JP2023012559A - Novel medicament for treating inflammatory disease - Google Patents

Abstract

To provide a medicament for treating and/or preventing inflammatory disease.SOLUTION: The present invention provides a medicament, comprising 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3-quinolinecarboxylic acid or a pharmaceutically acceptable salt thereof or a metabolite thereof as an active ingredient.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a therapeutic and/or preventive agent for inflammatory diseases, and more particularly to a therapeutic and/or preventive agent for inflammatory diseases containing a quinolone compound as an active ingredient.

Many intractable inflammatory diseases such as systemic lupus erythematosus (SLE) and psoriasis remain in remission even after treatment with medication, and are diseases that repeatedly relapse. , strong side effects, etc., and there is a need for a new drug that can solve these problems.

As a treatment for inflammatory diseases, anti-IL-17A antibodies show a therapeutic effect on psoriasis (Non-Patent Document 1), but the effect is due to the neutralizing action against IL-17A and decreases IL-17-producing cells. It is not due to action. Various studies have been conducted to reduce IL-17-producing cells, such as RORγt inhibitors and bacterial administration, but effective drug discovery has not been achieved (Non-Patent Documents 2 to 6).

Patent Document 1 discloses specific quinolone antibacterial agents, and that these antibacterial agents exhibit antibacterial activity against Clostridioides difficile living in the intestinal tract.

WO2013/029548

Langley RG, et al., N.Engl.J.Med. 2014; 371(4): 326-338. Bassolas-Molina H, et al., Front. Immunol. 2018; 9: 2307. Ogita T, et al., J. Biomed. Biotechnol. 2011;2011:378417. Mu Q, Tavella VJ, et al., Sci.Rep. 2017; 7(1): 13675. Wu HJ, Ivanov II, et al., Immunity. 2010; 25; 32(6): 815-827. Krebs CF, et al., Immunity. 2016; 45(5): 1078-1092.

An object of the present invention is to provide a new drug for treating and/or preventing intractable inflammatory diseases.

As a result of intensive studies, the present inventors found that 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5, a known quinolone antibacterial agent -pyridyl)-4-oxo-3-quinolinecarboxylic acid can reduce IL-17-producing cells, which are known to be involved in the exacerbation of many inflammatory diseases. The present inventors have completed the present invention by discovering that it can be effective in treatment.

That is, the present invention is as follows.
[Item 1] 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3-quinolinecarboxylic acid or its A drug for treating and/or preventing inflammatory diseases, containing a pharmaceutically acceptable salt or a metabolite thereof as an active ingredient.

[Item 2] 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-carbamoylpyridine- 3-yl)-1-cyclopropyl-6-fluoro-8-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, or 7-(6-amino-5-cyanopyridin-3-yl )-ethyl-1-cyclopropyl-6-fluoro-8-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate.

[Item 3] The drug according to Item 1 or 2, wherein the inflammatory disease is an inflammatory disease involving IL-17-producing cells.

[Item 4] The drug 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 drug according to any one of Items 1 to 4, which is an oral formulation.

[Item 6] The drug 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 - A method of treating and/or preventing inflammatory diseases, which comprises administering a quinoline 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) for manufacturing a drug for treating and/or preventing inflammatory diseases -cyano-5-pyridyl)-4-oxo-3-quinolinecarboxylic acid or a pharmaceutically acceptable salt or metabolite thereof.

[Item 9] 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5 for use in treating and/or preventing an inflammatory disease -pyridyl)-4-oxo-3-quinolinecarboxylic acid or a pharmaceutically acceptable salt or metabolite thereof.

Since the compounds of the present invention can reduce IL-17-producing cells, which are known to be involved in the exacerbation of many inflammatory diseases, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, and multiple sclerosis. , is expected as a new drug for treating and/or preventing intractable inflammatory diseases such as psoriasis.

The results of Example 1 are shown. The results of Example 2 are shown. The results of Example 2 are shown.

1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3-quinolinecarboxylic acid of the compound of the present invention is as follows. and is disclosed as Compound No. 2-18 in Patent Document 1, and its production method and antibacterial activity against Clostridioides difficile are also disclosed. (2S,3S,4S,5R,6R)-6-((7-amino-5-cyanopyridin-3-yl)-1, whose metabolites have the structures of 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 7-(6 -amino-5-cyanopyridin-3-yl)-1-cyclopropyl-6-fluoro-8-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate, which are also of the present invention. are included in the compounds of

Since the compounds of the present invention may exist in the form of hydrates and/or solvates, these hydrates and/or solvates are also included in the compounds of the present invention.
Also included in the compounds of the present invention are deuterium conversion products obtained by converting ¹ H in any one or more of the compounds of the present invention to ² H(D).
The compound of the present invention obtained as a crystal and its pharmaceutically acceptable salt may have crystal polymorphs, and such crystal polymorphs are also included in the present invention.

"Pharmaceutically acceptable salt" means acid addition salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, perchlorate, inorganic acid salts such as phosphate, oxalate, malonate, maleate, fumarate, lactate, malate, citrate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonate, p-toluene organic acid salts such as sulfonates and trifluoromethanesulfonates; and amino acid salts such as glutamates and aspartates. Various bases and salts include alkali metal salts such as sodium salts and potassium salts; Examples thereof include alkaline earth metal salts such as salts, or ammonium salts.

In the present invention, the term "inflammatory disease" is not particularly limited as long as it is an inflammatory disease, but preferably means an inflammatory disease involving IL-17-producing cells. Specific examples include systemic lupus erythematosus, rheumatoid arthritis, scleroderma, multiple sclerosis, psoriasis, and the like.

The administration route of the compound of the present invention may be oral administration, parenteral administration or rectal administration, and the daily dose varies depending on the type of compound, administration method, symptoms and age of the patient, and the like. For example, in the case of oral administration, it is usually about 0.125 mg to 400 mg, preferably about 0.25 mg to 200 mg, more preferably about 0.5 mg to 100 mg, still more preferably about 1 mg to 50 mg per 1 kg body weight of a human or mammal. can be administered in 1 to several divided doses, and specifically, the daily dose for humans is about 7.5 mg to 24000 mg, preferably about 15 mg to 12000 mg, more preferably about 30 mg to 6000 mg, more preferably About 60 mg to 3000 mg.

Dosage forms include tablets, capsules, granules, powders, syrups, suspensions, injections, suppositories, eye drops, ointments, ointments, patches, inhalants and the like. These formulations can be prepared according to a conventional method. Liquid preparations may be dissolved or suspended in water, a suitable aqueous solution, or other suitable medium at the time of use. Tablets and granules may also be coated by well-known methods. Formulations are manufactured by known methods using pharmaceutically acceptable additives.
Additives include excipients, disintegrants, binders, fluidizers, lubricants, coating agents, coloring agents, solubilizers, solubilizers, thickeners, dispersants, and stabilizers, depending on the purpose. , sweeteners, flavors and the like can be used. Specifically, for example, lactose, mannitol, calcium hydrogen phosphate, crystalline cellulose, low-substituted hydroxypropyl cellulose, corn starch, partially pregelatinized starch, carmellose calcium, croscarmellose sodium, crospovidone, sodium starch glycolate. , hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, calcium stearate, sodium stearyl fumarate, polyethylene glycol, propylene glycol, titanium oxide, talc, ferric oxide, yellow ferric oxide, etc. mentioned.

When the compound of the invention is prepared in a single formulation, for example, but not limited to, the compound of the invention may comprise from 0.1 to 85% by weight of the total composition of the formulation. Preferably, the compound of the invention is 10-70% by weight of the total composition of the formulation.

Furthermore, the compounds of the present invention can be used in combination or in combination with other drugs for the purpose of enhancing their effects and/or reducing side effects. Other drugs that can be used in combination or in combination include, for example, steroids such as prednisolone and budesonide, immunosuppressants such as azathioprine, cyclophosphamide and tacrolimus, and biological agents such as rituximab and belimumab.

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited thereto. The acquisition of the compound of the present invention (hereinafter referred to as "test substance") used here is shown below.
Test substance [1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3-quinolinecarboxylic acid]: Otsuka Obtained from Pharmaceutical Co., Ltd.

Example 1. Effects on IL-17A-producing cells in normal mice Test substances were administered to normal BALB/c mice, and the effects on IL-17A-producing CD4-positive T cells in mesenteric lymph nodes, inguinal lymph nodes, and spleen were evaluated. .
(Preparation of test substance)
A test substance was weighed and suspended in a 5% aqueous gum arabic solution to a concentration of 2 mg/mL. After preparation, it was stored at 4°C in the dark.
(drug administration)
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 administration group were as shown in Table 1.

(Preparation of mesenteric lymph node cells, inguinal lymph node cells, and spleen cells)
After 21 days of drug administration, mice were euthanized by cervical dislocation and mesenteric lymph nodes, inguinal lymph nodes and spleen were isolated. Each isolated tissue was ground on a plate containing PBS with the back of a syringe and transferred to a 50 mL tube fitted with a cell strainer. After centrifugation at 500 g for 5 minutes at room temperature, the cell pellet was suspended in RPMI-1640 medium containing 10% FBS and used for subsequent experiments.
(Evaluation of percentage of IL-17A-producing CD4-positive T cells)
Each cell suspension was seeded in a 96-well round bottom plate and stimulated with PMA/ionomycin in the presence of Breferdin A. After culturing for 4 hours, the cell surface was stained with BV510-labeled anti-CD4 antibody, fixed and permeabilized with BD Cytofix/Cytoperm, and intracellular IL-17A was stained with APC-labeled anti-IL-17A antibody. The cells after staining were analyzed with a flow cytometer, and the ratio of IL-17A-positive cells among CD4-positive T cells was evaluated.

(result)
FIG. 1 shows the ratio of IL-17A-positive cells among CD4-positive T cells when mesenteric lymph node cells, inguinal lymph node cells, and spleen cells were stimulated with PMA/ionomycin. As a result, a decrease in IL-17A-producing cells was observed in all tissues in the test substance-administered group, indicating that the test substance has an effect of decreasing IL-17A-producing cells.

Example 2. Effects on IL-17A-producing cells in the kidney of SLE model mice
A test substance was administered to SLE model mice, and the effect on IL-17A-producing cells in the kidney was evaluated.
(Preparation of imiquimod-induced SLE model)
0.03 mL of imiquimod-containing drug Veselna Cream 5% (Mochida Seiyaku) (containing 1.5 mg of imiquimod) was applied to the inside of the right auricle of BALB/c mice under isoflurane inhalation anesthesia using a small brush three times a week. Imiquimod was applied continuously from the start of the test to the end of the test.
(Preparation of test substance)
A test substance was weighed and suspended in a 5% aqueous gum arabic solution to a concentration of 2 mg/mL. After preparation, it was stored at 4°C and shielded from light.

(drug administration)
For 42 days from the 14th day after the start of imiquimod application, the test substance was orally administered at a dose of 20 mg/kg once a day. The settings for each administration group were as shown in Table 2.

(Dissection/collecting)
After 42 days of drug administration, mice were euthanized by cervical dislocation and kidneys were isolated. One of the isolated kidneys was flash-frozen in liquid nitrogen for gene expression analysis, and the other was stored on a PBS-supplemented plate for IL-17A-producing cell evaluation until use in the experiment.
(Preparation of immune cells from kidney)
The kidney for evaluation of IL-17A-producing cells was transferred to a plate supplemented with RPMI-1640 medium containing 10% FBS for each administration group and cut with scissors into 1 to 2 mm squares. Collagenase D was added to a concentration of 5 mg/mL, incubated at 37° C., 5% CO ₂ for 40 minutes, and transferred to a 50 mL tube fitted with a 70 μm cell strainer. The kidney mass was ground on the cell strainer with the back of a syringe, and the cell strainer was washed with PBS. The resulting cell suspension was centrifuged at 500 g at room temperature for 10 minutes, the supernatant was removed, and PBS was added to suspend the cells. A 40% Percoll cell suspension was prepared by adding an equal volume of 80% Percoll solution to the PBS in which the cells were suspended, and layered on a 50 mL tube containing the 80% Percoll solution. After centrifugation at 1500 g for 30 minutes at room temperature, the layer between 80% Percoll and 40% Percoll was recovered and diluted with PBS. After centrifuging at 500 g for 10 minutes at room temperature and removing the supernatant, the cells were suspended in RPMI-1640 medium containing 10% FBS and centrifuged at 500 g for 5 minutes at 4°C. The supernatant was removed, suspended in RPMI-1640 medium containing 10% FBS, and used for subsequent experiments.

(Evaluation of IL-17A-producing T cell ratio)
Cell suspensions from each treatment group were seeded into 96-well round bottom plates and stimulated with PMA/ionomycin in the presence of Breferdin A. After culturing for 4 hours, the cell surface was stained with FITC-labeled anti-TCRβ antibody, PerCP/Cy5.5-labeled anti-CD8α antibody, BV421-labeled anti-CD3 antibody, and BV510-labeled anti-CD4 antibody, fixed with BD Cytofix/Cytoperm, and permeabilized. After that, intracellular IL-17A was stained with APC-labeled anti-IL-17A antibody. The cells after staining were analyzed with a flow cytometer, CD4 positive T cells (CD3 ⁺ TCRβ ⁺ CD4 ⁺ CD8α ^- ), CD4 negative CD8 negative T cells (CD3 ⁺ TCRβ ⁺ CD4 ^- CD8α ^- ), TCR β chain negative T cells The percentage of IL-17A-positive cells in each (CD3 ⁺ TCRβ ⁻ ) was evaluated.
(Evaluation of IL-17A gene expression)
Cryopreserved kidneys were disrupted in ISOGEN and RNA was extracted. The extracted RNA was reverse transcribed to prepare cDNA, and expression analysis of Actb gene and Il17a gene was performed using the obtained cDNA as a template. The samples were treated individually, and the analysis results were expressed by fold change when the Actb gene was used as an endogenous control and the average value of the untreated group as a control.

(result)
CD4-positive T cells (CD3 ⁺ TCRβ ⁺ CD4 ⁺ CD8α ^- ), CD4-negative CD8-negative T cells (CD3 ⁺ TCRβ ⁺ CD4 ^- CD8α ^- ), TCRβ chain when immune cells prepared from kidney were stimulated with PMA/ionomycin FIG. 2 shows the percentage of IL-17A positive cells in each negative T cell (CD3 ⁺ TCRβ ⁻ ), and FIG. 3 shows the results of IL-17A gene expression analysis in the kidney.
Both the results of analysis of the proportion of IL-17A-producing cells in each cell type and the results of analysis of IL-17A gene expression provided data indicating that IL-17A-producing cells in the kidney were reduced in the test substance-administered group. It was shown that the test substance has the effect of reducing IL-17A-producing cells in the kidney of the SLE model.

Example 3. Effects on renal function in SLE model mice
A test substance is administered to SLE model mice, and the effect on renal function is evaluated.
(Preparation of test substance)
A test substance is weighed and suspended in a 5% aqueous gum arabic solution at concentrations of 0.5, 1 and 2 mg/mL. After preparation, store at 4°C in the dark.
(drug administration)
According to Table 3, the test substance is orally administered once daily. Drug administration continues until the end of the study.

(Preparation of imiquimod-induced SLE model)
From the 29th day after the start of drug administration, 0.03 mL of imiquimod-containing drug Veselna Cream 5% (Mochida Pharmaceutical Co., Ltd.) (containing 1.5 mg of imiquimod) was applied to the inside of the right auricle of BALB/c mice under isoflurane inhalation anesthesia. 3 times a week with Application of imiquimod is continued until the end of the study.
(Evaluation of renal function)
On the day before the start of drug administration, the day before the start of imiquimod application, and 14 days, 28 days, 42 days, and 56 days after the start of imiquimod application, overnight urine is collected using metabolic cages. The obtained urine is centrifuged at 3000 g and 4° C. for 10 minutes, the supernatant is collected, and albumin concentration and creatinine concentration are measured. Based on the obtained results, calculate the albumin/creatinine ratio and use it for analysis.

Example 4. Effect on Rheumatoid Arthritis Model Mice A test substance is administered to rheumatoid arthritis model mice, and the effects on pathological conditions are evaluated.
(Preparation of test substance)
A test substance is weighed and suspended in a 5% aqueous gum arabic solution at concentrations of 0.5, 1 and 2 mg/mL. After preparation, store at 4°C in the dark.
(drug administration)
According to Table 4, the test substance is orally administered once daily. Drug administration continues until the end of the study.

(Preparation of rheumatoid arthritis model, evaluation of pathology)
From the 29th day after drug administration, the mice are immunized with type II collagen to induce type II collagen-induced arthritis. A method for inducing arthritis and a method for evaluating pathological conditions are performed with reference to the report by Depis et al. (Arthritis Rheum. 2012 Oct;64(10):3189-98.).

Example 5. Effect on scleroderma model mice A test substance is administered to scleroderma model mice, and the effects on pathological conditions are evaluated.
(Preparation of test substance)
A test substance is weighed and suspended in a 5% aqueous gum arabic solution at concentrations of 0.5, 1 and 2 mg/mL. After preparation, store at 4°C in the dark.
(drug administration)
According to Table 5, the test substance is orally administered once daily. Drug administration continues until the end of the study.

(Creation of scleroderma model, evaluation of pathology)
From the 29th day after administration of the drug, bleomycin is subcutaneously administered to induce scleroderma-like symptoms. A method for inducing scleroderma-like symptoms and a method for evaluating pathological conditions are performed with reference to the report by Park et al. (Front Immunol. 2018 Jul 10;9:1611.).

Example 6. Effect on multiple sclerosis model mice A test substance is administered to multiple sclerosis model mice, and the effects on pathological conditions are evaluated.
(Preparation of test substance)
A test substance is weighed and suspended in a 5% aqueous gum arabic solution at concentrations of 0.5, 1 and 2 mg/mL. After preparation, store at 4°C in the dark.
(drug administration)
According to Table 6, the test substance is orally administered once daily. Drug administration continues until the end of the study.

(Generation of multiple sclerosis model mice, evaluation of pathological conditions)
From day 29 after drug administration, mice are immunized with a myelin protein-derived peptide to induce experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. A method for inducing EAE and a method for evaluating pathological conditions are performed with reference to a report by Chiba et al. (Int Immunopharmacol. 2011 Mar;11(3):366-72).

Example 7 Effect on Psoriasis Model Mouse A test substance is administered to a psoriasis model mouse, and the effect on pathological conditions is evaluated.
(Test method)
(Preparation of test substance)
A test substance is weighed and suspended in a 5% aqueous gum arabic solution at concentrations of 0.5, 1 and 2 mg/mL. After preparation, store at 4°C in the dark.
(drug administration)
According to Table 7, the test substance is orally administered once daily. Drug administration continues until the end of the study.

(Preparation of psoriasis model mice, evaluation of pathological conditions)
From the 29th day after administration of the drug, 5% imiquimod-containing drug Beselna cream (Mochida Seiyaku Co., Ltd.) is applied to the back and right auricle to induce psoriasis-like symptoms. A method for inducing psoriasis-like symptoms and a method for evaluating pathological conditions are performed with reference to a report by van der Fits et al. (J Immunol. 2009 May 1;182(9):5836-45.).

Claims (9)

1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3-quinolinecarboxylic acid or pharmaceutically acceptable thereof A drug for treating and/or preventing inflammatory diseases, containing as an active ingredient a salt or a metabolite thereof. 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 7-(6-amino-5-cyanopyridin-3-yl)-1- The agent of claim 1, which is ethyl cyclopropyl-6-fluoro-8-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate. 3. The agent of claim 1 or 2, wherein the inflammatory disease is an inflammatory disease involving IL-17-producing cells. The agent 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 drug according to any one of claims 1 to 4, which is an oral drug. Medicament according to any one of claims 1 to 5, characterized in that the daily dose of active ingredient is between 7.5 mg and 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-quinolinecarboxylic acid A method for treating and/or preventing an inflammatory disease, which comprises administering to a patient in need of such treatment, 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 for the manufacture of a medicament for treating and/or preventing inflammatory diseases -pyridyl)-4-oxo-3-quinolinecarboxylic acid or a pharmaceutically acceptable salt or metabolite thereof. 1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)- for use in treating and/or preventing inflammatory diseases 4-oxo-3-quinolinecarboxylic acid or a pharmaceutically acceptable salt or metabolite thereof.

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