CN113876779B - Application of UTR antagonist GSK1562590 - Google Patents

Abstract

The invention provides a new application of UT antagonist GSK1562590, which belongs to the technical field of medicines, after GSK1562590 is used for treatment, the mouse itch-like behavior induced by MC903 is obviously weakened, time dependence exists, the scratching times are obviously reduced, the chronic itch-like behavior is effectively reduced by GSK1562590, and meanwhile, GSK156290 also obviously reduces itch related receptors Mrgpra2b, CCR2, CCR6, IL-13 Ra 1, IL-13 Ra2, NTRK1, IL-31 Ra, CXCR3 and IL24 Ra, and the transcription levels of itch and inflammatory cytokines such as Nppb, IL-24, IL-19, CXCL5, fasL, IL-1b, IL-13, CCL22, CCL17 and carcinostatin M (OSM) are also obviously reduced. Therefore, GSK1562590 can effectively treat pruritus and can be used as a new means for drug intervention of pruritus treatment.

Description

Application of UTR antagonist GSK1562590

Technical Field

The invention relates to the technical field of medicines, in particular to a new application of UTR antagonist GSK 1562590.

Background

In patients with skin, pruritus is one of the most distressing symptoms that lead to the development of insomnia, anxiety and depression. The pruritus comprises acute pruritus and chronic pruritus, the acute pruritus is characterized by rapid occurrence and rapid fading, while the chronic pruritus is characterized by skin inflammation, barrier dysfunction, repeated attack and long pathological cycle (the pruritus lasts for more than or equal to 6 weeks). Chronic pruritus may be caused by various pathological diseases, such as eczema, renal failure, liver cirrhosis, nervous system diseases and cancer, and may also be caused by side effects after the use of the medicine, affecting the quality of life. Chronic pruritus is one of the main diagnostic symptoms of Atopic Dermatitis (AD), accompanied by the inflammatory process of some skin diseases.

It is reported that drugs are often used in clinic to relieve pruritus, such as gabapentin can relieve pruritus of diabetic neuropathy patients, but the dosage is high, the treatment period is as long as 4 weeks, and further such as lamotrigine can relieve neuropathic pruritus, and such as pregabalin can relieve pruritus induced by cetuximab, but drug resistance is easy to appear in the traditional drug treatment process, the chronic pruritus treatment is obviously poor, serious side effects are easy to generate, and although IgE antibodies or other TH2 cytokine specific antibodies have good early effects, the price is very expensive, and no specific drug approved by FDA for treating chronic pruritus exists at present. While the treatment of chronic itch remains extremely challenging due to the complex pathogenesis and the many causes of chronic itch.

Urotensin II (UII, or UTS 2) is believed to be a somatostatin-like cyclic peptide (Arman, bozgeyik, dagli, & Igci, 2017) which is a stronger endogenous vasoactive contractile peptide than endothelin 1 (ET-1) (Chiu, wang, & Shyu,2014 tomiyama, nakamachi, uchiyama, matsuda, & Konno, 2015), endothelin-1 is an inducer of pain and itch in humans and animals. The receptor for UII, GPR14, is a G protein-coupled receptor, termed the UII receptor (UTR), involved in the regulation of various physiological processes including vasoconstriction, cardiac myomechanical effects, regulation of immune function, neurotrophic effects, and thermal hyperalgesia and mechanical ectopic pain. However, to date, the distribution and function of GPR14 in sensory neuron systems and skin physiology is unclear, and it is unclear whether UII and GRP14 are involved in the occurrence of itch perception.

Disclosure of Invention

The invention aims to provide application of a high-affinity and selective urotensin II receptor UTR antagonist GSK1562590 in preparing a medicine for treating pruritus.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of UTR antagonist GSK1562590 in preparation of a medicine for treating pruritus.

Preferably, the pruritus comprises chronic pruritus.

Preferably, the chronic pruritus comprises chronic pruritus caused by chronic skin diseases and/or pathological diseases; the chronic skin diseases include atopic dermatitis, psoriasis and prurigo; the pathological diseases include renal failure, liver cirrhosis, nervous system diseases, and cancer.

Preferably, the GSK1562590 inhibits UII-GPR14 signaling in chronic pruritus.

Preferably, the GSK1562590 inhibits the expression of itch and inflammation-associated cytokines.

Preferably, the GSK1562590 inhibits the expression of inflammation-related peptides.

Preferably, the itch and inflammation-associated cytokines include Nppb, IL-24, IL-19, CXCL5, fasL, IL-1b, IL-13, CCL22, CCL17, carcinostatin M.

Preferably, the itch-associated receptors include Mrgpra2b, CCR2, CCR6, IL-13R α 1, IL-13R α 2, NTRK1, IL-31R α, CXCR3, IL27R α.

The invention also provides a medicine for treating pruritus, which comprises an active ingredient GSK1562590 and a pharmaceutically acceptable carrier.

Preferably, the content of the active ingredient in the medicament is 1-99%.

The invention has the following beneficial effects:

the invention verifies the treatment effect of the GSK1562590 on chronic pruritus through a mouse model of MC903 mediated chronic pruritus, and the result shows that the UTR antagonist GSK1562590 obviously weakens pruritus-like behaviors induced by MC903 on the 10 th treatment day, time dependence exists, scratching times are obviously reduced, the GSK1562590 effectively reduces the chronic pruritus-like behaviors, meanwhile, the GSK156290 also obviously reduces the transcription levels of Mrgpra2b, CCR2, CCR6, IL-13R alpha 1, IL-13R alpha 2, NT1, IL-31R alpha, CXCR3 and IL27R alpha, inflammatory cytokines such as Nppb, IL-24, IL-19, CXCL5, RKL, IL-1b, IL-13, CCL22, CCL17 and carcinostatic M (OSM). Therefore, GSK1562590 is effective in treating pruritus.

Drawings

FIG. 1 is a graph of the results of dose-dependent pruritus scratching and induced pain in mice caused by intradermal injection of different doses of UII solutions in the transient pruritus model constructed in example 1, A is the result of the number of scratching times at different doses within 60min, B is the number of scratching times at higher doses and high doses over a period of 0-10 min, 10-20 min, 20-30 min, 30-40 min, 40-50 min, 50-60 min, C is the number of swabs at different doses within 60min, and light swabs indicate a lower degree of pain behavior; d is the comparison result of scratching and wiping behaviors within 60 min;

FIG. 2 is a graph of immunohistochemical analysis of the expression and distribution of UII and GPR14 in atopic dermatitis mice and healthy mice, wherein A is a graph of the expression and distribution of UII in atopic dermatitis mice and healthy mice, and B is a statistical comparison of immunofluorescence intensity of UII in atopic dermatitis mice and healthy mice (Control); c is a graph of the expression and distribution of GPR14 in atopic dermatitis mice and healthy mice, and D is a statistical comparison graph of immunofluorescence intensity of GPR14 in atopic dermatitis mice and healthy mice (Control);

FIG. 3 is a graph of immunohistochemical analysis of the expression and distribution of UII and GPR14 in mTGN in atopic dermatitis mice and mTGN in healthy mice, wherein A is a graph of the expression and distribution of UII in atopic dermatitis mice and healthy mice, and B is a statistical comparison of immunofluorescence intensities of UII in mTGN in atopic dermatitis mice and healthy mice (Control) mTGN; c is the expression and distribution chart of GPR14 in an atopic dermatitis mouse mTGN and a healthy mouse (Control) mTGN, and D is the immunofluorescence intensity statistical comparison chart of GPR14 in the atopic dermatitis mouse mTGN and the healthy mouse (Control) mTGN; e is UII ⁺ Neuronal subtype in atopic dermatitis mouse diameter: (<10 μm), medium and small diameters (10-20 μm), large diameters (> 20 μm) (PGP 9.5 ⁺ ) Expression proportion map in mTGN; f is UII ⁺ Neuronal subtypes differ in diameter (PGP 9.5) in healthy mice (Control) ⁺ ) Expression proportion maps in mTGN; g is GPR14 ⁺ Neuronal subtypes differ in diameter in atopic dermatitis mice (PGP 9.5) ⁺ ) Expression proportion maps in mTGN; h is GPR14 ⁺ Neuronal subtypes differ in diameter (PGP 9.5) in healthy mice (Control) ⁺ ) Expression proportion maps in mTGN; wherein mTGN represents the trigeminal ganglion;

FIG. 4 shows the behavioral test results of GSK1562590 for treating MC 903-induced chronic pruritus in mice, wherein A is a flow chart of the treatment; b is the statistics of the scratching times of mice after coating MC903 and EtOH on day 1, and the scratching times of mice of a control group and a treatment group after application on day 10, wherein a circle on day 1 represents the scratching times of the mice of the control group (the mice treated by MC903 are not applied with vehicle), a square represents the scratching times of the mice of the treatment group (the mice treated by MC903 are not applied with GSK 1562590), a circle on day 10 represents the scratching times of the mice of the control group (the mice treated by MC903 are applied with vehicle), and a square represents the scratching times of the mice of the treatment group (the mice treated by MC903 are applied with GSK 1562590); c is a statistical chart of scratching times of mice in the control group and the treatment group every day during the treatment period;

FIG. 5 is the chronic itchy skin expression and distribution of UII and GPR14 on the same side of the carpel (left side) induced by MC903 after treatment by the control and treatment groups, wherein A is the chronic itchy skin expression and distribution of UII on the same side of the carpel (left side) induced by MC903 after treatment by the control and treatment groups; b is the chronic itchy skin expression and distribution of GPR14 on the ipsilateral (left) ear skin induced by MC903 after treatment by control and treatment groups; c is a statistical comparison of immunofluorescence intensity of UII in chronically itchy skin ipsilateral (left) to the ear skin induced by MC903 after treatment by control and treatment groups; d is a statistical comparison of immunofluorescence intensities of GPR14 in MC903 induced chronically itchy skin ipsilateral to the ear (left) following treatment by control and treatment groups;

FIG. 6 is the EtOH-induced chronic itchy skin expression and distribution of UII and GPR14 on the ipsilateral (right) ear skin following treatment by control and treatment groups, wherein A is the EtOH-induced chronic itchy skin expression and distribution of UII on the contralateral (right) ear skin following treatment by control and treatment groups; b is the EtOH-induced chronic itchy skin expression and distribution of GPR14 on the contralateral (right) side of the ear skin after treatment by the control and treatment groups; c is a statistical comparison of immunofluorescence intensity of UII in EtOH-induced chronically itchy skin on the contralateral (right) side of the ear skin after treatment by control and treatment groups; d is a statistical comparison of immunofluorescence intensities of GPR14 in EtOH-induced chronically itchy skin on the contralateral (right) side of the ear skin after treatment by control and treatment groups;

FIG. 7 shows the effect of GSK1562590 on ear skin, wherein A is the pathological change of ear skin after GSK1562590 treatment; b is a graph of the change of the thickness of the ear skin after GSK1562590 treatment; c is the result of enrichment change of inflammatory cells (mainly eosinophils and basophils) in the dermis of the ear of the mouse after GSK1562590 treatment; d is a graph of inflammatory cell infiltration in the epidermis of the mouse ear after GSK1562590 treatment;

FIG. 8 is a graph of the immunohistochemical results for Mcpt 8-specific labeled basophils following GSK1562590 treatment;

FIG. 9 is a graph showing transcriptional expression profiles of itch related receptors and itch related genes in mouse ear skin subjected to RNA-Seq assay on ear skin after GSK1562590 treatment, and A is a graph showing transcriptional expression profiles of itch related receptors in mouse ear skin subjected to RNA-Seq assay on ear skin after GSK1562590 treatment; b is a transcription expression condition graph of itch related genes in the skin of the mouse ears after GSK1562590 treatment and RNA-Seq detection on the ear skins;

FIG. 10 is a diagram of the Urotensin 2-GPR14 signaling pathway.

Detailed Description

The invention provides application of UTR antagonist GSK1562590 in preparation of a medicine for treating pruritus.

In the invention, by constructing a mouse chronic pruritus model induced by MC903, the GSK1562590 obviously weakens pruritus-like behaviors induced by MC903 on the 10 th treatment day, has time dependence, obviously reduces scratching times and can effectively treat chronic pruritus. In the present invention, pruritus preferably includes chronic pruritus, said chronic pruritus preferably includes chronic pruritus caused by atopic dermatitis, said GSK1562590 is purchased from (RD, cat. No. 5110), said GSK1562590 has the following structural formula:

in the present invention, GSK1562590 is distinguished from previous antagonists of poor potency, selectivity and/or limited intrinsic activity, is a high affinity and selective UTR antagonist and exhibits sustained UTR residence time and better preclinical efficacy in vivo, treating chronic pruritus by selectively inhibiting GPR14, acting on the UII/GPR14 signaling pathway associated with pruritic sensory neuron-dermoepidermal immunity. The GSK1562590 preferably inhibits the expression of inflammation-related cytokines, and the pruritus and inflammation-related cytokines preferably comprise Nppb, IL-24, IL-19, CXCL5, fasL, IL-1b, IL-13, CCL22, CCL17 and carcinostatin M. The GSK1562590 preferably inhibits expression of itch-related receptors, preferably including Mrgpra2b, CCR2, CCR6, IL-13 Ra 1, IL-13 Ra2, NTRK1, IL-31 Ra, CXCR3, IL27 Ra.

In the invention, the medicament preferably comprises an active ingredient GSK1562590 and a pharmaceutically acceptable carrier, wherein the content of the active ingredient in the medicament is 1-99%. In the present invention, the pharmaceutically acceptable carrier includes one or more of a colorant, a diluent, a sweetener, a disintegrant, a cosolvent, a binder, a dispersant, and a wetting agent, and the carrier is conventional in the art, provided that the carrier does not react with the active ingredient of the present invention or affect the therapeutic effect of the drug of the present invention. The dosage form of the medicine can be conventional tablets, capsules, controlled release preparations, sustained release preparations, granules, solutions and injections. The medicine of the invention can be applied through oral administration, subcutaneous administration, muscle and vein, the administration dosage of the medicine of the invention can be changed properly according to the administration object, the administration route or the preparation form of the medicine, but the premise is to ensure that the medicine composition can achieve effective blood concentration in the body of a mammal. The preparation method of the medicament of the present invention is not particularly limited, and the medicament may be prepared by a method well known in the art, and then packaged in a manner conventional in the art.

In the present invention, all the raw material components are commercially available products well known to those skilled in the art unless otherwise specified.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 mouse cheek intradermal injection of UII induced transient pruritus model

(1) Laboratory animal

6-8 weeks healthy C57BL/6J female mice, weighing 16-18 g, purchased from Beijing Sibefu Biotech, inc., were bred in 8 mice per cage, and had free access to water. The temperature of the animal room is constant at 25 ℃, the ventilation is good, and the illumination and the dark environment are alternately carried out for 12h period.

(2) Preparation of UII solution

The UII solution is prepared into UII solutions with high dose of 2.43mg/mL, higher dose of 1.215mg/mL, medium dose of 0.729mg/mL and low dose of 0.405mg/mL respectively. Firstly, dissolving 2.43mg of UII in 1mL of Vehicle (namely PBS 7.2-7.4) to prepare a high-dose (2.43 mg/mL) UII solution, then dissolving the 2.43mg/mL UII solution in 2mL of Vehicle for dilution to obtain a higher-dose (1.215 mg/mL) UII solution, dissolving the 1.215mg/mL UII solution in 1.7mL of Vehicle to obtain a medium-dose (0.729 mg/m 1) UII solution, and diluting the 0.729mg/mL UII solution in 1.8mL of Vehicle to obtain a low-dose (0.405 mg/mL) UII solution. The concentration ratio is calculated according to the weight of the mouse.

(3) The mouse cheek intracutaneous injection of UII induces the transient pruritus model as the following steps:

1) The mice are bred in a mouse animal room to adapt for 3-5 days, and the mice are divided into 2 groups according to the weight: the treatment group and the control group, the treatment group is 8 mice respectively, namely a UII solution group, a medium-dose UII solution group, a higher-dose UII solution group and a high-dose UII solution group, and the control group is 8 mice in a Vehicle group;

2) Treatment groups mice were treated in batches with 10 μ L each of the right cheek intradermally injected low, medium, higher and high doses of UII solutions, control groups injected with only the same dose of Vehicle (i.e., PBS solution pH 7.2-7.4), immediately after cheek injection was complete, video observations were made, and the scratching behavior of the mice was recorded for 1 hour and counted (hind limb scratching behavior toward the injection site was considered an indication of itching, one scratching (scratching bout) was defined as the mice lifting their paws and continued scratching for a period of time until they returned to the floor.

3) Mice were injected intradermally into the right cheek 1 time every 1 day as described in step 2) and video was observed.

As shown in FIG. 1, the results of the behavioral tests show that the intracutaneous injection of different doses of UII solution into the cheek of mice induces the scratching response of the mice and also induces slight rubbing, which indicates a lower level of pain behavior, and the comparison of the average number of scratching times with the average number of rubbing times shows that UII is dose-dependent on the induction of itching and pain.

Example 2 histopathological examination of AD-like mouse model skin, mouse Trigeminal Ganglion (TG) section samples

The immunohistochemical (paraffin section staining) experimental method comprises the following steps:

(1) Dewaxing and antigen retrieval

Firstly, removing paraffin on a section, putting a sample into a dye vat glass container filled with dimethylbenzene in a fume hood, and putting for 2 times, wherein each time is 10min, the paraffin on the section is removed as much as possible, and uneven dyeing caused by incomplete paraffin removal is prevented, wherein the sample is taken from the skin on the back of the neck of a mouse;

placing the slices in dye vat containers prepared by 100%, 95%, 70% and 50% of ethanol in advance respectively for 10min, taking out the slices, placing the slices in pure water for washing twice, wherein water flow cannot be too fast to prevent the samples from being washed off, placing the slices in pure water after washing is finished, pouring prepared 1X antigen repairing liquid (DAKO) into the dye vat, placing the dye vat into an autoclave for heating, placing the antigen repairing liquid into the antigen repairing liquid for boiling for 30min after the temperature of the solution exceeds 95 ℃ after the antigen repairing liquid is boiled, wherein the time cannot be too long, otherwise, the samples can be boiled to be rotten, taking out the dye vat, and directly placing the dye vat into a refrigerator at 4 ℃ for cooling until the antigen repairing liquid is cooled and clarified;

(2) Dyeing process

Placing slices in a refrigerator at 4 ℃ at normal temperature to return to the room temperature, carefully wiping antigen repairing liquid around each tissue by using absorbent paper, delineating the edge of the sample by using a PAP pen (hydrophobic), placing the delineated slices into a dye vat containing PBS for washing for 3 times, 5min each time, placing the washed slices into a wet dish (for preventing the sample from drying), adding 0.2% Triton-PBS into each sample for permeation for 60-90 min, placing the samples into the dye vat containing PBS for washing for 1 time and 5min after permeation, adding 200 mu L of sealing liquid containing 10% fetal bovine serum and 1 BSA-PBS into each sample after washing, and sealing for 1-2 h at the room temperature;

after blocking, UII (1).

From the results of immunohistochemical analysis in fig. 2 and 3, it can be seen that UII and GPR14 were significantly up-regulated in atopic skin mice, the signals of UII and GPR14 were enhanced, and the signals were mostly localized to the microvascular wall and keratinocytes, and the dermatitis skin was significantly increased. Furthermore, in the mouse MC 903-induced AD-like model group, UII expression was significantly up-regulated in AD-mTGN, while GPR14 expression was down-regulated, compared to the healthy control group, indicating a correlation between UII and GPR14 expression and atopic dermatitis skin.

Example 3 mouse model of chronic pruritus constructed by MC903

(1) Laboratory animal

6-8 weeks healthy C57BL/6J female mice, weighing 16-18 g, purchased from Beijing Sibefu Biotech, inc., were bred in 8 mice per cage, and had free access to water. The temperature of the animal room is constant at 25 ℃, the ventilation is good, and the illumination and the dark environment are alternately carried out for 12h period.

(2) The steps of the mouse chronic pruritus model constructed by MC903 are as follows:

1) The mice are bred in a mouse animal room to adapt for 3-5 days, and the mice are divided into 2 groups according to the weight: treatment groups and control groups, 8 mice per group;

2) Mice were subjected to MC903 (4 nM, 20. Mu.L) in the left ear and anhydrous ethanol (20. Mu.L) in the right ear, wherein MC903 was purchased from (sigma, C4369-10 MG), all mice were subjected to behavior video immediately after daily administration for 60 minutes each time, the scratching behavior of the mice was recorded for 1 hour and the results were counted, and the scratching of the mice during construction of the chronic pruritus model by MC903 was shown in Table 1.

Table 1 scratching of mice during construction of a chronic pruritus model by MC903

As can be seen from Table 1, the chronic pruritus model was successfully established for 7 consecutive days.

Example 4 assay of GSK1562590 for the treatment of MC903 induced chronic itching in mice

(1) According to the weight and the itching scratching condition of the mice, the mice successfully established in the chronic pruritus model in the example 3 are averagely divided into 2 groups, namely a treatment group MC903+ GSK1562590 and a control group MC903+ Vehicle, and each group comprises 8 mice.

(2) Coating the left and right ears of mice with MC903 (4nM, 20 μ L) and absolute ethanol (20 μ L) each day, then performing gavage treatment 1 time daily immediately with the treatment group and the control group, applying 5mg/kg GSK1562590 (1.7% DMSO solution, 20% hydroxypropyl- β -cyclodextrin, dose 100 μ L), applying vehicle (1.7% DMSO solution, 20% hydroxypropyl- β -cyclodextrin, dose 100 μ L) to the control group, observing animal behavior by video before each administration, and recording the administration time after administration;

5mg/kg GSK1562590 (1.7% DMSO20% -hydroxypropyl-. Beta. -cyclodextrin solution, dosage 100. Mu.L). The calculation method comprises the following steps: the stock solution was frozen in 100mM DMSO prepared by dissolving GSK1562590 as purchased. The concentration of the GSK1562590 mother liquor obtained from M = C × V × M is about 10mg/166ul =60.24 μ g/μ L.5mg/kg, calculated as 18g of body weight per mouse. That is, one mouse needs 0.09mg of dosage, calculated by 10 mice, that is, 0.9mg of mother liquor is needed in total, calculated by 1mg of total amount, about 17 mu L of GSK1562590 mother liquor is needed each time. The treatment group required a total dose of 1000 μ L of 5mg/kg GSK1562590 (1.7% dmso20% -hydroxypropyl- β -cyclodextrin solution) per treatment group. The control group required a total dose of 1000uL of vehicle (1.7% DMSO solution-20% hydroxypropyl-. Beta. -cyclodextrin solution).

Preparing 20% hydroxypropyl-beta-cyclodextrin solution:

0.2g of hydroxypropyl-beta-cyclodextrin powder is dissolved in 1mL of sterile water or deionized water to obtain a hydroxypropyl-beta-cyclodextrin solution with the concentration of 20%.

5mg/kg GSK1562590 (1.7% DMSO-20% hydroxypropyl-. Beta. -cyclodextrin solution, 100. Mu.L dose): adding corresponding volume (17 μ L) of GSK1562590 mother liquor prepared in advance with DMSO into 20% hydroxypropyl- β -cyclodextrin solution, mixing thoroughly, standing at room temperature to obtain 5mg/kg GSK1562590 (1.7% DMSO20% -20% hydroxypropyl- β -cyclodextrin solution); vehicle (1.7% DMSO-20% hydroxypropyl-. Beta. -cyclodextrin solution, dose 100. Mu.L) preparation: adding only 17 μ l of LDMSO to a 20% hydroxypropyl- β -cyclodextrin solution, mixing thoroughly, and standing at room temperature to obtain a solution with a content of (1.7% dmso-20% hydroxypropyl- β -cyclodextrin solution).

(3) Carrying out balance treatment on two groups of mice for 1h, carrying out behavior video recording on the mice after the balance of 1h is finished, wherein each video recording lasts for 60min, the drug application process lasts for 5 days, the scratching behavior of the mice for 1 hour is recorded every day, and the results are counted, wherein the weight of the two groups of mice is balanced, and the diet is healthy;

(4) After the drug application process is continued for 5 days, the MC903 and the absolute ethyl alcohol are not coated, the drug administration treatment is not continued, and only two groups of mice are subjected to video observation;

(5) Mice were harvested on day 6 post-administration for RNA-Seq, H & E staining and immunohistochemical analysis of the ear skin, ipsilateral and contralateral Trigeminal Ganglia (TG).

The result B in fig. 4 shows that the scratching times of the mice in the treatment group are obviously lower than those of the control group at the 10 th day (namely, the 3 rd day after the administration), the statistical analysis result shows that the scratching times of the mice in the treatment group are obviously inhibited, the scratching times of the mice in the treatment group are still obvious compared with those of the control group at the 5 th day after the administration, and the ear thicknesses of the two groups of mice have different degrees of dermatitis skin lesion, so that the chronic pruritus behavior induced by MC903 is effectively reduced by the GSK1562590 inhibitor; the results of C in fig. 4 show that the mice started to be treated by drug application on day 8, that the treated group and the control group did not have a significant difference in itch-like behavior between day 8 and day 9, that the statistical analysis results on day 10 of treatment showed a significant inhibition of the number of scratching times in the treated group compared to the control group, that UII was involved in the later stage of MC 903-induced chronic itch, and that GSK1562590 significantly attenuated MC 903-induced itch-like behavior on day 10 and was time-dependent.

Example 5H & E staining and immunohistochemistry Chronic itchy skin was analyzed for pathology and infiltration of skin epidermal immune cells.

(1) Immunohistochemistry (Paraffin section staining) method same as example 2

(2) H & E staining

Ear skin samples taken on day 13 of the MC903 chronic pruritus model were prepared in advance and H & E stained as follows:

1) Washing the sample with distilled water for two minutes;

2) Dyeing the processed sample with hematoxylin for 5-10 minutes, and washing away residual dyeing liquid with pure water after dyeing;

3) Differentiating with 0.5% hydrochloric acid alcohol (75% alcohol) for 30 s, and washing with pure water;

4) Soaking the glass slide in pure water for 5-10 min to make it turn blue;

5) Dyeing with eosin for 1-5 minutes, and flushing residual dyeing liquid with pure water after dyeing is finished;

6) Immersing the glass slide into absolute ethyl alcohol for dehydration for 2 times, and dehydrating for 2 minutes each time;

7) Immersing the glass slide into dimethylbenzene for transparent treatment;

8) And sealing and performing microscopic examination.

(3) Mast cell test detection by C-kit

An ear skin section sample extracted on day 13 of an MC903 chronic pruritus model is prepared in advance, and the detection steps are as follows:

1) Slices were dewaxed conventionally to water: xylene → 100% absolute ethanol → 95% absolute ethanol → 70% absolute ethanol → 50% absolute ethanol (10 min for each step) → distilled water (5 min for each step);

2) A heat-repairing antigen: preparing an antigen repairing liquid (the original concentration is 10 x) and pure water in a dye vat according to a ratio of 1;

3) Carefully wiping the antigen repair solution around each tissue with absorbent paper, delineating the sample edge with PAP pen (hydrophobic), washing the delineated slide with PBS for 5min for 3 times;

4)3％H ₂ O ₂ incubating at room temperature for 25min to inactivate endogenous peroxidase (coated with tin foil paper and protected from light), washing with PBS for 3 times, each for 5min;

5) Permeability: adding 0.2% Triton-PBS into each sample, performing permeation for 60min, and washing with PBS for 1 time and 5min after permeation;

6) And (3) sealing: adding 5% BSA blocking solution dropwise, incubating at room temperature for 20min, and wiping off the excess solution for washing-free;

7) Diluting primary antibody (c-Kit) with blocking solution according to a ratio of 1;

8) Diluting the Bio-goat anti-immune IgG concentrated solution into working solution by using confining liquid according to the ratio of 1 to 50, dripping the Bio-goat anti-rabbit IgG working solution, incubating for 30min at the temperature of 20 to 37 ℃, or selecting room temperature incubation for 1h, washing for 4 times by PBS (5 min each time);

9) Diluting the SABC-POD concentrated solution into a working solution by using a confining solution according to a ratio of 1;

10 Preparing DAB mixed solution, sequentially adding 4 drops of reagent 1,8 drops of reagent2,4 drops of reagent 3 and 4 drops of reagent 4 (blue Nickel) into 5ml of distilled water to ensure complete mixing, dropwise adding DAB mixed solution, wrapping with tinfoil paper, incubating in a dark place for 30min (preventing background color from becoming dark), observing the dyeing condition of a sample under a mirror, and cleaning with distilled water for 1 time and 5min after finishing.

11 DAPI chromogenic labeling nuclei: dripping DAPI to cover the tissue sample, and incubating for 5min;

12 Sealing sheet): after DAPI staining is complete, a dehydration step may also be performed: 50% absolute ethyl alcohol → 70% absolute ethyl alcohol → 95% absolute ethyl alcohol → 100% absolute ethyl alcohol → xylene, and sealing piece preservation.

13. And (4) photographing and observing, and storing the slide at-80 ℃ after photographing.

As can be seen from fig. 5, the MC903 mouse ear skin immunohistochemical staining results showed that the chronic itchy skin expression of UII and GPR14 was significantly enhanced on the MC 903-induced ipsilateral (left) ear skin, and that the epidermal fluorescence signal was significantly attenuated on the ipsilateral side of UII and GPR14 by GSK1562590 treatment compared to control treatment; however, as can be seen from FIG. 6, there was no significant difference in the intensity of the fluorescence signals of the epidermis on the opposite side (right side) of the ear skin of mice induced by EtOH between UII and GPR14, which indicates that UII and GPR14 are involved in the construction of MC 903-induced chronic itchy skin of mice.

The results of fig. 7 and 8 show that the thickness of the mouse ear epidermis is significantly reduced after the treatment of GSK1562590, the infiltration of eosinophilic granulocytes and basophilic granulocytes in the mouse ear epidermis is significantly reduced, but not reduced in the dermis, and the results show that GSK1562590 affects the pathological changes of the ear skin, and the treatment of GSK1562590 can effectively relieve the atopic dermatitis skin inflammation.

Example 6MC 903-induced high throughput sequencing assay of ear skin samples from mouse model of chronic itching

1) Tissue total RNA extraction

Preparing ear skin samples extracted on day 13 of an MC903 chronic pruritus model in advance;

total RNA was extracted from tissues using Trizol (Invitrogen, USA) according to the instructions; taking about 60mg of tissues, grinding the tissues into powder in a 2mL tube by using liquid nitrogen, homogenizing for 2 minutes, and standing for 5 minutes at normal temperature;

following centrifugation at 13000g,4 ℃ for 5 minutes, the supernatant was transferred to a new EP tube with 0.3mL chloroform/isoamyl alcohol (24), shaken vigorously for 15s (1 time wash, left to stand for about 3min, vortexed again), allowed to stand at room temperature for 15min, and then centrifuged at 13000g for 10min at 4 ℃;

after centrifugation, the upper aqueous phase, which retains the RNA, was transferred to a new tube, an equal volume of isopropanol supernatant was added, and then centrifuged at 13000 rpm for 20 minutes at 4 ℃.

The supernatant was removed, 1mL of 75% ethanol was added to the resuspended solution, which was then centrifuged at 13000g for 10min at 4 ℃, 75% ethanol in the tube was discarded, and RNase-free water 25. Mu.L-100. Mu.L was added to solubilize the RNA, after which total RNA was identified and quantified using nanodroplets and an Agilent 2100 bioanalyzer (Thermo Fisher Scientific, USA).

2) mRNA library construction

mRNA was purified by adsorbing magnetic beads with Oligo (dT). The purified mRNA is cleaved into small fragments at an appropriate temperature using fragment buffer. Reverse transcription was performed using random hexamer primers to generate first strand cDNA, followed by synthesis of second strand cDNA. End repair was performed by adding A-Tailing Mix and RNA IndexAdapters. The cDNA fragment obtained in the previous step is amplified by PCR, and the product is dissolved in EB solution after being purified by Ampure XP Beads. And (3) performing heating denaturation and splint oligonucleotide sequence cyclization on the double-stranded PCR product obtained in the last step to obtain a final library. Single-stranded circular DNA (ssCir DNA) is formatted into the final library. The final library was amplified with phi29 to make DNA Nanospheres (DNBs) with more than 300 copies of one molecule, and the DNBs were loaded into patterned nanoarrays to generate single-ended 50 base reads on a BGIseq500 platform (BGI-Shenzhen, china).

As can be seen from the RNA-Seq analysis results of fig. 10, GSK156290 significantly reduced Mrgpra2b, CCR2, CCR6, IL-13 ra 1, IL-13 ra2, NTRK1, IL-31 ra, CXCR3, CXCR6, and IL27 ra in MC903 induced chronic itchy ear skin, which significantly down-regulated genes are the basis for reducing the itch-like response. In addition, the transcription level of inflammation-related cytokines such as Nppb, IL-24, IL-19, CXCL5, fasL, IL-1b, IL-13, CCL22, CCL17 and carcinostatin M (OSM) is also obviously reduced, the transcription level is related to AD diseases, and IL-13 and IL-31 play a key role in AD. Therefore, UII represents a new source of pruritus in mice and may promote human dermatitis-associated pruritus, highlighting its role as a target for chronic pruritus.

Typical cases are as follows:

case 1

One atopic dermatitis patient (age 35) was treated with daily oral administration of GSK1562590 (5 mg/kg) and after three days of treatment had a significant reduction in scratch response, 4mm of the patient's skin was sampled without injury, and a significant reduction in epidermal thickness and a significant reduction in intradermal eosinophil and basophil infiltration was seen via H & E staining results and immunohistochemical analysis.

Case 2

One atopic dermatitis patient (56 years old) was observed to have a significant reduction in daily scratch response 5 days after oral GSK1562590 (5 mg/kg) treatment compared to five days ago, and a significant reduction in epidermal thickness and a significant reduction in intradermal eosinophil and basophil infiltration was seen on a 4mm minimally invasive sample of the patient's skin via H & E staining results and immunohistochemical analysis.

The invention can effectively reduce the pruritus frequency and relieve the dermatitis caused by atopic dermatitis by GSK 1562590.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (7)

1. The application of UTR antagonist GSK1562590 in preparing a medicament for treating pruritus is characterized in that the pruritus is chronic pruritus or pruritus caused by atopic dermatitis.

2. The use of claim 1, wherein GSK1562590 inhibits UII-GPR14 signaling in chronic pruritus.

3. The use according to claim 1, wherein GSK1562590 inhibits the expression of itch and inflammatory-related cytokines.

4. The use according to claim 1, wherein GSK1562590 inhibits the expression of itch-related receptors.

5. The use according to claim 3, wherein the pruritus and inflammation-associated cytokines comprise Nppb, IL-24, IL-19, CXCL5, fasL, IL-1b, IL-13, CCL22, CCL17, carcinostatin M.

6. The use according to claim 4, wherein the itch-related receptors include Mrgpra2b, CCR2, CCR6, IL-13 Ra 1, IL-13 Ra2, NTRK1, IL-31 Ra, CXCR3, IL27 Ra.

7. The use of claim 1, wherein GSK1562590 is present in a drug in an amount of 1% to 99%.

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