant proliferation and activation in DNFB-induced chronic pruritus model mice.

[0115] (2) The activation of microglia was inhibited by injecting a microglia inhibitor (minocycline) into the spinal cord of mice with chronic pruritus model by pharmacological methods to assess whether the scratching behavior of mice was inhibited, and to explore the role of microglia activation in chronic pruritus.

[0116] On day 12 of establishing the chronic pruritus model in mice, allergic contact dermatitis mouse models derived from wild-type mice of each sex were randomly divided into groups. Details of the number of mice in each group can be found in [link to relevant documentation]. Figure 13 (A and B). The solvent group received an intrathecal injection of 10 μL of solvent; the treatment group received an intrathecal administration of 10 μL of minocycline (50 μg). The number of spontaneous scratches in mice was counted at 1–2 h, 4–5 h, and 23–24 h after administration.

[0117] On day 12 of mouse model establishment, the allergic contact dermatitis mouse models established from wild-type mice of each sex were randomly divided into groups. Details of the number of mice in each group can be found in [link to relevant documentation]. Figure 13 (C and D). Mice were administered the drug once daily from days 5 to 11; the solvent group received an intrathecal injection of 10 μL of solvent per dose; the treatment group received an intrathecal injection of 10 μL of minocycline (50 μg) per dose. From days 6 to 30, mouse behavior was recorded for 1 hour every two days to count the number of scratches.

[0118] On day 12 of mouse model establishment, the allergic contact dermatitis mouse models established from wild-type mice of each sex were randomly divided into groups. Details of the number of mice in each group can be found in [link to relevant documentation]. Figure 13 (E and F). Mice were administered the drug every two days from day 12 to day 23; the solvent group received an intrathecal injection of 10 μL of solvent per dose; the treatment group received an intrathecal injection of 10 μL of minocycline (50 μg) per dose. From day 12 to day 30, mouse behavior was recorded for one hour every two days to count the number of scratches.

[0119] The results are as follows Figure 13 As shown.

[0120] In the absence of significant differences in baseline scratching levels (BL), acute intrathecal injection of minocycline produced a significant antipruritic effect in both male and female mice compared to the solvent group. In particular, it significantly reduced the number of spontaneous scratches in the chronic pruritus model mice at 1-2 hours and 23-24 hours post-administration. This indicates that the activation of microglia at the spinal cord level plays an important role in chronic pruritus, and their activation can mediate a significant antipruritic effect.

[0121] Compared with the solvent group, continuous intrathecal minocycline administration significantly reduced the number of scratches in both male and female mice, promoting their rapid recovery.

[0122] In summary, these findings indicate that microglia activated at the spinal cord level play an indispensable role in the development and maintenance of chronic pruritus signals, and that inhibiting microglia activation can suppress chronic pruritus in mice. The antipruritic effect of CB2R targeting spinal cord microglia may be mediated by regulating the activity and function of spinal cord microglia in a chronic pruritus model mouse.

[0123] Inhibiting mitogen-activated protein kinase p-p38 in microglia can treat chronic pruritus.

[0124] p-38 primarily mediates the transmission of extracellular signals to the cell nucleus. Its activation (phosphorylation) can regulate the activity of intracellular transcription factors and further regulate the synthesis and release of pro-inflammatory factors such as IL-1β.

[0125] (1) Wild-type normal mice were used as the untreated group; the DNFB-induced chronic pruritus model was injected intrathecally with the solvent every day from day 5 to 11 as the DNFB control group; the DNFB-induced chronic pruritus model was injected intrathecally with 10 μg AM1241 every day from day 5 to 11 as the DNFB AM1241 group. The expression of phosphorylated p38 (p-p38) in microglia of mice in different treatment groups was detected by tissue immunofluorescence, and the number of microglia per unit area and the number of p-p38 cells in the dorsal horn were counted.

[0126] The results are as follows Figure 14 As shown.

[0127] Compared to the untreated group, the number of microglia per unit area was significantly increased in the DNFB-treated groups (DNFB control group and DNFB AM1241 group). This indicates that microglia underwent significant reactive proliferation under chronic itching conditions. However, compared to the DNFB control group, the number of microglia in the dorsal horn of the spinal cord of mice in the DNFB AM1241-treated group was not significantly reduced. This indicates that CB2R activation did not inhibit microglia proliferation. Figure 14 As shown in Figures A and B.

[0128] Compared to the untreated group, the number of p-p38-positive cells per unit area was significantly increased in the DNFB control group. This indicates that p38 is significantly activated in microglia under conditions of chronic itching. Compared to the DNFB control group, the number of p-p38-positive cells in the dorsal horn of the mouse spinal cord was significantly reduced in the DNFB AM1241-treated group. This indicates that CB2R activation on microglia can significantly inhibit p38 activation. Figure 14As shown in Figures A and C.

[0129] (2) Establish wild-type and CB2R - / - A mouse model of allergic contact dermatitis was established, and the number of microglia and p-p38-positive cells in the spinal cord tissue of different mice was detected by tissue immunofluorescence. The origin of p-p38 at the spinal cord level was detected by immunofluorescence colocalization (Tmem119 and Ai9 mice were used for co-labeling experiments).

[0130] The results are as follows Figure 15 As shown.

[0131] Compared to wild-type mice, CB2R - / - The number of microglia and p-p38 positive cells in the spinal cord of mice was significantly increased. This suggests that activation of p38 specifically expressed in microglia may be crucial for the generation and maintenance of chronic pruritus signals.

[0132] (3) On day 12 of establishing the allergic contact dermatitis mouse model, wild-type mice of each sex were randomly divided into groups. The number of mice in each group is detailed in [link to relevant documentation]. Figure 16 Figures A and B are shown. The solvent group received an intrathecal injection of 10 μL of solvent; the treatment group received intrathecal administration of different doses of the p-p38 inhibitor (SB203580). The number of spontaneous scratches by mice was counted at 1–2 h, 4–5 h, and 23–24 h after administration to assess the degree of itching.

[0133] Wild-type allergic contact dermatitis mouse models of each sex were constructed. The number of mice in each group is detailed in [link to relevant documentation]. Figure 16 Figures C, D, E, and F are shown. Different doses of the p-p38 inhibitor (SB203580) were administered intrathecally once daily during either the chronic pruritus model establishment phase (days 5-11) or the chronic pruritus maintenance phase (days 12-23). ​​The solvent group received 10 μL of solvent intrathecally. The number of spontaneous scratches per hour was recorded every two days to assess the degree of pruritus.

[0134] The results are as follows Figure 16 As shown.

[0135] In the absence of a significant difference in baseline scratching frequency (BL) before administration, acute intrathecal administration of SB203580 demonstrated a good antipruritic effect in both male and female mice compared to the solvent group. Figure 16 As shown in Figures A and B, this indicates that p38 activation is closely related to chronic pruritus in mice, and that inhibiting p38 activation significantly reduced the number of scratches and alleviated chronic pruritus in mice.

[0136] During the establishment phase (days 5-11) and maintenance phase (days 12-23) of the chronic pruritus model, compared with the solvent group, intrathecal administration of SB203580 significantly reduced the number of scratches in both male and female mice with chronic pruritus, promoting rapid recovery. Figure 16 Figures C, D, E, and F show this. This directly demonstrates the important function of p-p38 in chronic pruritus and further proves that the activation of the CB2R receptor at the spinal cord level, i.e., the antipruritic effect of intrathecal AM1241, is produced by inhibiting the activation of p38.

[0137] The inhibitory effect of AM1241 on p-p38 may mediate the antipruritic effect following CB2R activation. Similarly, CB2R... - / - The more severe itching exhibited by the mice may be closely related to the increased microglia and p-p38 at the level of their spinal cord.

[0138] The therapeutic effect of IL-1β signaling blockers on chronic pruritus

[0139] (1) Wild-type normal mice were used as the untreated group; DNFB-induced chronic pruritus model was treated with intrathecal injection of solvent daily from day 5 to day 11 as the DNFB control group; DNFB-induced chronic pruritus model was treated with intrathecal injection of 10 μg AM1241 daily from day 5 to day 11 as the DNFB AM1241 group. The protein expression changes of more than 40 inflammation-related factors in the spinal cord tissue of mice in each group were detected using antibody microarray. The mRNA level of IL-1β in different treatment groups was detected by qRT-PCR, and the source of IL-1β in the spinal cord was detected by immunofluorescence colocalization (Tmem119; Ai9 mice were used for co-labeling experiments).

[0140] The results are as follows Figure 17 As shown.

[0141] Compared to the untreated group, the expression of some inflammatory factors (such as IL-1β, ICAM-1, IL-7, BLC, etc.) was significantly increased in the DNFB control group; compared to the DNFB control group, the expression of some inflammatory factors (such as IL-1β, IL-12p70, IL-7, IL-6, etc.) in the DNFB AM1241 group was significantly reduced in the mouse intrathecal expression. Among these, the change was most significant in IL-1β. Figure 17 As shown in Figures A and B. Studies have shown that the expression and secretion of IL-1β depend on the activation of p-38, which is consistent with the changes in p-38 activation in the above examples.

[0142] qRT-PCR results showed that under chronic pruritus conditions, IL-1β expression was significantly increased in the spinal cord, and tissue inflammation levels were elevated. However, IL-1β expression levels were significantly reduced after intrathecal treatment with the CB2R agonist AM1241. Figure 17 As shown in Figure C, this is consistent with the detection results from the antibody chip.

[0143] The green fluorescent labeling of IL-1β and the red fluorescent protein labeling of tdTomato(Ai9) microglia were completely co-labeled, indicating that IL-1β in the spinal cord tissue of the chronic itch model mouse mainly originated from microglia.

[0144] (2) A wild-type male rat model of allergic contact dermatitis was established and the rats were randomly divided into groups. The number of mice in each group is detailed in [link to relevant documentation]. Figure 18 On day 12 of the trial, the treatment group received an acute intrathecal injection of 100 ng / mL (10 μL) of the IL-1β signaling blocker IL-1ra, while the solvent group received an intrathecal injection of 10 μL of solvent. The number of spontaneous scratches by mice was counted at 1–2 h, 4–5 h, and 23–24 h after administration to assess the degree of itching.

[0145] The results are as follows Figure 18 As shown.

[0146] In the absence of a significant difference in baseline scratch count (BL) before administration, intrathecal IL-1ra showed a superior antipruritic effect compared to the solvent group. This indicates that the microglia-derived inflammatory cytokine IL-1β plays an important role in chronic pruritus in mice, and that intrathecal administration of a CB2R agonist reduces IL-1β synthesis. Furthermore, blocking the binding of IL-1β to its downstream receptors with IL-1ra inhibits chronic pruritus signaling and reduces scratch count in mice.

[0147] The therapeutic effect of GRPR receptor antagonist PD176252 on chronic pruritus

[0148] (1) Wild-type normal mice were used as the untreated group; the DNFB-induced chronic pruritus model was injected intrathecally with the solvent daily from day 5 to day 11 as the DNFB control group; the DNFB-induced chronic pruritus model was injected intrathecally with 10 μg AM1241 daily from day 5 to day 11 as the DNFB AM1241 group. The activation changes of pruritus-specific neurons (mainly gastrin-releasing peptide (Grp) positive neurons and its receptor Grpr positive neurons) in the spinal cord tissue of each group of mice were detected by RNAscope in situ hybridization, with c-Fos as the marker gene for neuronal excitation.

[0149] The results are as follows Figure 19 As shown.

[0150] Compared to the untreated group, the number of c-Fos-positive neurons in the dorsal horn of the spinal cord was significantly increased in DNFB-induced chronic pruritus model mice (DNFB control group). However, after AM1241 treatment (DNFB AM1241 group), the number of c-Fos-positive neurons was significantly reduced. This indicates that neurons in the dorsal horn of the spinal cord were significantly activated in the chronic pruritus mouse model, but the activity of these excited neurons was significantly inhibited after intrathecal administration of a CB2R agonist. Simultaneously, this suggests that under chronic pruritus conditions, some downstream neurons are also significantly activated due to the activation and proliferation of microglia. However, when AM1241 activates CB2R on microglia and inhibits p-p38 in microglia, microglia activity is suppressed, and the secretion of inflammatory factors is reduced. This further acts on receptors expressed on downstream neurons, leading to a decrease in the activation of downstream neurons.

[0151] In-situ co-labeling of c-Fos with Grp and Grpr revealed that c-Fos + Grp + and c-Fos + Grpr + The number of double-positive cells was significantly increased in mice with a chronic pruritus model, but decreased significantly after AM1241 treatment. This indicates that Grp and Grpr-positive neurons are significantly activated in the chronic pruritus model, while their excitability is significantly reduced after AM1241 treatment. Furthermore, among the neurons activated under these chronic pruritus conditions, 43.04% were Grp-positive and 38.43% were Grpr-positive. These two types of neurons, associated with pruritus signaling, constituted the vast majority of these activated neurons.

[0152] In summary, under conditions of chronic pruritus, the relationship between spinal microglia and downstream neurons can be enhanced by activating CB2R on microglia, inhibiting microglia, and simultaneously inhibiting downstream GRP. + and GRPR + The activity of itch-specific neurons is reduced, thereby weakening the transmission of itch signals.

[0153] (2) A wild-type male rat model of allergic contact dermatitis was established and the rats were randomly divided into groups. The number of mice in each group is detailed in [link to relevant documentation]. Figure 20 On day 12 of the trial, the treatment group received an acute intrathecal injection of 10 μg of the GRPR receptor antagonist PD176252 to block the interaction between GRP and GRPR, while the solvent group received an intrathecal injection of 10 μL of solvent. The number of spontaneous scratches was counted at 1–2 h, 4–5 h, and 23–24 h post-administration to assess the degree of itching.

[0154] The results are as follows Figure 20 As shown.

[0155] With no significant difference in baseline scratching frequency before administration, intrathecal administration of PD176252 demonstrated a highly effective antipruritic effect compared to the solvent group. This indicates that GRPR does indeed mediate the transmission of pruritus signals under chronic pruritus conditions and plays an indispensable role in chronic pruritus.

[0156] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. The use of a CB2R agonist in the preparation of a medicament for the prevention and / or treatment of chronic pruritus; wherein the CB2R agonist is AM1241.

2. The application according to claim 1, characterized in that, The drug may also include other active ingredients that prevent and / or treat chronic pruritus.

3. The application according to claim 1, characterized in that, The dosage form of the drug includes at least one of suspension, granules, capsules, powders, tablets, pills, injections, suppositories, aerosols, or drops.

4. The application according to claim 1, characterized in that, The medication is used to reduce the frequency of spontaneous scratching.

5. The application according to claim 1, characterized in that, The CB2R agonist is present in the drug at a concentration of 1 wt% to 99 wt%.

6. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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