阅读说明：本技术 用于预防或治疗瘙痒症的、包含吡唑类衍生物作为有效成分的药物组合物以及用于其检测的筛选方法 (Pharmaceutical composition for preventing or treating pruritus comprising pyrazole derivative as active ingredient and screening method for detection thereof ) 是由 吴禹泽 李智煐 妟成爱 于 2018-03-06 设计创作，主要内容包括：本发明涉及一种用于预防或治疗瘙痒症的、包含吡唑类衍生物作为有效成分的药物组合物以及用于其检测的筛选方法。根据本发明的用于预防或治疗瘙痒的药物组合物可以通过抑制细胞内Mrgpr X1的活性来缓解瘙痒症状,且可以通过抑制细胞内hH1R的活性来缓解瘙痒症状,从而也可以用作预防或治疗组胺介导的瘙痒症的药物。另外,通过皮肤干燥小鼠模型实验已经证实,该组合物还对皮肤干燥具有显著的缓解作用,从而可以用作治疗皮肤干燥的药物。此外,根据本发明的药物组合物可以缓解由牛皮癣引起的瘙痒症状,因此也可以用作治疗牛皮癣的药物。而且,根据本发明的药物组合物即使在体内给药期间也可以保持稳定的活性,而不会引起不良反应。(The present invention relates to a pharmaceutical composition for preventing or treating pruritus, which comprises a pyrazole derivative as an active ingredient, and a screening method for detecting the same. The pharmaceutical composition for preventing or treating pruritus according to the present invention can relieve pruritus symptoms by inhibiting the activity of intracellular Mrgpr X1 and can relieve pruritus symptoms by inhibiting the activity of intracellular hH1R, and thus can also be used as a medicament for preventing or treating histamine-mediated pruritus. In addition, the composition has obvious relieving effect on the xerosis cutis through experiments of a skin dryness mouse model, so that the composition can be used as a medicine for treating the xerosis cutis. In addition, the pharmaceutical composition according to the present invention can alleviate itching symptoms caused by psoriasis, and thus can also be used as a medicine for treating psoriasis. Also, the pharmaceutical composition according to the present invention can maintain stable activity even during in vivo administration without causing adverse reactions.)

1. A pharmaceutical composition for preventing or treating pruritus, comprising a compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

[ formula 1]

In the formula 1, the first and second groups,

R¹And R²independently is a straight or branched chain C₁-C₆Alkyl, straight or branched C₁-C₆alkoxy, -NO₂、-NR⁴R⁵Or unsubstituted or substituted C₆-C₁₂Aryl, wherein, said substituted C₆-C₁₂the aryl group may be substituted with one or more substituents selected from: halogen, straight or branched C₁-C₆Alkyl, straight or branched C₁-C₆Alkoxy, -NO₂and-NR⁴R⁵At this time, R⁴And R⁵Independently hydrogen, or straight or branched C₁-C₆An alkyl group;

R³Is hydrogen, - (C ═ O) OR⁶Or unsubstituted or substituted 3-to 10-membered heterocycloalkyl or heterocycloalkenyl containing one or more heteroatoms selected from N, O and S, in which case R⁶May be hydrogen, or C which may be straight or branched₁-C₆Alkyl, wherein the substituted heterocycloalkyl or heterocycloalkenyl may be substituted with one or more substituents selected from the group consisting of: halogen, straight or branched C₁-C₆Alkyl, and straight or branched C₁-C₆An alkoxy group;

A is-NH-, -O-, -S-, - (C ═ O) NH-, -NH (C ═ O) -, - (C ═ O) O-, or-O (C ═ O) -; and is

m and n may be independently an integer of 0 to 8.

2. The pharmaceutical composition for preventing or treating pruritus according to claim 1, wherein:

R¹And R²independently is a straight or branched chain C₁-C₃Alkyl, straight or branched C₁-C₃Alkoxy, -NO₂、-NR⁴R⁵Or unsubstituted or substituted C₆-C₁₀Aryl, wherein, said substituted C₆-C₁₀The aryl group may be substituted with one or more substituents selected from: halogen, straight or branched C₁-C₃Alkyl, straight or branched C₁-C₃Alkoxy, -NO₂and-NR⁴R⁵at this time, R⁴And R⁵Independently hydrogen, or straight or branched C₁-C₃An alkyl group;

R³Is hydrogen, - (C ═ O) OR⁶Or unsubstituted or substituted 3-8 membered heterocycloalkyl or heterocycloalkenyl containing one or more heteroatoms selected from N, O and S, in which case R⁶May be hydrogen, or C, straight or branched₁-C₃Alkyl, wherein the substituted heterocycloalkyl or heterocycloalkenyl may be substituted with one or more substituents selected from the group consisting of: halogen, straight or branched C₁-C₃Alkyl, and straight or branched C₁-C₃An alkoxy group;

A is-NH-, -O-, -S-, - (C ═ O) NH-, -NH (C ═ O) -, - (C ═ O) O-, or-O (C ═ O) -; and is

m and n may be independently an integer of 0 to 6.

3. The pharmaceutical composition for preventing or treating pruritus according to claim 1, wherein:

R¹And R²Independently is a straight or branched chain C₁-C₃Alkyl, -NO₂Or unsubstituted or substituted phenyl, wherein the substituted phenyl may be substituted with one or more substituents selected from:

Straight or branched C₁-C₃Alkyl, straight or branched C₁-C₃Alkoxy and-NO₂；

R³Is hydrogen, - (C ═ O) OR⁶Or unsubstituted or substituted 5-to 7-membered heterocycloalkyl containing one or more heteroatoms selected from N, O and S, in which case the substituted heterocycloalkyl may be substituted by one or more straight or branched C₁-C₃Alkyl substitution;

a is- (C ═ O) NH-, -NH (C ═ O) -, - (C ═ O) O-, or-O (C ═ O) -; and is

m and n may be independently an integer of 0 to 5.

4. The pharmaceutical composition for preventing or treating pruritus according to claim 1, wherein:

R¹And R²Independently methyl, or unsubstituted or substituted phenyl, wherein said substituted phenyl may be substituted with one or more groups selected from methyl, methoxy and-NO₂Substituted with the substituent(s);

R³Is- (C ═ O) OH or piperazine substituted by one or more methyl groupsA pyridyl group;

A is- (C ═ O) NH-or-NH (C ═ O) -; and is

m and n may be independently an integer of 0 to 3.

5. The pharmaceutical composition for preventing or treating pruritus according to claim 1, wherein the pruritus is histaminic pruritus.

6. The pharmaceutical composition for preventing or treating pruritus according to claim 1, wherein the pruritus is non-histaminic pruritus.

7. the pharmaceutical composition for preventing or treating pruritus according to claim 1, wherein: the pruritus is pruritus caused by one or more diseases selected from the group consisting of: psoriasis (Psoriatic), Dry skin (Dry skin), neurodermatitis, contact dermatitis, seborrheic dermatitis, atopic dermatitis, trichomonas dermatitis, asteatosis (asteatosis), senile pruritus, insect bites, photosensitive dermatitis, urticaria, prurigo, herpes, impetigo (impetigo), eczema (eczema), psoriasis (tinea), lichen (lichen), scabies (scabies), and acne vulgaris.

8. A health-care functional food composition for preventing or relieving pruritus, comprising a compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

[ formula 1]

In the formula 1, the first and second groups,

R¹And R²Independently is a straight or branched chain C₁-C₆Alkyl, straight or branched C₁-C₆Alkoxy, -NO₂、-NR⁴R⁵Or unsubstituted or substituted C₆-C₁₂Aryl, wherein, said substituted C₆-C₁₂The aryl group may be substituted with one or more substituents selected from: halogen, straight or branched C₁-C₆Alkyl, straight or branched C₁-C₆Alkoxy, -NO₂and-NR⁴R⁵At this time, R⁴And R⁵Independently hydrogen, or straight or branched C₁-C₆An alkyl group;

R³Is hydrogen, - (C ═ O) OR⁶Or unsubstituted or substituted 3-to 10-membered heterocycloalkyl or heterocycloalkenyl containing one or more heteroatoms selected from N, O and S, in which case R⁶May be hydrogen, or C which may be straight or branched₁-C₆Alkyl, wherein the substituted heterocycloalkyl or heterocycloalkenyl may be substituted with one or more substituents selected from the group consisting of: halogen, straight or branched C₁-C₆Alkyl, and straight or branched C₁-C₆An alkoxy group;

A is-NH-, -O-, -S-, - (C ═ O) NH-, -NH (C ═ O) -, - (C ═ O) O-, or-O (C ═ O) -; and is

m and n may be independently an integer of 0 to 8.

9. A method of screening for a compound for preventing or treating pruritus comprising the steps of:

Treating cells expressing MRGPR X1 (Mas-associated G protein-coupled receptor) with an agent inducing itch, and culturing the cells (step 1); and

cells with pruritus were treated with candidate materials and the inhibitory effect on MRGPR X1 activity was measured (step 2).

10. The method for screening a compound for the prevention or treatment of pruritus according to claim 9,

Wherein the substance inducing pruritus of step 1 is chloroquine.

11. A method of screening for an active material for the prevention or treatment of pruritus comprising the steps of:

Treating cells expressing hH1R (human histamine 1 receptor) with an agent that triggers pruritus and culturing the cells (step 1); and

Cells with pruritus were treated with candidate materials and the inhibitory effect on hH1R activity was measured (step 2).

12. The method for screening a compound for use in the prevention or treatment of pruritus according to claim 11,

wherein the substance inducing pruritus in step 1 is selected from one or more of the following substances: interleukin-1 (interleukin-1), cytokines (cytokine), serotonin (serotonin), acetylcholine (acetylcholine), substance P (substance P), leukotrienes (leukotrine), and prostaglandins (prostaglandin).

Technical Field

The present invention relates to a pharmaceutical composition for preventing or treating pruritus, which comprises a pyrazole derivative as an active ingredient, and a screening method for detecting the same.

Background

Pruritus (or pruritus) is an obvious symptom that appears systemically in various skin cases. Pruritus causes itching to protect the skin from insects, toxic plants, or other harmful irritants. However, pruritus does not always play a beneficial role as described above. Chronic pruritus is associated with eczema, kidney disease, cirrhosis and skin diseases, including certain cancers. Many nervous system diseases, including, for example, multiple sclerosis, diabetic neuropathy, and postherpetic neuralgia (shingles), etc., also cause severe itching. Pruritus is developed from sensory nerve cells, the cell bodies of which are known to be in the dorsal root ganglia.

many types of pruritus are known to be mediated by histamine (histaminic pruritus). Chloroquine is an antimalarial agent and is known to cause pruritus independent of histamine (non-histaminic pruritus).

The Mas-associated G protein-coupled receptor (Mrgprs) is known to be a non-histaminic scrapie receptor, a GPCR (G protein-coupled receptor) expressed only in peripheral sensory neurons. These receptors are present in various tissues of adults, especially in neurons (Dong et al, 2001). Wherein Mrgpr X1 is distributed in the human dorsal root ganglion.

human histamine receptor 1 antagonists are known to inhibit histamine-induced pruritus (in 2009, sensory neuron-specific GPCR Mrgpr is the pruritus receptor mediating chloroquine-induced pruritus. cell 139, 1353-1365 (2009)). However, the antagonist had no effect on non-histaminic pruritus. Allergic pruritus is mediated by histamine, accounting for only one third of all pruritus. Thus, allergic pruritus can be treated with antihistamines, but most pruritus cannot be treated with antihistamines.

As a therapeutic agent for pruritus, agents for cooling the skin, including calamine lotion or 1% menthol lotion, steroids, and antihistamines, are currently being used. However, these preparations for cooling the skin only work temporarily. On the other hand, steroids show strong anti-inflammatory activity, and thus they are excellent in relieving symptoms of various diseases such as joint disease, cerebrovascular disease, inflammatory disease and allergic disease, but have been limitedly used due to their side effects upon long-term administration or overdose. Antihistamines are mainly used for oral administration. Generally, drugs have been developed that only inhibit the H1 receptor directly associated with pruritus. However, the use of these drugs is also limited because they cause side effects such as general decline in cognitive ability and motor nerves. As mentioned above, there is no general treatment for pruritus. Therefore, there is a need to develop a safe and effective drug for pruritus.

Therefore, the present inventors have studied to develop a safe and effective therapeutic agent effective not only for the treatment of histamine-type pruritus but also for non-histamine-type pruritus. In the course of research, the present inventors have confirmed that pyrazole derivative compounds can effectively inhibit the activities of Mrgpr X1 and hH1R (human histamine receptor subtype 1), thereby completing the present invention.

disclosure of Invention

An object of the present invention is to provide a pharmaceutical composition for preventing or treating pruritus.

another object of the present invention is to provide a functional health food composition for preventing or treating pruritus.

it is still another object of the present invention to provide a method for screening a compound for preventing or treating pruritus.

In order to accomplish the above objects, the present invention provides a pharmaceutical composition for preventing or treating pruritus, which comprises a compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

(in formula 1, R¹、R²、R³a, m and n are as defined in the specification. )

The present invention also provides a health functional food composition for preventing or treating pruritus, which comprises a compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

(in formula 1, R¹、R²、R³A, m and n are as defined in the specification. )

Further, the present invention provides a screening method for a compound for preventing or treating pruritus, which includes the steps of:

Treating cells expressing MRGPR X1 (Mas-associated G protein-coupled receptor) with an agent inducing itch, and culturing the cells (step 1); and

Cells with pruritus were treated with candidate materials and the inhibitory effect on MRGPR X1 activity was measured (step 2).

In addition, the present invention provides a screening method of an active material for preventing or treating pruritus, which comprises the steps of:

Treating cells expressing hH1R (human histamine 1 receptor) with an agent that triggers pruritus and culturing the cells (step 1); and

Cells with pruritus were treated with candidate materials and the inhibitory effect on hH1R activity was measured (step 2).

Advantageous effects

The pharmaceutical composition for preventing or treating pruritus according to the present invention can be effectively used as a prophylactic or therapeutic agent for non-histaminic pruritus, because it can alleviate the symptoms of pruritus by inhibiting the activity of intracellular Mrgpr X1. The pharmaceutical composition for preventing or treating pruritus according to the present invention can be effectively used as a prophylactic or therapeutic agent for histaminic pruritus because it can alleviate the symptoms of pruritus by inhibiting the activity of intracellular hH 1R. In addition, the composition has obvious relieving effect on the xerosis cutis through experiments of a skin dryness mouse model, so that the composition can be used as a medicine for treating the xerosis cutis. Furthermore, the pharmaceutical composition according to the present invention can alleviate itching symptoms caused by psoriasis, and thus can also be used as a medicine for treating psoriasis. Also, the pharmaceutical composition according to the present invention can maintain stable activity even during in vivo administration without causing adverse reactions.

Drawings

Fig. 1 is a graph showing the results of fluorescence analysis performed after Mrgpr X1 cells were treated with chloroquine alone.

Fig. 2 is a graph showing the results of fluorescence analysis performed after Mrgpr X1 cells were treated with a pharmaceutical composition containing the pyrazole derivative of example 1 and chloroquine.

FIG. 3 is a graph showing the results of fluorescence analysis performed after treating hH1R cells with histamine alone.

FIG. 4 is a graph showing the results of fluorescence analysis performed after treatment of hH1R cells with histamine and diphenhydramine.

Fig. 5 is a graph showing the results of fluorescence analysis performed after hH1R cells were treated with a pharmaceutical composition comprising the pyrazole derivative of example 1 and histamine.

Fig. 6 is a graph illustrating evaluation of the itch therapeutic effect in a histamine mouse model treated with a pharmaceutical composition comprising the pyrazole derivative of example 1.

Fig. 7 is a graph illustrating evaluation of the therapeutic effect on pruritus when a mouse model of chloroquine is treated with a pharmaceutical composition containing the pyrazole derivative of example 1.

Fig. 8 is a graph illustrating evaluation of the therapeutic effect on pruritus in a skin-dry mouse model treated with a pharmaceutical composition containing the pyrazole derivative of example 1.

Fig. 9 is a graph showing the results of a rotarod movement state test of mice treated with a pharmaceutical composition comprising the pyrazole derivative of example 1.

Fig. 10 is a graph illustrating evaluation of the therapeutic effect on pruritus when a mouse model of psoriasis is treated with a pharmaceutical composition comprising the pyrazole derivative of example 1.

Detailed Description

Hereinafter, the present invention is described in detail.

The present invention provides a pharmaceutical composition for preventing or treating pruritus, which comprises a compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

In the above formula 1, the above formula (ii),

R¹and R²Independently is a straight or branched chain C₁-C₆Alkyl, straight or branched C₁-C₆alkoxy, -NO₂、-NR⁴R⁵Or unsubstituted or substituted C₆-C₁₂Aryl, wherein, said substituted C₆-C₁₂Aryl radicals may be substituted by oneOr a plurality of substituents selected from: halogen, straight or branched C₁-C₆Alkyl, straight or branched C₁-C₆Alkoxy, -NO₂and-NR⁴R⁵At this time, R⁴And R⁵Independently hydrogen, or straight or branched C₁-C₆An alkyl group;

R³Is hydrogen, - (C ═ O) OR⁶Or unsubstituted or substituted 3-to 10-membered heterocycloalkyl or heterocycloalkenyl containing one or more heteroatoms selected from N, O and S, in which case R⁶may be hydrogen, or C which may be straight or branched₁-C₆Alkyl, wherein the substituted heterocycloalkyl or heterocycloalkenyl may be substituted with one or more substituents selected from the group consisting of: halogen, straight or branched C₁-C₆Alkyl, and straight or branched C₁-C₆an alkoxy group;

A is-NH-, -O-, -S-, - (C ═ O) NH-, -NH (C ═ O) -, - (C ═ O) O-, or-O (C ═ O) -; and is

m and n may be independently an integer of 0 to 8.

Further, in the above formula 1,

R¹And R²Independently is a straight or branched chain C₁-C₃Alkyl, straight or branched C₁-C₃Alkoxy, -NO₂、-NR⁴R⁵Or unsubstituted or substituted C_6-C₁₀Aryl, wherein, said substituted C_6-C₁₀The aryl group may be substituted with one or more substituents selected from: halogen, straight or branched C₁-C₃Alkyl, straight or branched C₁-C₃Alkoxy, -NO₂and-NR⁴R⁵At this time, R⁴And R⁵Independently hydrogen, or straight or branched C₁-C₃an alkyl group;

R³Is hydrogen, - (C ═ O) OR⁶or unsubstituted or substituted 3-8 membered heterocycloalkyl or heterocycloalkenyl containing one or more heteroatoms selected from N, O and S, in which case R⁶May be hydrogen, or C, straight or branched₁-C₃Alkyl, wherein the substituted heterocycloalkyl or heterocycloalkenyl may be substituted with one or more substituents selected from the group consisting of: halogen, straight or branched C₁-C₃Alkyl, and straight or branched C₁-C₃An alkoxy group;

A is-NH-, -O-, -S-, - (C ═ O) NH-, -NH (C ═ O) -, - (C ═ O) O-, or-O (C ═ O) -; and is

m and n may be independently an integer of 0 to 6.

further, in the above formula 1,

R¹And R²Independently is a straight or branched chain C₁-C₃Alkyl, -NO₂Or unsubstituted or substituted phenyl, wherein the substituted phenyl may be substituted with one or more substituents selected from: straight or branched C₁-C₃Alkyl, straight or branched C₁-C₃Alkoxy and-NO₂；

R³Is hydrogen, - (C ═ O) OR⁶Or unsubstituted or substituted 5-to 7-membered heterocycloalkyl containing one or more heteroatoms selected from N, O and S, in which case the substituted heterocycloalkyl may be substituted by one or more straight or branched C₁-C₃Alkyl substitution;

a is- (C ═ O) NH-, -NH (C ═ O) -, - (C ═ O) O-, or-O (C ═ O) -; and is

m and n may be independently an integer of 0 to 5.

further, in the above formula 1,

R¹And R²Independently methyl, or unsubstituted or substituted phenyl, wherein said substituted phenyl may be substituted with one or more groups selected from methyl, methoxy and-NO₂Substituted with the substituent(s);

R³Is- (C ═ O) OH or piperidinyl substituted with one or more methyl groups;

A is- (C ═ O) NH-or-NH (C ═ O) -; and is

m and n may be independently an integer of 0 to 3.

The pruritus may be histaminic pruritus, non-histaminic pruritus, pruritus induced by chloroquine, pruritus induced by dry skin, or psoriatic pruritus (pruritus induced by psoriasis). Pruritus may also be caused by the following diseases: neurodermatitis, contact dermatitis, seborrheic dermatitis, self-allergic dermatitis, trichomonas dermatitis, seborrhoeic deficiency (seborrhoeic deficiency), senile pruritus, insect bites, photosensitive dermatitis, urticaria, prurigo, herpes, impetigo, eczema, tinea, lichen, scabies or acne vulgaris. At this time, in the case of the histaminic pruritus, the pharmaceutical composition of the present invention can block the activity of histamine by reversible/competitive antagonism against hH1R (human histamine 1 receptor), thereby effectively treating or preventing the pruritus.

The pharmaceutical composition of the present invention may also be effective in treating or preventing pruritis caused by chloroquine by blocking chloroquine activity via reversible/competitive antagonism against MRGPR X1.

Pharmaceutically acceptable salts of the compounds of formula 1 may be prepared by conventional methods known to those skilled in the art. For example, pharmaceutically acceptable salts include: salts of inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid, sodium hydrogen sulfate, phosphoric acid, nitric acid, and carbonic acid; salts of organic acids such as formic, acetic, propionic, oxalic, succinic, benzoic, citric, maleic, malonic, tartaric, gluconic, lactic, gastric, fumaric, lactobionic, salicylic and acetylsalicylic (aspirin); salts of amino acids such as glycine, alanine, vanillin, isoleucine, serine, cysteine, cystine, aspartic acid, glutamine, lysine, arginine, tyrosine, and proline; salts of sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and toluenesulfonic acid; metal salts formed by reaction with alkali metals such as sodium and potassium; or ammonium ion salts.

Acid addition salts may be prepared by conventional methods known to those skilled in the art. For example, the derivative represented by formula 1 is dissolved in an organic solvent such as methanol, ethanol, acetone, dichloromethane, and acetonitrile, and an organic acid or an inorganic acid is added thereto to induce precipitation. Then, the precipitate was filtered and dried to obtain a salt. Or the solvent and the excess acid are distilled off under reduced pressure, followed by drying in an organic solvent and crystallization to obtain a salt.

in addition, pharmaceutically acceptable metal salts can be prepared by using bases. The alkali metal or alkaline earth metal salt is obtained by the following method: dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution; filtering the insoluble complex salt; the remaining solution was evaporated and dried. In this case, the metal salt is preferably prepared in a pharmaceutically suitable form of a sodium, potassium or calcium salt. The corresponding silver salt is then prepared by reacting the alkali or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

Also, the compound represented by formula 1 may be used in the form of solvates, optical isomers, hydrates, and the like prepared therefrom, and pharmaceutically acceptable salts thereof.

The pharmaceutical composition of the present invention can be formulated according to a conventional method by adding non-toxic and pharmaceutically acceptable carriers, adjuvants and excipients. For example, the pharmaceutical compositions of the present invention may be formulated for oral or parenteral administration in the form of tablets, capsules, lozenges, solutions, suspensions and the like.

The compound represented by formula 1 or a pharmaceutically acceptable salt thereof may be administered in various oral and parenteral formulations during clinical administration. When the compound represented by formula 1 or a pharmaceutically acceptable salt thereof is formulated, commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, are used. Solid preparations for oral administration are tablets, pills, powders, granules and capsules. These solid formulations are prepared by mixing one or more compounds with one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may also be used. Liquid preparations for oral administration are suspensions, solutions, emulsions and syrups, which may contain, in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatic compounds and preservatives. Formulations for parenteral administration are sterile aqueous solutions, water-insoluble excipients, suspensions and emulsions. Water-insoluble excipients and suspensions may contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), injectable esters (such as ethyl oleate), and the like.

The pharmaceutical composition comprising the compound represented by formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient may be administered parenterally, including subcutaneous injection, intravenous injection, intramuscular injection, or intrapleural injection.

to prepare a formulation of a compound represented by formula 1 or a pharmaceutically acceptable salt thereof for parenteral administration, the compound represented by formula 1 or a pharmaceutically acceptable salt thereof is mixed with a stabilizer or a buffer in water to generate a solution or a suspension, which is then formulated into an ampoule or a vial. The compositions herein may be sterilized and may also contain preservatives, stabilizers, wettable powders or emulsifiers, salts and/or buffers for adjusting the osmotic pressure, and other therapeutically useful materials, and the compositions may be formulated by conventional mixing, granulating or coating methods.

Formulations for oral administration include tablets, pills, hard/soft capsules, solutions, suspensions, emulsions, syrups, granules, elixirs, and lozenges, and the like. In addition to the active ingredient, these formulations may include diluents (e.g., lactose, glucose, sucrose, mannitol, sorbitol, cellulose and/or glycine) and lubricants (e.g., silica, talc, stearate and its magnesium or calcium salts, and/or polyethylene glycol). Tablets may contain binders such as magnesium aluminium silicate, starch paste, gelatin, methyl cellulose, sodium carboxymethyl cellulose and/or polyvinylpyrrolidone and, if desired, disintegrating agents such as starch, agarose, alginic acid or a sodium salt thereof or azeotropic mixtures and/or absorbents, colorants, flavors and sweeteners.

In addition, excipients that may be used in the pharmaceutical composition according to the present invention include sweeteners, binders, solubilizers, dissolution aids, wetting agents, emulsifiers, isotonic agents, adsorbents, disintegrants, antioxidants, preservatives, lubricants, fillers, flavors, and the like. For example, lactose, glucose, sucrose, mannitol, sorbitol, cellulose, glycine, silicon dioxide, talc, stearic acid, glyceryl stearate, magnesium aluminum silicate, starch, gelatin, tragacanth, alginic acid, sodium alginate, methylcellulose, sodium carboxymethylcellulose, agar, water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, citrus essence, strawberry essence, vanilla essence, and the like may be used as excipients.

The effective dose of the pharmaceutical composition of the present invention can be determined according to age, body weight, sex, administration method, health condition and severity of disease. The dose is usually 0.01 to 5000 mg/day based on an adult patient having a body weight of 70 kg, and may be administered once or several times per day at regular intervals according to the judgment of a doctor or pharmacist.

The present invention also provides a health functional food composition for preventing or relieving pruritus, which comprises a compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In the above formula 1, the above formula (ii),

R¹And R²Independently is a straight or branched chain C₁-C₆alkyl, straight or branched C₁-C₆Alkoxy, -NO₂、-NR⁴R⁵or unsubstituted or substituted C₆-C₁₂Aryl, wherein, said substituted C₆-C₁₂The aryl group may be substituted with one or more substituents selected from: halogen, straight or branched C₁-C₆Alkyl, straight or branched C₁-C₆Alkoxy, -NO₂and-NR⁴R⁵At this time, R⁴And R⁵Independently of each otherIs hydrogen, or C being straight or branched₁-C₆An alkyl group;

R³Is hydrogen, - (C ═ O) OR⁶Or unsubstituted or substituted 3-to 10-membered heterocycloalkyl or heterocycloalkenyl containing one or more heteroatoms selected from N, O and S, in which case R⁶May be hydrogen, or C which may be straight or branched₁-C₆Alkyl, wherein the substituted heterocycloalkyl or heterocycloalkenyl may be substituted with one or more substituents selected from the group consisting of: halogen, straight or branched C₁-C₆Alkyl, and straight or branched C₁-C₆An alkoxy group;

A is-NH-, -O-, -S-, - (C ═ O) NH-, -NH (C ═ O) -, - (C ═ O) O-, or-O (C ═ O) -; and is

m and n may be independently an integer of 0 to 8.

Further, in the above formula 1,

R¹and R²Independently is a straight or branched chain C₁-C₃Alkyl, straight or branched C₁-C₃Alkoxy, -NO₂、-NR⁴R⁵Or unsubstituted or substituted C_6-C₁₀Aryl, wherein, said substituted C_6-C₁₀The aryl group may be substituted with one or more substituents selected from: halogen, straight or branched C₁-C₃Alkyl, straight or branched C₁-C₃alkoxy, -NO₂and-NR⁴R⁵At this time, R⁴And R⁵Independently hydrogen, or straight or branched C₁-C₃an alkyl group;

R³Is hydrogen, - (C ═ O) OR⁶or unsubstituted or substituted 3-8 membered heterocycloalkyl or heterocycloalkenyl containing one or more heteroatoms selected from N, O and S, in which case R⁶may be hydrogen, or C, straight or branched₁-C₃Alkyl, wherein the substituted heterocycloalkyl or heterocycloalkenyl may be substituted with one or more substituents selected from the group consisting of: halogen, straight or branched C₁-C₃Alkyl, and straight or branched chainC of a branched chain₁-C₃An alkoxy group;

a is-NH-, -O-, -S-, - (C ═ O) NH-, -NH (C ═ O) -, - (C ═ O) O-, or-O (C ═ O) -; and is

m and n may be independently an integer of 0 to 6.

Further, in the above formula 1,

R¹and R²Independently is a straight or branched chain C₁-C₃Alkyl, -NO₂Or unsubstituted or substituted phenyl, wherein the substituted phenyl may be substituted with one or more substituents selected from: straight or branched C₁-C₃Alkyl, straight or branched C₁-C₃Alkoxy and-NO₂；

R³Is hydrogen, - (C ═ O) OR⁶Or unsubstituted or substituted 5-to 7-membered heterocycloalkyl containing one or more heteroatoms selected from N, O and S, in which case the substituted heterocycloalkyl may be substituted by one or more straight or branched chain C₁-C₃Alkyl substitution;

A is- (C ═ O) NH-, -NH (C ═ O) -, - (C ═ O) O-, or-O (C ═ O) -; and is

m and n may be independently an integer of 0 to 5.

Further, in the above formula 1,

R¹And R²Independently methyl, or unsubstituted or substituted phenyl, wherein said substituted phenyl may be substituted with one or more groups selected from methyl, methoxy and-NO₂Substituted with the substituent(s);

R³Is- (C ═ O) OH or piperidinyl substituted with one or more methyl groups;

A is- (C ═ O) NH-or-NH (C ═ O) -; and is

m and n may be independently an integer of 0 to 3.

The health-care functional food composition according to the present invention may be prepared by adding the compound of formula 1 described above or a pharmaceutically acceptable salt thereof to food or drink products for the purpose of preventing or relieving pruritus.

The food herein is not limited. For example, the composition of the present invention may be added to beverages, meats, sausages, bread, cookies, rice cakes, chocolates, candies, snacks, pizza, korean instant noodles, flour products, chewing gums, dairy products including ice cream, soups, drinks, alcoholic beverages, vitamin complexes, etc., and almost all health functional foods may be included in a broad sense.

The compound represented by formula 1 of the present invention may be used as a food additive. In this case, the compound may be added as such or mixed with other food ingredients in accordance with a conventional method. The mixing ratio of the active ingredients can be adjusted depending on the purpose of use (prevention or alleviation). Generally, the compound represented by formula 1 of the present invention may be added in an amount of 0.1 to 90 parts by weight based on the total weight of the food. However, if long-term administration is required for health and hygiene or to regulate health conditions, the content may be lower than the above, but higher contents are also acceptable, since the compound has proven to be very safe.

In addition to the compound, the composition for health drink of the present invention may further comprise various flavors or natural carbohydrates, etc. like other drinks. The natural carbohydrate may be one of the following: monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol and erythritol. In addition, natural sweeteners (thaumatin, stevia extract such as rebaudioside a, glycyrrhizin, etc.) and synthetic sweeteners (saccharin, aspartame, etc.) may also be included as sweeteners. The natural carbohydrate content is preferably 1-20g, more preferably 5-12g, in 100g of the composition of the invention.

In addition to the above ingredients, the compound represented by formula 1 of the present invention may include various nutrients, vitamins, minerals (electrolytes), essences including natural essences and synthetic essences, colorants and extenders (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid tackifiers, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents once added to soda water, and the like. The pyrazole derivatives represented by formula 1 of the present invention may also include natural fruit juices, fruit drinks, and pulps that may be added to vegetable drinks. All the mentioned ingredients can be added separately or together. The mixing ratio of these components is not critical in practice, but each component may be usually added in an amount of 0.1 to 20 parts by weight based on 100 parts by weight of the pyrazole derivative represented by the formula 1 of the present invention.

Also, the present invention provides a method for screening a compound for preventing or treating pruritus, which includes the steps of:

Treating cells expressing MRGPR X1 (Mas-associated G protein-coupled receptor) with an agent inducing itch, and culturing the cells (step 1); and

cells with pruritus were treated with candidate materials and the inhibitory effect on MRGPR X1 activity was measured (step 2).

At this time, the substance inducing itch of step 1 may be chloroquine, and any substance inducing itch that is not mediated by histamine and is independent of histamine may be used without limitation. In another aspect, the substance that causes pruritus may be an acute pruritus-causing substance.

In addition, the present invention provides a screening method of an active material for preventing or treating pruritus, which comprises the steps of:

Treating cells expressing hH1R (human histamine 1 receptor) with an agent that triggers pruritus and culturing the cells (step 1); and

cells with pruritus were treated with candidate materials and the inhibitory effect on hH1R activity was measured (step 2).

At this time, the substance inducing itch of step 1 is selected from one or more of the following substances: histamine, interleukin-1, cytokines, serotonin, acetylcholine, substance P, leukotriene and prostaglandin, and any substance that causes itch depending on histamine may be used without limitation.

In the screening method according to the present invention, the measurement of step 2 may be performed by one or more methods. The method is selected from: fluorescence assay, fluorescence resonance energy transfer assay, bioluminescence resonance energy transfer assay, fluorescence polarization assay, western blot, immunoprecipitation assay, dual luciferase reporter assay, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry, and any measurement method commonly used in the art can be used without limitation.

practical and presently preferred embodiments of the present invention are shown in the following examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

< example 1> pharmaceutical composition for preventing or treating pruritus

In experimental example 1, a pharmaceutical composition comprising the pyrazole derivative represented by formula 2 for preventing or treating pruritus was prepared. At this time, the pyrazole derivative represented by formula 2 was purchased from Com Genex (CAS No. 1023449-28-6).

< Experimental example 1> screening of active Material for preventing or treating pruritus

In order to find a compound suitable for preventing or treating pruritus, the following experiment was performed using the screening method of the present invention.

Preparation of a stable cell line MRGPR X1

The human MRGPRX1 gene was subcloned into pcDNA5 FRT vector and transfected into HEK 293T FRT cells. Cells were incubated at 37 ℃ with 5% CO₂Was cultured in an incubator for 48 hours.

to confirm the number of well-formed single colonies, the transfected cells were serially diluted, then transferred and evenly distributed on 100mm dishes. The medium was replaced every 3-4 days with fresh medium containing 50. mu.g/ml hygromycin. The cells were cultured until colonies formed.

Once colonies were formed, only a single colony was transferred to a 24-well plate and then cultured. Then, well-grown cells were transferred to 6-well plates and then cultured. Antibiotic selection was performed. In order to select those cells that did not survive in Zeocin medium but grew well in hygromycin medium, these cells were seeded in 6-well plates. Media containing 50. mu.g/ml Zeocin was added to one plate and media containing 50. mu.g/ml hygromycin was added to the other plate. Cells that died in Zeocin medium but grew well in hygromycin medium were selected and PCR and β -galactosidase assays were performed to confirm successful transfection.

MRGPR X1 screening assay

The following experiment was performed using the Mrgpr X1 stable cell line prepared above to find materials effective for pruritus.

MRGPR X1 stable cells were first counted to 8000 cells/well and then seeded into 384-well black/transparent poly-D-lysine coated plates. Then, the cells were incubated at 37 ℃ with 5% CO₂Was cultured overnight in an incubator.

The subsequent steps were performed using Biomex FX (beckmann). After removal of the medium, the solution was dissolved at a concentration of 2.5. mu.M in Na-HEPES buffer (5mM KCl, 2mM MgCl)₂Flo-3AM dye in 140mM NaCl, 10mM NaOH-HEPES, pH7.2) was added to the plates (25. mu.l/well) and then at 37 ℃ with 5% CO₂Was cultured in the incubator of (1) for 30 minutes.

During the incubation period, compound plates were prepared. Specifically, 6mM original stock plates (96 plate type) were thoroughly pre-mixed, and then intermediate plates were prepared. Na-HEPES buffer having 1mM chloroquine dissolved therein was distributed into 384-well transparent V-plates (49. mu.l/well) using Multidrop. Then, 1. mu.l was taken out from the original stock plate (6mM, 100% DMSO), and diluted in an intermediate plate (120uM compound, 1mM chloroquine, 2% DMSO), thereby preparing a final compound plate.

Na-HEPES buffer having 1mM chloroquine dissolved therein was dispensed into 384-well transparent plates (40. mu.l/well) using Multidrop, and 10. mu.l was taken out of the intermediate plate and diluted in the final compound plate (24. mu.M compound, 1mM chloroquine, 0.4% DMSO).

After completion of the incubation, the dye was discarded, Na-HEPES buffer (25. mu.l/well) was added thereto for washing, and then buffer (25. mu.l/well) was added thereto again. 25 μ l of the primary candidate material was added to each well of the assay plate containing 25 μ l of buffer, followed by reading of the assay plate (final 12 μ M compound, 500 μ M chloroquine, 0.2% DMSO). The vehicle was treated by dissolving 500 μ M chloroquine in 0.2% DMSO.

using a microplate reader (Flexstation II)³⁸⁴Meigu molecular instruments corporation) analyzed the experimental results. Fluorometry (Ex 488nm, Em 535nm) was performed at 3.2 second intervals for 80 seconds. As a result, compounds with good test results were classified, and on this basis 910 secondary candidates for treatment of pruritus were selected.

3. Human histamine 1 receptor antagonist assay

Human histamine 1 receptor antagonist assays were performed using 910 secondary candidate materials selected in the MRGPR X1 experiment above.

HEK 293T cells were transiently transfected with 2 μ g of human histamine 1 receptor and then seeded at a density of 4000 cells/well. Cells were incubated at 37 ℃ with 5% CO₂Was cultured in an incubator for 48 hours.

Subsequent steps were performed using Biomex FX. After removal of the medium, the solution was dissolved at a concentration of 2.5. mu.M in Na-HEPES buffer (5mM KCl, 2mM MgCl)₂140mM NaCl, 10mM NaOH-HEPES, pH7.2) was added to the vehicle and the plate of the experimental group (25. mu.l/well), and the flo-3AM dye dissolved in Na-HEPES buffer solution (containing 5. mu.M diphenhydramine dissolved therein) at a concentration of 2.5. mu.M was added to the plate of the control group (25. mu.l/well), followed by 5% CO at 37 ℃ and 5%₂Was cultured in the incubator of (1) for 30 minutes.

During the incubation period, compound plates were prepared. Specifically, 6mM original stock plates (96 plate type) were thoroughly pre-mixed, and then intermediate plates were prepared. Na-HEPES buffer having 2. mu.M histamine dissolved therein was distributed into 384-well transparent V-plates (49. mu.l/well) using Multidrop. Then, 1 μ l was taken out from the original stock plate (6mM, 100% DMSO), and diluted in an intermediate plate (120 μ M compound, 2 μ M histamine, 2% DMSO), thereby preparing a final compound plate.

Na-HEPES buffer containing 2. mu.M histamine dissolved therein was distributed into 384-well transparent plates (40. mu.L/well) using Multidrop, and 10. mu.L was taken out of the intermediate plate and diluted in the final compound plate (24. mu.M compound, 2. mu.M histamine, 0.4% DMSO, wells excluding control).

After the end of the incubation, the dye was discarded, Na-HEPES buffer (25. mu.l/well) was added thereto for washing, and then buffer (25. mu.l/well) was added thereto again (wells excluding the control group). Na-HEPES buffer containing 5. mu.M diphenhydramine and 2. mu.M histamine dissolved therein was added to the wells of the control group (25. mu.l/well). Finally, 25 μ l of compound was added to each well of the assay plate containing 25 μ l buffer, followed by reading of the assay plate (final 12 μ M compound, 1 μ M histamine, 0.2% DMSO).

Using a microplate reader (Flexstation II)³⁸⁴meigu molecular instruments corporation) analyzed the experimental results. Fluorometry (Ex 488nm, Em 535nm) was performed at 3.2 second intervals for 80 seconds. As a result, 25 compounds having excellent effects were selected from the 910 compounds. Several skeletons representing their structural similarity were identified. Among them, 4 compounds having representative structures were derived. Finally, compound #3, which proved to be excellent in the treatment of pruritus and at the same time had excellent solubility in blood, was selected as the final treatment material for pruritus. Compound #3 is a compound represented by formula 2 of example 1.

4. Analysis of Experimental results

to evaluate the activity of Mrgpr X1-expressing cells and hH 1R-expressing cells based on compound #3 (the ultimate treatment material for pruritus), the data collected from the above experiments were compared and analyzed.

Fig. 1 is a graph showing the results of fluorescence analysis performed after treating Mrgpr X1 cells with chloroquine alone;

Fig. 2 is a graph showing the results of fluorescence analysis performed after Mrgpr X1 cells were treated with a pharmaceutical composition containing the pyrazole derivative of example 1 and chloroquine.

As shown in fig. 1 and 2, the fluorescence analysis results were decreased by about 54% when the pharmaceutical composition of the present invention was co-treated with chloroquine, as compared to treatment with chloroquine alone.

Therefore, the pharmaceutical composition for preventing or treating pruritus according to the present invention can be effectively used as a prophylactic or therapeutic agent for non-histaminic pruritus, because it can alleviate the symptoms of pruritus by inhibiting the activity of intracellular Mrgpr X1.

FIG. 3 is a graph showing the results of fluorescence analysis performed after treating hH1R cells with histamine alone;

FIG. 4 is a graph showing the results of fluorescence analysis performed after treatment of hH1R cells with histamine and diphenhydramine;

Fig. 5 is a graph showing the results of fluorescence analysis performed after hH1R cells were treated with a pharmaceutical composition comprising the pyrazole derivative of example 1 and histamine.

as shown in fig. 3 to 5, if the inhibitory effect of the treatment with histamine and diphenhydramine (H1 receptor antagonist) together is considered to be 100%, the pharmaceutical composition of the present invention can be reduced by about 94%.

Therefore, the pharmaceutical composition for preventing or treating pruritus according to the present invention can be effectively used as a prophylactic or therapeutic agent for histaminic pruritus, because it can alleviate the symptoms of pruritus by inhibiting the activity of intracellular hH 1R.

< Experimental example 2> animal model experiment of therapeutic agent for pruritus of the present invention

In order to confirm the in vivo therapeutic effect of the pharmaceutical composition for the treatment of pruritus of the present invention, evaluation of pruritus in a histamine mouse model, evaluation of pruritus in a chloroquine mouse model, evaluation of pruritus in a mouse model with dry skin, evaluation of pruritus in a psoriasis mouse model, and mouse exercise ability test were performed.

1. Evaluation of itch in Histamine mouse model

In order to evaluate the itch treating effect of the pharmaceutical composition of example 1 in the histamine mouse model, the following experiment was conducted.

C57BL6 mice, 7 weeks old, were placed in new cages and acclimated for 30 minutes before starting the experiment. For the control group, 3% DMSO was dissolved in physiological saline (0.9% saline) and injected intraperitoneally (50. mu.l/mouse). The animal model mice were injected intraperitoneally with the pharmaceutical composition of example 1 (30mg/kg) dissolved in physiological saline (0.9% saline) containing 3% DMSO (50 μ l/mouse). The mice were also placed in the same cages as above for 30 minutes.

Then, 500. mu.g/50. mu.l of histamine was subcutaneously injected into the dorsal side of the control group and animal model mice, and the behavior thereof was recorded with a digital video camera for 30 minutes. And recording the scratching times when the recorded video file is played on the computer. The action of scratching the skin continuously from the time the mouse lifts the hind foot from the floor until the mouse returns the hind foot to the floor was regarded as 1 scratching, and the number of scratching counted in 30 minutes was used as an index for evaluating itching.

Fig. 6 is a graph illustrating evaluation of the itch therapeutic effect in a histamine mouse model treated with a pharmaceutical composition comprising the pyrazole derivative of example 1.

As shown in fig. 6, the total number of scratches accumulated in 30 minutes in the control group was 118.38 times on average, whereas the number of scratches in the experimental group to which the pharmaceutical composition of the present invention was administered was 39.90 times on average.

Therefore, the pharmaceutical composition comprising a pyrazole derivative of the present invention exhibits an effect of reducing histaminic pruritus, and thus can be effectively used as a pharmaceutical composition for treating or preventing pruritus.

2. Evaluation of itch in chloroquine mouse model

in order to evaluate the itch treating effect of the pharmaceutical composition of example 1 in the chloroquine mouse model, the following experiment was conducted.

c57BL6 mice, 7 weeks old, were placed in new cages and acclimated for 30 minutes before starting the experiment. For the control group, 3% DMSO was dissolved in physiological saline (0.9% saline) and injected intraperitoneally (50. mu.l/mouse). The animal model mice were injected intraperitoneally with the pharmaceutical composition of example 1 (30mg/kg) dissolved in physiological saline (0.9% saline) containing 3% DMSO (50 μ l/mouse). The mice were also placed in the same cages as above for 30 minutes.

Then, 200. mu.g/50. mu.l of chloroquine was subcutaneously injected into the dorsal side of the control group and animal model mice, and the behavior thereof was recorded with a digital video camera for 30 minutes. And recording the scratching times when the recorded video file is played on the computer. The action of scratching the skin continuously from the time the mouse lifts the hind foot from the floor until the mouse returns the hind foot to the floor was regarded as 1 scratching, and the number of scratching counted in 30 minutes was used as an index for evaluating itching.

Fig. 7 is a graph illustrating evaluation of the therapeutic effect on pruritus when a mouse model of chloroquine is treated with a pharmaceutical composition containing the pyrazole derivative of example 1.

As shown in fig. 7, the total number of scratches accumulated for 30 minutes in the control group was 425.00 times, whereas the average number of scratches of the experimental group to which the pharmaceutical composition of the present invention was administered was 150.50 times.

Therefore, it can be confirmed from the above results that the pharmaceutical composition comprising the pyrazole derivative compound of the present invention is effective not only in alleviating histamine-induced pruritus but also in alleviating non-histamine-induced pruritus, indicating that the composition of the present invention can be effectively used for treating non-histamine-induced pruritus.

3. Evaluation of itch in a mouse model of xeroderma

In order to evaluate the itch treating effect of the pharmaceutical composition of example 1 in the skin-dry mouse model, the following experiment was conducted.

First, a skin-dry mouse model was constructed. According to Jpn J Pharmacol, 3 months 2002; 88(3): 285-92, the mouse model with dry skin was prepared. Will be provided withMixed with physiological saline (0.9% saline) at a ratio of 1: 1, and then injected intraperitoneally into 7-week-old C57BL6 mice (20 μ l/mouse) to anesthetize the mice. Then, hair on the back of the right neck was shaved off using a scissors. Mixing the raw materials in a ratio of 1: 1 ratio of diethyl ether and acetone. The mixture was dipped into absorbent cotton and the right nape of the neck was rubbed with absorbent cotton for 1 minute. The absorbent cotton soaked with water was then wiped in the same manner. The process is carried out one in the morningNext, the treatment was performed once in the afternoon for 5 days.

Then, evaluation of itch in a skin-dry mouse model was performed. On day 5 afternoon, control mice were injected intraperitoneally with 3% DMSO (50 μ l/mouse) in physiological saline (0.9% saline). The animal model mice were injected intraperitoneally with the pharmaceutical composition of example 1 (30mg/kg) dissolved in physiological saline (0.9% saline) containing 3% DMSO (50 μ l/mouse). The mice were also placed in the same cage for 30 minutes. After 30 minutes, their behavior was recorded with a digital camera for 30 minutes. And recording the scratching times when the recorded video file is played on the computer. The action of scratching the skin continuously from the time the mouse lifts the hind foot from the floor until the mouse returns the hind foot to the floor was regarded as 1 scratching, and the number of scratching counted in 30 minutes was used as an index for evaluating itching.

fig. 8 is a graph illustrating evaluation of the therapeutic effect on pruritus in a skin-dry mouse model treated with a pharmaceutical composition containing the pyrazole derivative of example 1.

As shown in fig. 8, the average number of total scratches accumulated in 30 minutes in the control group was 130.00 times, whereas the average number of total scratches of the experimental group to which the pharmaceutical composition of the present invention was administered was 14.00 times.

Therefore, it was confirmed from the above results that the pharmaceutical composition comprising the pyrazole derivatives according to the present invention has a significant effect in relieving itching caused by dry skin, which indicates that the composition of the present invention can be effectively used for treating itching caused by dry skin or dry skin.

4. Rotating shaftStickEvaluation of motion state

To evaluate the in vivo stability of the pharmaceutical composition according to the present invention, a rotarod exercise status test was performed using a mouse model.

The test mice were placed on a rotarod apparatus and then the speed was increased from 4 to 40 until the mice dropped. When the mice dropped, they were moved into the cage. Repeated 3 times daily for 3 consecutive days, mice were acclimated to the rotarod apparatus. Three days after acclimation, mice were transferred to cages for stabilization.

For the control group, 3% DMSO was dissolved in physiological saline (0.9% saline) and injected intraperitoneally (50. mu.l/mouse). The animal model mice were injected intraperitoneally with the pharmaceutical composition of example 1 (30mg/kg) dissolved in physiological saline (0.9% saline) containing 3% DMSO (50 μ l/mouse). The mice were also placed in the same cages as above for 30 minutes. The above experiment was repeated three times as in the above adaptability test, and the average drop time was calculated.

fig. 9 is a graph showing the results of a rotarod movement state test of mice treated with a pharmaceutical composition comprising the pyrazole derivative of example 1.

As shown in fig. 9, when the exercise state ability was compared between the control group and the experimental group to which the pharmaceutical composition of the present invention was administered, there was no significant difference between the groups.

Therefore, it was confirmed that the pharmaceutical composition containing the pyrazole derivatives of the present invention does not cause any adverse reaction in vivo upon administration, but maintains stable activity, and thus the composition can be effectively used as a pharmaceutical composition for preventing or treating pruritus.

5. Evaluation of itch in psoriasis mouse model

In order to evaluate the itch treating effect of the pharmaceutical composition of example 1 in the psoriasis mouse model, the following experiment was conducted.

First, a mouse model of psoriasis was constructed. According to Pain, 2016 for 11 months; 2536, 2543 to prepare a mouse model of psoriasis. Mixing Sutai (15mg/kg) with normal saline according to the ratio of 1: 1, then 5mg/kg of Pencapone, and the mixture was injected intraperitoneally into 7-week-old C57BL6 mice to anesthetize the mice. The back of the mouse was dehaired with a scissors with a depilatory area size of 2.5 x 2cm and then dehaired twice with a straight blade. The petrolatum cream was wiped with a cotton swab over the dehaired areas of the normal group for 7 days. Imiquimod (Aldara) cream (62.5mg, 5% imiquimod) was swabbed on the unhaired areas of the control and experimental groups using cotton swabs for 7 days.

Then, evaluation of pruritus in a mouse model of psoriasis was performed. On day 8, 3% DMSO (50. mu.l/mouse) in physiological saline (0.9% saline) was injected intraperitoneally into normal and control mice. Mice in the experimental group were injected intraperitoneally with the pharmaceutical composition of example 1 (30mg/kg) dissolved in physiological saline (0.9% saline) containing 3% DMSO (50 μ l/mouse). Mice were placed in the same cage for 30 minutes. After 30 minutes, their behavior was recorded with a digital camera for 30 minutes. And recording the scratching times when the recorded video file is played on the computer. The action of scratching the skin continuously from the time the mouse lifts the hind foot from the floor until the mouse returns the hind foot to the floor was regarded as 1 scratching, and the number of scratching counted in 30 minutes was used as an index for evaluating itching.

Fig. 10 is a graph illustrating evaluation of the therapeutic effect on pruritus when a mouse model of psoriasis is treated with a pharmaceutical composition comprising the pyrazole derivative of example 1.

As shown in fig. 10, the total number of scratches accumulated in the normal group was 20.00, while the average number of scratches in the control group (imiquimod cream-treated group) was 71.00. In the group to which the pharmaceutical composition containing the pyrazole derivative according to the present invention was administered, the average total number of scratches was 1.00, indicating a significant reduction in the number of scratches.

therefore, it can be confirmed from the above results that the pharmaceutical composition comprising the pyrazole derivative according to the present invention is significantly effective in relieving pruritus caused by psoriasis, indicating that the composition of the present invention can be effectively used for treating pruritus or psoriasis caused by psoriasis.

< production example 1> preparation of powder

2g of Compound represented by formula 1

Lactose 1g

The powder is prepared by mixing all the above components (filled in a gas-tight package) according to conventional methods for preparing powders.

< production example 2> preparation of tablet

Tablets are prepared by mixing all the above ingredients using conventional methods for preparing tablets.

< production example 3> preparation of capsules

Capsules were prepared by mixing all the above ingredients (filled in gelatin capsules) according to a conventional method for preparing capsules.

< production example 4> preparation of injection solution

The injection solution is prepared by including all the above components in the indicated amounts according to a conventional method for preparing an injection solution.

< production example 5> preparation of ointment

The ointment is prepared by including all the above components in the indicated amounts according to conventional methods for preparing ointments.

< preparation example 6> preparation of health functional food

Vitamins and minerals suitable for health functional foods are mixed in a preferred mixing ratio, but the composition ratio thereof may be arbitrarily adjusted. Mixing the above components according to conventional method for preparing functional health food, preparing into granule, and using the granule for preparing functional health food according to conventional method.

< preparation example 7> preparation of health drink

Mixing the above components according to conventional method for preparing health beverage. The mixture was heated at 85 ℃ for 1 hour with stirring, and then filtered. The filtrate was filled into a 2-liter sterilized container, sealed and sterilized again, and stored in a refrigerator until it was used to prepare a health drink composition.

The ingredients suitable for the favorite beverage are mixed in a preferred mixing ratio, but the composition ratio may be adjusted according to regional and ethnic preferences (e.g., demand class, demand country, and purpose of use, etc.).

Industrial applicability

The pharmaceutical composition for preventing or treating pruritus according to the present invention can be effectively used as a prophylactic or therapeutic agent for non-histaminic pruritus, a prophylactic or therapeutic agent for histaminic pruritus, a therapeutic agent for dry skin, and a therapeutic agent for psoriasis.