KR20220140562A - New drugs for the treatment of inflammatory diseases - Google Patents

Abstract

The present invention relates to a medicament for the treatment and/or prophylaxis of inflammatory diseases comprising a quinolone compound as an active ingredient.

Description

New drugs for the treatment of inflammatory diseases

The present invention relates to a medicament for the treatment and/or prophylaxis of an inflammatory disease, and more particularly, to a medicament for the treatment and/or prophylaxis of an inflammatory disease comprising a quinolone compound as an active ingredient.

Many refractory inflammatory diseases, such as systemic lupus erythematosus (SLE) and psoriasis, for example, medications can only achieve remission, patients have multiple relapses, no curative therapy, and existing medications are severe. It has a problem of treatment, such as having side effects, and therefore there is a demand for the development of a new drug that does not have such a problem.

As one of the treatments for inflammatory diseases, the anti-IL-17A antibody exhibits a therapeutic effect on psoriasis (Non-Patent Document 1), but the effect is only a neutralizing effect on IL-17A, and IL-17-production There is no effect of reducing cells. Although many studies have been made to reduce IL-17-producing cells, for example, RORγt inhibitors or bacteriotherapy, no effective drug-development has yet been achieved (Non-Patent Documents 2 to 6).

Patent Document 1 discloses a specific quinolone antimicrobial agent that exhibits antibacterial activity against Clostridium difficile that inhabits the intestinal tract.

Citation List

patent literature

[PL 1] WO2013/029548

non-patent literature

[NPL 1] Langley RG, et al., N. Engl. J. Med. 2014; 371(4): 326-338.

[NPL 2] Bassolas-Molina H, et al., Front. Immunol. 2018; 9: 2307.

[NPL 3] Ogita T, et al., J. Biomed. Biotechnol. 2011;2011:378417.

[NPL 4] Mu Q, Tavella VJ, et al., Sci. Rep. 2017; 7(1): 13675.

[NPL 5] Wu HJ, Ivanov II, et al., Immunity. 2010; 25; 32(6): 815-827.

[NPL 6] Krebs CF, et al., Immunity. 2016; 45(5): 1078-1092.

technical problem

The main object of the present invention is to provide a novel medicament for the treatment and/or prevention of refractory inflammatory diseases.

solution to the problem

The inventors have studied extensively, followed by the known quinolone antimicrobial agent 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5- Found that pyridyl)-4-oxo-3-quinoline-carboxylic acid can reduce IL-17-producing cells, which is known to be associated with exacerbation of many inflammatory diseases, and is effective in treating refractory inflammatory diseases. did. Based on the new findings, the present invention has been completed.

The present invention includes the following embodiments.

(Item 1)

1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3-quinoline- as active ingredient A medicament for the treatment and/or prophylaxis of an inflammatory disease, comprising a carboxylic acid or a pharmaceutically acceptable salt thereof or a metabolite thereof.

(Item 2)

The method of item 1, wherein the metabolite is (2S,3S,4S,5R,6R)-6-((7-amino-5-cyanopyridin-3-yl)-1-cyclopropyl-6-fluoro-8 -Methyl-4-oxo-1,4-dihydroquinoline-3-carbonyl)oxo)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, 7-(6- Amino-5-carbamoylpyridin-3-yl)-1-cyclopropyl-6-fluoro-8-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, or ethyl 7- (6-amino-5-cyanopyridin-3-yl)-1-cyclopropyl-6-fluoro-8-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate.

(Item 3)

The medicament according to item 1 or item 2, wherein the inflammatory disease is an inflammatory disease associated with IL-17-producing cells.

(Item 4)

The medicament according to any one of items 1 to 3, wherein the inflammatory disease is systemic lupus erythematosus, rheumatoid arthritis, scleroderma, multiple sclerosis, or psoriasis.

(Item 5)

The medicament according to any one of items 1 to 4, for oral administration.

(Item 6)

The medicament according to any one of items 1 to 5, wherein the daily dose of the active ingredient is 7.5 mg to 24000 mg.

(Item 7)

A therapeutically effective amount of 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3-quinoline- A method for treating and/or preventing an inflammatory disease comprising administering a carboxylic acid or a pharmaceutically acceptable salt thereof or a metabolite thereof to a patient in need thereof.

(Item 8)

1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5- in the manufacture of a medicament for the treatment and/or prophylaxis of inflammatory diseases Use of pyridyl)-4-oxo-3-quinoline-carboxylic acid or a pharmaceutically acceptable salt thereof or a metabolite thereof.

(Item 9)

1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl for use in the treatment and/or prophylaxis of inflammatory diseases )-4-oxo-3-quinoline-carboxylic acid or a pharmaceutically acceptable salt thereof or a metabolite thereof.

Effects of the Invention

The compounds of the present invention can decrease IL-17-producing cells, which are known to be associated with exacerbation of many inflammatory diseases. Therefore, new drugs for treating and/or preventing refractory inflammatory diseases such as systemic lupus erythematosus, rheumatoid arthritis, scleroderma, multiple sclerosis and psoriasis are expected. Furthermore, the compounds of the present invention are poorly absorbed drugs, whereby they are distributed in high concentrations in the intestinal tract when administered orally, but have low blood transmissibility. Accordingly, the compounds of the present invention also have an advantage, namely a low risk of systemic side effects, which is a problem with existing quinolone antibacterial agents.

[Figure 1] Figure 1 shows the results of Example 1.
[Figure 2] Figure 2 shows the results of Example 2.
[Fig. 3] Fig. 3 shows the results of Example 2.
[Fig. 4] Fig. 4 shows the results of Example 8.
[Fig. 5] Fig. 5 shows the results of Example 8.

Compounds of the Invention 1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3-quinoline -Carboxylic acid has the structure of Formula (1), which is disclosed as Compound Nos. 2-18 in Patent Document 1, which also discloses its process against Clostridium difficile and its antibacterial activity. Its metabolites are (2S,3S,4S,5R,6R)-6-((7-amino-5-cyanopyridin-3-yl)-1 having the structures of the following formulas (2) to (4), respectively -Cyclopropyl-6-fluoro-8-methyl-4-oxo-1,4-dihydroquinoline-3-carbonyl)oxo)-3,4,5-trihydroxytetrahydro-2H-pyran-2 -carboxylic acid, 7-(6-amino-5-carbamoylpyridin-3-yl)-1-cyclopropyl-6-fluoro-8-methyl-4-oxo-1,4-dihydroquinoline- 3-carboxylic acid, and ethyl 7-(6-amino-5-cyanopyridin-3-yl)-1-cyclopropyl-6-fluoro-8-methyl-4-oxo-1,4-dihydro quinoline-3-carboxylate, which is included in the compounds of the present invention.

[Chem. One]

[Chem. 2]

[Chem. 3]

[Chem. 4]

The compounds of the present invention may be in the form of hydrates and/or solvates, and therefore the compounds of the present invention also encompass hydrates and/or solvates thereof.

Furthermore, also within the scope of the present invention are compounds of the present invention in which any one or more ¹ H atoms have been replaced by ² H(D) atoms.

Polymorphisms may exist in crystals of a compound of the present invention or a pharmaceutically acceptable salt thereof, and therefore such crystal polymorphisms are also within the scope of the present invention.

"Pharmaceutically acceptable salts" are acid addition salts, including salts with inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulfate, perchlorate, and phosphate; salts with organic acids such as oxalates, malonates, maleates, fumarates, lactates, maleates, citrates, tartrates, benzoates, trifluoroacetates, acetates, methanesulfonates, p-toluenesulfonates, and trifluoromethanesulfonate; and salts with amino acids such as glutamate and aspartate; and salts with bases, alkali metal salts such as sodium salts and potassium salts; alkaline-earth metal salts such as calcium salts; and ammonium salts.

As used herein, "inflammatory disease" is not limited as long as it is an inflammatory disease, preferably it refers to an inflammatory disease involving IL-17-producing cells. For example, it includes systemic lupus erythematosus, rheumatoid arthritis, scleroderma, multiple sclerosis and psoriasis.

The compounds of the present invention may be administered via any route selected from oral administration, parenteral administration and rectal administration. The daily dose varies depending on the compound, the route of administration, the patient's condition, the patient's age, and the like. For example, for oral administration, it is generally from about 0.125 mg to about 400 mg, preferably from about 0.25 mg to about 200 mg, and more preferably from about 0.5 mg to about 100 mg per kg body weight of a human or mammal. , even more preferably in a dose of about 1 mg to about 50 mg, may be administered once to several times. For example, a daily dose for humans is from about 7.5 mg to about 24000 mg, preferably from about 15 mg to about 12000 mg, more preferably from about 30 mg to about 6000 mg, even more preferably from about 60 mg to about 3000 mg.

Dosage forms of the present invention include tablets, capsules, granules, powders, syrups, suspensions, injections, suppositories, eye drops, ointments, liniments, patches, and inhalants. These dosage forms may be prepared in a conventional manner. When the dosage form is a liquid, it may be a formulation that prepares a solution or suspension for use by mixing it with water, a suitable aqueous solution, or other suitable solvent. Tablets and granules can be coated in a well known manner. Dosage forms may be prepared in a known manner using pharmaceutically acceptable excipients.

As used herein, additives include excipients, disintegrants, binders, glidants, lubricants, coatings, colorants, solubilizers, solubilizers, thickeners, dispersants, stabilizers, sweeteners and flavoring agents, depending on the intended use. For example, they are lactose, mannitol, calcium hydrogen phosphate, microcrystalline cellulose, low-substituted hydroxypropylcellulose, corn starch, partially pregelatinized starch, carmellose calcium, croscarmellose sodium, crospovidone, sodium starch glycol Late, hydroxypropylcellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, soft silicic acid anhydride, magnesium stearate, calcium stearate, sodium stearyl fumarate, polyethylene glycol, propylene glycol, titanium oxide, talc, iron sesquioxide and yellow ferric oxide.

When the compound of the present invention is formulated in a single dosage form, the dosage form may comprise 0.1 to 85% (w/w) of the compound of the present invention per total composition, although the present invention is not limited thereto. Preferably, it is from 10 to 70% (w/w) per total composition.

Furthermore, the compounds of the present invention may be used in combination with another drug or in combination with another drug for the purpose of enhancing the effect and/or alleviating the side effects. Other drugs that may be used in combination include, for example, steroids such as prednisolone and budesonide, immunosuppressants such as azathioprine, cyclophosphamide and tacrolimus, biopharmaceuticals such as rituximab and belimumab.

Example

The present invention will be described in more detail below by reference to examples, but the present invention should not be limited thereto. As used herein, a compound of the present invention (hereinafter referred to as "test substance") and a reference drug were obtained as set forth below.

Test substance [1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3-quinoline- carboxylic acid]: obtained from Otsuka Pharmaceutical Co., Ltd.

Example 1. Effect on IL-17A-producing cells in normal mice

Normal BALB/c mice were administered the test substance to evaluate the effect of the test substance on IL-17A-producing CD4-positive T cells in mesenteric lymph nodes, inguinal lymph nodes and spleen.

(Preparation of test substance)

The test substance was weighed and the concentration was adjusted to 2 mg/mL by suspending it in 5% aqueous gum arabic. The prepared suspension was stored at 4°C after blocking light.

(drug injection)

The test substance was orally administered to normal BALB/c mice at a dose of 20 mg/kg once a day for 21 days. The settings for each group are presented in Table 1.

(Production of mesenteric lymph node cells, inguinal lymph node cells and splenocytes)

Mice that received drug administration for 21 days were euthanized by cervical dislocation, and then mesenteric lymph nodes, inguinal lymph nodes and spleens were isolated. Each isolated tissue was crushed with the lower part of a syringe on a PBS-added plate, and the crushed tissue was placed in a 50 mL tube equipped with a cell strainer. The crushed tissue in the tube was centrifuged at 500 g for 5 minutes at room temperature, and the obtained cell pellet was suspended in RPMI-1640 medium containing 10% FBS and used in the experiments shown below.

(Evaluation of the percentage of IL-17A-producing CD4-positive T cells)

Each cell suspension prepared above was seeded in 96-well round-bottom plates and stimulated with PMA/ionomycin in the presence of breperdin A. After a 4-hour incubation, the cell surface was stained with BV510-labeled anti-CD4 antibody, cells were fixed, and permeabilized with BD Cytofix/Cytopem, and then intracellular IL-17A was transfected with APC-labeled anti-IL. Stained with -17A antibody. Stained cells were analyzed by flow cytometry, thereby assessing the proportion of IL-17A-positive cells among CD4-positive T cells.

(result)

Each ratio of IL-17A-positive cells among CD4-positive T cells obtained by stimulating mesenteric lymph node cells, inguinal lymph node cells or splenocytes with PMA/ionomycin is shown in FIG. 1 . According to the obtained results, IL-17A-producing cells were decreased in the test substance administration group for all tissues, suggesting that the test substance may have an action of reducing IL-17A-producing cells.

Example 2. Effects on IL-17A-producing cells in the kidney of a mouse model of SLE

A mouse model for SLE was administered the test substance, and the effect of the test substance on IL-17A-producing cells in the kidney was evaluated.

(Preparation of imiquimod-induced SLE model)

Imiquimod-containing drug, BESELNA Cream 5% (MOCHIDA PHARMACEUTICAL CO., LTD.) (0.03 mL, containing 1.5 mg of imiquimod) 3 times a week It was applied to the inside of the right outer ear of BALB/c mice with a small brush under inhalation anesthesia of isoflurane. Application of imiquimod was continued from the beginning of the trial to the end of the trial.

(Preparation of test substance)

The test substance was weighed and the concentration was adjusted to 2 mg/mL by suspending it in 5% aqueous gum arabic. The prepared suspension was stored at 4°C after blocking light.

(drug injection)

From the 14th day after the start of imiquimod-application, the test substance was orally administered to mice at a dose of 20 mg/kg once a day for 42 days. The settings for each group are presented in Table 2.

(dissection and preprocessing)

Mice that received drug administration for 42 days were euthanized by cervical dislocation, and then a pair of kidneys were isolated. One of the isolated kidneys was rapidly frozen in liquid nitrogen for gene expression analysis and the other was stored on PBS-supplemented plates until initiation of testing, which was used for evaluation of IL-17A-producing cells.

(Production of immune cells from the kidneys)

Kidneys for evaluation of IL-17A-producing cells were transferred onto a plate supplemented with RPMI-1640 medium containing 10% FBS per each group, and cut into 1-2 mm cubes with scissors. Collagenase D was added to the plate to adjust the concentration to 5 mg/mL. Samples were incubated at 37° C. under 5% CO ₂ for 40 min and transferred to a 50 mL tube equipped with a 70 μm cell strainer. The kidney block was crushed with the lower part of the syringe on a cell strainer, and the cell strainer was washed with PBS. The resulting cell suspension was centrifuged at 500 g for 10 minutes at room temperature. The supernatant was removed and PBS was added to the residue to suspend it. A 40% Percoll cell-suspension was prepared by adding an 80% Percoll solution in the same volume as the PBS used to suspend the cells to the suspension, and stacked in a 50 mL tube to which the 80% Percoll solution had already been added. The sample in the tube was centrifuged at 1500 g for 30 minutes at room temperature, and a layer between 80% Percoll and 40% Percoll was collected, which was diluted with PBS. The diluted samples were centrifuged at 500 g for 10 min at room temperature, the supernatant removed, the residue suspended in RPMI-1640 medium containing 10% FBS, and the suspension centrifuged at 500 g for 5 min at 4°C. did. The supernatant was removed and the residue was suspended in RPMI-1640 medium containing 10% FBS, which was used for the following tests.

(Evaluation of the proportion of IL-17A-producing T cells)

Cell-suspensions of each dose group were seeded onto 96-well round-bottom plates, which were stimulated with PMA/ionomycin in the presence of breperdin A. After 4-hour incubation, the cell surface was stained with FITC-labeled anti-TCRβ antibody, PerCP/Cy5.5-labeled anti-CD8α antibody, BV421-labeled anti-CD3 antibody, BV510-labeled anti-CD4 antibody. Cells were fixed, permeabilized with BD Cytofix/Cytopem, and intracellular IL-17A was stained with APC-labeled anti-IL-17A antibody. Stained cells were analyzed by flow cytometry, whereby CD4-positive T cells (CD3 ⁺ TCRβ ⁺ CD4 ⁺ CD8α ⁻ ), CD4-negative CD8-negative T cells (CD3 ⁺ TCRβ ⁺ CD4 ⁻ CD8α ⁻ ), or TCRβ The proportion of IL-17A-positive cells in chain-negative T cells (CD3 ⁺ TCRβ ⁻ ) was assessed.

(Evaluation of IL-17A gene expression)

Cryopreserved kidneys were homogenized with ISOGEN and RNA was extracted. The extracted RNA was reverse transcribed to prepare cDNA. Expression analysis of the Actb gene and Il17a gene was performed by using the obtained cDNA as a template. Each sample was treated individually, and the analysis results were expressed as fold changes, where the Actb gene was the endogenous control and the mean of the untreated group was the standard control.

(result)

CD4-positive T cells (CD3 ⁺ TCRβ ⁺ CD4 ⁺ CD8α ⁻ ), CD4-negative CD8-negative T cells (CD3 ⁺ TCRβ ⁺ CD4 ⁻ CD8α ⁻ ) obtained by stimulating immune cells prepared from kidneys with PMA/ionomycin ), each ratio of IL-17A-positive cells in TCRβ chain-negative T cells (CD3 ⁺ TCRβ ⁻ ) is shown in FIG. 2 , and the results of analysis of IL-17A gene expression in the kidney are shown in FIG. 3 .

According to the analysis result of the IL-17A-producing cell ratio in each cell line, the data show that the IL-17A-producing cell in the kidney decreased in the test substance administration group, which was also analyzed by the IL-17A gene expression The results also show that the test substance has an action of reducing IL-17A-producing cells in the kidney of the SLE model.

Example 3. Effect on renal function of mouse model on SLE

A mouse model for SLE was administered the test substance, and the effect of the test substance on renal function was evaluated.

(Preparation of test substance)

The test substance was weighed and the concentration was adjusted to 0.5, 1, and 2 mg/mL by suspending it in 5% aqueous gum arabic. The prepared suspension was stored at 4°C after blocking light.

(drug injection)

According to Table 3, the test substance was orally administered to mice once a day. Dosing of the substance was continued until the end of the trial.

(Preparation of imiquimod-induced SLE model)

From the 29th day after starting drug-administration, the imiquimod-containing drug Bezelna Cream 5% (Mochida Pharmaceutical Co., Ltd.) (0.03 mL, containing 1.5 mg of imiquimod) was administered with isoflu 3 times a week It was applied to the inside of the right outer ear of BALB/c mice with a small brush under inhalation anesthesia of the egg. Application of imiquimod was continued until the end of the test.

(Evaluation of renal function)

Urine excreted overnight was returned to the metabolic cage on the eve of drug-administration, on the eve of initiating imiquimod-administration, and on days 14, 28, 42, and 56 after initiation of imiquimod-administration. collected. The collected urine was centrifuged at 3000 g for 10 minutes at 4° C., and then the concentration of albumin and the concentration of creatinine were measured. Based on the obtained results, the ratio of albumin/creatinine was calculated and used for analysis.

Example 4. Effect on mouse model for rheumatoid arthritis

The test substance was administered to a mouse model for rheumatoid arthritis, and the effect of the test substance on the pathological condition was evaluated.

(Preparation of test substance)

The test substance was weighed and the concentration was adjusted to 0.5, 1, and 2 mg/mL by suspending it in 5% aqueous gum arabic. The prepared suspension was stored at 4°C after blocking light.

(drug injection)

According to Table 4, the test substance was orally administered to mice once a day. Dosing of the substance was continued until the end of the trial.

(Preparation of rheumatoid arthritis model and evaluation of pathological conditions)

From day 29 after starting drug-administration, type II collagen was immunized to create type II collagen-induced arthritis. Methods for inducing arthritis and methods for evaluating pathological conditions thereof are described in Depis, et al. (Arthritis Rheum. 2012 Oct; 64(10): 3189-98).

Example 5. Effect on mouse model for scleroderma

The test substance was administered to a mouse model for scleroderma, and the effect of the test substance on the pathological condition was evaluated.

(Preparation of test substance)

The test substance was weighed and the concentration was adjusted to 0.5, 1, and 2 mg/mL by suspending it in 5% aqueous gum arabic. The prepared suspension was stored at 4°C after blocking light.

(drug injection)

According to Table 5, the test substance was orally administered to mice once a day. Dosing of the substance was continued until the end of the trial.

(Preparation of scleroderma model and evaluation of pathological condition)

From the 29th day after starting drug-administration, bleomycin was administered to mice subcutaneously to induce scleroderma-like symptoms. Methods for inducing scleroderma-like symptoms and methods for evaluating their pathological conditions are described in Park, et al. (Front Immunol. 2018 Jul 10; 9: 1611)].

Example 6. Effect on mouse model for multiple sclerosis

The test substance was administered to a mouse model for multiple sclerosis, and the effect of the test substance on the pathological condition was evaluated.

(Preparation of test substance)

The test substance was weighed and the concentration was adjusted to 0.5, 1, and 2 mg/mL by suspending it in 5% aqueous gum arabic. The prepared suspension was stored at 4°C after blocking light.

(drug injection)

According to Table 6, the test substance was orally administered to mice once a day. Dosing of the substance was continued until the end of the trial.

(Preparation of a mouse model for multiple sclerosis and evaluation of pathological conditions)

From day 29 after starting drug-administration, a known experimental autoimmune encephalomyelitis (EAE) was induced as a mouse model for multiple sclerosis by immunization with peptides derived from myelin protein. Methods for inducing EAE or for evaluating pathological conditions thereof are described in Chiba, et al. (Int Immunopharmacol. 2011 Mar; 11(3): 366-72).

Example 7. Effect on mouse model for psoriasis

The test substance was administered to a mouse model for psoriasis, and the effect of the test substance on the pathological condition was evaluated.

(Test Methods)

(Preparation of test substance)

The test substance was weighed and the concentration was adjusted to 0.5, 1, and 2 mg/mL by suspending it in 5% aqueous gum arabic. The prepared suspension was stored at 4°C after blocking light.

(drug injection)

According to Table 7, the test substance was orally administered to mice once a day. Dosing of the substance was continued until the end of the trial.

(Preparation of a mouse model for psoriasis and evaluation of pathological conditions)

From the 29th day after starting drug-administration, an imiquimod-containing drug, Bezelna Cream 5% (Mochida Pharmaceutical Co., Ltd.) was applied to the back and right ear of the mouse to induce psoriasis-like symptoms. did. Methods for inducing psoriasis-like symptoms and methods for evaluating their pathological conditions are described in van der Fits, et al. (J Immunol. 2009 May 1; 182(9): 5836-45).

Example 8. Effect on mouse model for psoriasis

A mouse model for psoriasis was administered a test substance, and the effect of the test substance on the pathological condition and IL-17A-producing cells was evaluated.

(Test Methods)

(Preparation of test substance)

The test substance was weighed and the concentration was adjusted to 2 mg/mL by suspending it in 5% aqueous gum arabic. The prepared suspension was stored at 4°C after blocking light.

(drug injection)

According to Table 8, the test substance was orally administered to mice once a day. Dosing of the substance was continued until the end of the trial.

(Preparation of a mouse model for imiquimod-induced psoriasis)

From the 29th day after starting drug-administration, the imiquimod-containing drug Bezelna Cream 5% (Mochida Pharmaceutical Co., Ltd.) (0.03 mL, containing 1.5 mg of imiquimod) was administered with isoflu 3 times a week Psoriasis-like symptoms were induced by application to the inside of the right outer ear of BALB/c mice with a small brush under inhalation anesthesia of Ran.

(Measurement of outer ear thickness)

After imiquimod-application was started, the thickness of the outer ear was measured with a caliper once a week.

(dissection and collection)

On day 44 after initiation of imiquimod-application, mice were euthanized by cervical vertebral fracture dislocation and their right external ear was collected. The collected auricles were rapidly frozen in liquid nitrogen and stored in a freezer at -80°C.

(Evaluation of IL-17A gene expression in the outer ear)

The frozen outer ear was triturated with isogen, and RNA was extracted. The extracted RNA was reverse transcribed to prepare cDNA. Using the obtained cDNA as a template, the expression of 18S rRNA gene and IL-17A gene was analyzed by real-time PCR. Each sample was treated individually, and the analysis results were expressed as fold changes, where the 18S rRNA gene was the endogenous control and the mean of the untreated group was the standard control.

(result)

The result of the measured outer ear thickness is shown in FIG. 4 , and the evaluation result of IL-17A gene expression in the outer ear is shown in FIG. 5 .

The result of continuous-measurement of the thickness of the outer ear to which imiquimod was applied showed that the hyperplasia of the outer ear was suppressed in the test substance administration group. The evaluation result of IL-17A gene expression in the outer ear showed that IL-17A gene expression was lowered in the test substance administration group. Thus, this indicated that the test substance was able to reduce IL-17A-producing cells and inhibit the hyperplasia of the outer ear in a model for psoriasis.

Claims (9)

1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3-quinoline- as active ingredient A medicament for the treatment and/or prophylaxis of an inflammatory disease, comprising a carboxylic acid or a pharmaceutically acceptable salt thereof or a metabolite thereof. 2. The method of claim 1, wherein the metabolite is (2S,3S,4S,5R,6R)-6-((7-amino-5-cyanopyridin-3-yl)-1-cyclopropyl-6-fluoro- 8-Methyl-4-oxo-1,4-dihydroquinoline-3-carbonyl)oxo)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, 7-(6 -amino-5-carbamoylpyridin-3-yl)-1-cyclopropyl-6-fluoro-8-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, or ethyl 7 -(6-amino-5-cyanopyridin-3-yl)-1-cyclopropyl-6-fluoro-8-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate . The medicament according to any one of claims 1 to 2, wherein the inflammatory disease is an inflammatory disease associated with IL-17-producing cells. The medicament according to any one of claims 1 to 3, wherein the inflammatory disease is systemic lupus erythematosus, rheumatoid arthritis, scleroderma, multiple sclerosis or psoriasis. The medicament according to any one of claims 1 to 4, for oral administration. 6. The medicament according to any one of claims 1 to 5, wherein the daily dose of the active ingredient is 7.5 mg to 24000 mg. A therapeutically effective amount of 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3-quinoline- A method for treating and/or preventing an inflammatory disease comprising administering a carboxylic acid or a pharmaceutically acceptable salt thereof or a metabolite thereof to a patient in need thereof. 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5- in the manufacture of a medicament for the treatment and/or prophylaxis of inflammatory diseases Use of pyridyl)-4-oxo-3-quinoline-carboxylic acid or a pharmaceutically acceptable salt thereof or a metabolite thereof. 1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl for use in the treatment and/or prophylaxis of inflammatory diseases )-4-oxo-3-quinoline-carboxylic acid or a pharmaceutically acceptable salt thereof or a metabolite thereof.

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