JP2016169178A - Chronic rhinosinusitis therapeutic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a chronic rhinosinusitis therapeutic agent that comprises an antiinflammatory agent that shows actual therapeutic effect on human chronic rhinosinusitis as an active ingredient.SOLUTION: A chronic rhinosinusitis therapeutic agent comprises at least one selected from the group consisting of a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof, or a compound represented by the following formula (2) or a pharmacologically acceptable salt thereof as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a therapeutic agent for chronic rhinosinusitis comprising a dihydropseudoerythromycin derivative having no antibacterial action and anti-inflammatory action as an active ingredient.

  Chronic rhinosinusitis is a chronic inflammation that occurs in the mucosa in the sinuses. As inflammation becomes chronic, pus accumulates in the sinuses and nasal polyps (inflammatory proliferative masses) are formed. As a treatment for chronic rhinosinusitis, macrolide antibiotics that are less susceptible to serious side effects than steroids and have stronger anti-bacterial and anti-inflammatory effects than other antibiotics are mainly used. Used.

  As macrolide antibiotics used for the treatment of chronic rhinosinusitis such as nasal polyps, 14-membered ring macrolides such as erythromycin, clarithromycin, and roxithromycin are mainly used. However, these 14-membered ring macrolides have an antibacterial action in addition to an anti-inflammatory action and have a risk of inducing resistant bacteria, and are therefore difficult to use as an anti-inflammatory agent. In order to solve this problem, a 12-membered pseudoerythromycin derivative having no antibacterial action and anti-inflammatory action (Kitasato Institute EM700 series, see Patent Documents 1 and 2) has been reported.

  Since the pseudoerythromycin derivative is partly decomposed by an acid and is relatively unstable, the pharmacological action may not be sufficiently exhibited by oral administration. Then, in order to improve the stability with respect to the acid of the said pseudo erythromycin derivative, it is reported that it reduces to a dihydro body (refer patent document 3 and nonpatent literature 1).

International Publication No. 2002/14338 International Publication No. 2004/39823 Japanese Patent No. 5118973

Sugawara, et al. , The Journal of Antibiotics, 2012, vol. 65, p. 487-490.

  The present invention provides a therapeutic agent for chronic rhinosinusitis comprising an anti-inflammatory agent that actually has a therapeutic effect on human chronic rhinosinusitis as an active ingredient.

  As a result of intensive studies to solve the above problems, the present inventor has found that, among dihydropseudoerythromycin derivatives, a specific compound has a growth inhibitory action and apoptosis on fibroblasts collected from human nasal fins. It has been found that it has an inducing action, and the present invention has been completed.

That is, the therapeutic agent for chronic rhinosinusitis according to the present invention is the following [1] to [3].
[1] Selected from the group consisting of a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof, or a compound represented by the following formula (2) or a pharmacologically acceptable salt thereof A therapeutic agent for chronic rhinosinusitis, comprising one or more of the active ingredients as active ingredients.

[2] The therapeutic agent for chronic rhinosinusitis according to [1], which has an action of suppressing the growth of nasal or sinus fibroblasts.
[3] The therapeutic agent for chronic rhinosinusitis according to [1] above, which has an effect of inducing apoptosis of nasal cavity or sinus fibroblasts.

  The therapeutic agent for chronic rhinosinusitis according to the present invention is a dihydropseudoerythromycin derivative that has an anti-inflammatory action but does not show an antibacterial action, and has a growth-inhibiting action and an apoptosis-inducing action on human nasal fibroblasts. Is a very effective therapeutic agent for chronic rhinosinusitis.

In Example 1, it is the figure which showed the calculation result of the relative cell number (%) of each drug treatment group which made the cell number (ATP amount) of the control (vehicle) 100%. In Example 2, it is the figure which showed the calculation result of the relative caspase activity value (%) of each drug treatment group which made the caspase activity of the control (vehicle) 100%.

  The therapeutic agent for chronic rhinosinusitis according to the present invention is a compound represented by the following formula (1) (9-dihydro-pseudoerythromycin A 6,9-epoxide: EM900) or a pharmacologically acceptable salt thereof, Or a compound represented by the following formula (2) (de (3′-N-methyl) -3′-N- (p-chlorobenzyl) -9-dihydro-pseudoerythromycin A 6,9-epoxide: EM905) or One or more selected from the group consisting of pharmacologically acceptable salts are used as active ingredients.

  In EM900 and EM905, the 8- and 9-position solids in Formula (1) or Formula (2) are not particularly limited. That is, EM900 and EM905 include all the stereoisomers at the 8th and 9th positions in formula (1) or formula (2).

  EM900 and EM905 not only have an anti-inflammatory effect, but also have a growth inhibitory action and an apoptosis-inducing action on nasal or sinus fibroblasts. That is, the proliferation of fibroblasts inflamed by EM900 or the like is suppressed, and as a result of apoptosis, the inflammation is cured and the nasal mucosa is reduced. Therefore, these compounds are suitable as an active ingredient of a therapeutic agent for chronic rhinosinusitis. In particular, EM900 and EM905 are not for cultured cell lines established so that subculture is possible, but for growth inhibition and apoptosis induction on fibroblasts actually isolated from human nasal polyps. Because of its action, the therapeutic agent for chronic rhinosinusitis according to the present invention can be expected to have a high therapeutic effect.

  Although the manufacturing method of EM900 and EM905 is not specifically limited, For example, it can manufacture by the method etc. which are specifically described below (refer patent document 3). Those skilled in the art can appropriately modify or modify starting materials, reaction conditions, reaction reagents, and the like as necessary.

  Examples of pharmaceutically acceptable salts that EM900 and EM905 can form include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, and succinic acid. And organic acid salts such as salts, fumarate, acetate, methanesulfonate, and toluenesulfonate.

  The active ingredient of the therapeutic agent for chronic rhinosinusitis according to the present invention may be a solvate of EM900 and EM905 or a pharmaceutically acceptable salt thereof. Examples of the solvent that forms the solvate include water, methanol, ethanol, isopropanol, acetone, and ethyl acetate.

  As an active ingredient of the therapeutic agent for chronic rhinosinusitis according to the present invention, one or more substances selected from the group consisting of EM900, EM905, and salts thereof, and hydrates and solvates thereof are used. be able to. The administration route of the therapeutic agent for chronic rhinosinusitis according to the present invention is not particularly limited, and can be administered orally or parenterally.

  As the therapeutic agent for chronic rhinosinusitis according to the present invention, the active ingredient may be directly administered to a patient, but preferably contains the active ingredient and a pharmacologically and pharmaceutically acceptable additive. It should be administered as a formulation in the form of a pharmaceutical composition. Examples of pharmacologically and pharmaceutically acceptable additives include excipients, disintegrating agents or disintegrating aids, binders, coating agents, dyes, diluents, bases, solubilizing agents or solubilizing aids, An isotonic agent, a pH adjuster, a stabilizer, a propellant, an adhesive, and the like can be used. Examples of preparations suitable for oral administration include tablets, capsules, powders, fine granules, granules, solutions, syrups, etc. Examples of preparations suitable for parenteral administration include, for example, powder spray Agents, injections, drops, ointments, creams, transdermal absorption agents, inhalants, suppositories, and the like, but the form of the preparation is not limited thereto. As the dosage form of the therapeutic agent for chronic rhinosinusitis according to the present invention, an oral preparation that is relatively easy to take, or a powder spray, ointment, cream, or nasal drop that can be directly applied to the affected area is preferable.

  For preparations suitable for oral administration, as additives, for example, excipients such as glucose, lactose, D-mannitol, starch, crystalline cellulose; disintegrants or disintegration aids such as carboxymethylcellulose, starch, carboxymethylcellulose calcium; Binders such as propylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and gelatin; lubricants such as magnesium stearate and talc; groups such as hydroxypropylmethylcellulose, sucrose, polyethylene glycol, gelatin, kaolin, glycerin, purified water, and hard fat An agent can be used. Preparations suitable for injection or infusion include aqueous solutions such as distilled water for injection, physiological saline, propylene glycol, or solubilizers or solubilizers that can constitute soluble injections for use; glucose, sodium chloride, D-mannitol Pharmaceutical additives such as isotonic agents such as glycerin; pH regulators such as inorganic acids, organic acids, inorganic bases or organic bases can be used.

  The dosage of the therapeutic agent for chronic rhinosinusitis according to the present invention should be appropriately increased or decreased according to conditions such as the age, weight, and symptoms of the patient. Is the amount of the active ingredient and is about 0.05 to 500 mg orally. In general, the above-mentioned dose can be administered once to several times per day, but may be administered every several days. When there are two or more active ingredients, the total amount is set within the range.

  EXAMPLES Hereinafter, although the Example and comparative example of this invention are shown and this invention is demonstrated in more detail, this invention is not limited to these.

Synthesis Example 1 Synthesis of EM900 EM900 was synthesized by the method described in Patent Document 3 using EMA (erythromycin A) as a raw material. Details are as follows.

A glacial acetic acid solution (710.0 mL) of EMA (104.4 g, 16.90 mmol) was stirred at room temperature for 2 hours, and then an aqueous NaHCO ₃ solution was slowly added to neutralize. The reaction solution was extracted with CHCl ₃ , and the organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to obtain a crude product (99.30 g). The obtained crude product was dissolved in CHCl ₃ (250 mL), hexane (50 mL) was added and recrystallized to obtain white powder EM201 (74.50 g, 71%).

EM201;
Rf = 0.63 (CHCl ₃ : MeOH: NH ₄ OH aq = 15: 1: 0.2)

K ₂ CO ₃ (1.400 g, 10.60 mmol) was added to a MeOH solution (150.0 mL) of EM201 (7.600 g, 10.60 mmol), and the mixture was heated to reflux for 2 hours. After returning to room temperature, the solvent was distilled off and the residue was dissolved in aqueous NaHCO ₃ solution. The reaction solution was extracted with CHCl ₃ , and the organic layer was dried over Na ₂ SO ₄ , filtered, and concentrated to obtain a crude product (9.300 g). The obtained crude product was separated and purified by flash column chromatography (CHCl ₃ : MeOH: NH ₄ OH aq = 10: 0.5: 0.01-10: 1: 0.05), and white powder EM701 ( 5.900 g, 78%).

EM701;
Rf = 0.47 (CHCl ₃ : MeOH: NH ₄ OH aq = 15: 1: 0.2)

PtO ₂ (476.2 mg, 2.100 mmol) and CF ₂ HCOOH (299.0 μL, 4.750 mmol) were added to acetic acid (AcOH; 7.000 mL), and the mixture was stirred at 5 atm and room temperature for 1 hour under H ₂ atmosphere. An AcOH solution (7.0000 mL) of EM701 (1.000 g, 1.400 mmol) was added, and the mixture was stirred for 4 hours at 5 atm and room temperature in an H ₂ atmosphere. Thereafter, CH ₃ CO ₂ NH ₄ (7.0000 g) was added, stirred, filtered and concentrated. The concentrate was extracted with CHCl ₃ and washed with saturated aqueous NaHCO ₃ and brine. The washed organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to obtain a crude product (968.4 mg). The obtained crude product was separated and purified by flash column chromatography (CHCl ₃ : MeOH: NH ₄ OH aq = 50: 1: 0.02-30: 1: 0.02), and white powder EM900 (767. 7 mg, 76%).

EM900;
Rf = 0.53 (CHCl ₃ : MeOH: NH ₄ OH aq = 15: 1: 0.2);
HR-MS m / z: 718.4767 [M + H] +, Calcd for C 37 H 68 NO 12: 718.4742 [M + H]

[Synthesis Example 2] Synthesis of EM905 EM905 was synthesized by the method described in Patent Document 3 using EM900 manufactured in Synthesis Example 1 as a raw material. Details are as follows.

EM900 (706.3 mg, 0.984 mmol) in methanol (MeOH) solution (9.840 mL) was added sodium acetate (AcONa; 403.6 mg, 4.920 mmol), I ₂ (499.5 mg, 1.968 mmol), saturated NaHCO 3. After adding ₃ solutions, it confirmed that it was basic with a universal test paper, and stirred at 50 degreeC for 20 minutes. After stirring, Na ₂ S ₂ O ₃ (400.0 mg) was added and the temperature was returned to room temperature. The reaction solution was extracted with CHCl ₃ and washed with a mixed solution of brine and NH ₄ OH aq. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to obtain a crude product (700.0 mg). The obtained crude product was separated and purified by flash column chromatography (CHCl ₃ : MeOH: NH ₄ OH aq = 100: 1: 0.1-30: 1: 0.1) to obtain white powder EM901 (546. 5 mg, 79%) was obtained.

EM901;
Rf = 0.53 (CHCl ₃ : MeOH: NH ₄ OH aq = 10: 1: 0.2)
HR-MS m / z: 704.4615 [M + H] +, Calcd for C 36 H 66 NO 12: 704.4585 [M + H]

EM901 (20.00 mg, 0.0280 mmol) in CHCl ₃ solution (280.0 μL) was added to i-Pr ₂ NEt (24.40 μL, 0.14 mmol), p-ClBnBr (p-chlorobenzyl bromide: 28.80 mg, 0 .1400 mmol) was added, and the mixture was stirred at room temperature for 2 hours under N ₂ atmosphere. After stirring, saturated Na ₂ S ₂ O ₃ solution (7.0000 mL) was added, extracted with CHCl ₃ , washed with saturated Na ₂ S ₂ O ₃ solution, saturated NH ₄ Cl solution, and brine, and then the organic layer was Na ₂ SO _4. After filtration, filtration and concentration gave a crude product (24.10 mg). The obtained crude product was separated and purified by flash column chromatography (CHCl ₃ : MeOH: NH ₄ OH aq = 100: 1: 0.1) to obtain a white powder EM905 (21.60 mg, 93%). .

EM905;
Rf = 0.59 (CHCl ₃ : MeOH: NH ₄ OH aq = 30: 1: 0.2)
HR-MS m / z: 828.4657 [M + H] +, Calcd for C 43 H 71 NO 12 Cl: 828.4665 [M + H]

[Comparative Synthesis Example 1] Synthesis of de (3′-dimethylamino) -3′-morpholino-9-dihydro-pseudoerythromycin A 6,9-epoxide (EM914) EM914 is based on EM901 produced in Synthesis Example 2. And synthesized by the method described in Patent Document 3. Details are as follows.

A solution of Na (21.80 mg, 0.9480 mmol) in MeOH (15.80 mL) was cooled to 0 ° C. and EM901 (111.5 mg, 0.1580 mmol), I ₂ (200.5 mg, 0.7900 mmol) was added, The mixture was stirred at 0 ° C. for 40 minutes in an Ar atmosphere. After stirring, Na ₂ S ₂ O ₃ (100.0 mg) was added and the temperature was returned to room temperature. The reaction solution was extracted with CHCl ₃ and washed with a mixed solution of brine and NH ₄ OH aq. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to obtain a crude product (100.0 mg). The obtained crude product was separated and purified by flash column chromatography (CHCl ₃ : MeOH: NH ₄ OH aq = 100: 1: 0.1-10: 1: 0.1), and white powder EM903 (98. 40 mg, 90%).

EM903;
Rf = 0.43 (CHCl3: MeOH: NH ₄ OH aq = 10: 1: 0.2)
HR-MS m / z: 690.4431 [M + H] +, Calcd for C 35 H 64 NO 12: 690.4429 [M + H]

In CH ₃ CN solution (7.0000 mL) of EM903 (24.20 mg, 0.0350 mmol), Ar-atmosphere i-Pr ₂ NEt (61.00 μL, 0.3500 mmol), bis (2-bromoethyl) ether (44.00 μL) , 0.3500 mmol) and stirred at 80 ° C. for 20 hours. After stirring, i-Pr ₂ NEt (61.00 μL, 0.3500 mmol) and bis (2-bromoethyl) ether (44.00 μL, 0.3500 mmol) were added, and the mixture was stirred at 80 ° C. for 6 hours. After stirring, saturated Na ₂ S ₂ O ₃ solution (7.0000 mL) was added, extracted with CHCl ₃ , washed with saturated Na ₂ S ₂ O ₃ solution, saturated NH ₄ Cl solution, and brine, and then the organic layer was Na ₂ SO _4. After filtration, filtration and concentration gave a crude product (36.50 mg). The obtained crude product was separated and purified by flash column chromatography (CHCl ₃ : MeOH: NH ₄ OH aq = 100: 1: 0.1-30: 1: 0.1), and white powder EM914 (23.23). 60 mg, 89%).

EM914;
Rf = 0.44 (CHCl ₃ : MeOH: NH ₄ OH aq = 30: 1: 0.2)
HR-MS m / z: 760.4885 [M + H] +, Calcd for C 39 H 70 NO 13: 760.4847 [M + H]

Comparative Synthesis Example 2 de (3′-N-methyl) -3′-N- (p-chlorobenzyl) -de (3-O-kuradinosyl) -9-dihydro-3-keto-pseudoerythromycin A 6, Synthesis of 9-epoxide 12,13-carbonate (EM939) EM939 was synthesized by the method described in Patent Document 3 using EM900 produced in Synthesis Example 1 as a raw material. Details are as follows.

To a solution of EM900 (5.004 g, 6.975 mmol) in EtOAc (69.80 mL) was added NaHCO ₃ (8.790 g, 104.6 mmol), and CbzCl (benzyloxycarbonyl chloride: 14.93 mL, 104.6 mmol) was added dropwise. The mixture was heated to 70 ° C. and stirred for 2 hours. After stirring, Et ₃ N was added and the temperature was returned to room temperature. The reaction solution was extracted with EtOAc and washed with brine. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to obtain 7.000 g of a crude product. The obtained crude product was separated and purified by flash column chromatography (CHCl ₃ : MeOH: NH ₄ OH aq = 50: 1: 0.1) to obtain a white powder EM930 (6.365 g, 94%). .

EM930;
HR-MS m / z: 994.5170 [M + Na] +, Calcd for C 52 H 77 NO 16 Na: 994.5140 [M + Na]

1.0N HCl aq (52.30 mL) was added to a CH ₃ CN solution (104.6 mL) of EM930 (5.081 g, 5.230 mmol), and the mixture was stirred for 4 hours. After stirring, saturated NaHCO ₃ solution (400.0 mL) was added, extracted with CHCl ₃ , washed with brine, dried over Na ₂ SO ₄ , filtered, and concentrated to obtain a crude product (4.312 g). It was. The obtained crude product was separated and purified by flash column chromatography (CHCl ₃ : MeOH: NH ₄ OH aq = 50: 1: 0.1) to obtain a white powder EM931 (4.028 g, 95%). .

EM931;
HR-MS m / z: 814.4384 [M + H] +, Calcd for C 44 H 64 NO 13: 814.4378 [M + H]

A solution of EM931 (2.027 g, 2.492 mmol) in CH ₂ Cl ₂ (49.80 mL) was cooled to −78 ° C. under N ₂ atmosphere, pyridine (2.420 mL, 29.90 mmol) was added, and triphosgene (1. (479 g, 4.984 mmol) in CH ₂ Cl ₂ (99.70 mL) was added dropwise, and the mixture was heated from −78 ° C. to room temperature and stirred for 0.5 hour. After stirring, saturated NH ₄ Cl solution (400.0 mL) was added, extracted with CH ₂ Cl ₂ , washed with saturated NaHCO ₃ solution and brine, the organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to a crude product A product (1.900 g) was obtained. The obtained crude product was separated and purified by flash column chromatography (CHCl ₃ : MeOH: NH ₄ OH aq = 100: 1: 0.1) to obtain a white powder EM936 (1.882 g, 90%). .

EM936;
HR-MS m / z: 862.4000 [M + Na] +, Calcd for C 45 H 61 NO 14 Na: 862.3990 [M + Na]

Dess-Martin periodinane (4.343 g, 10.24 mmol) was added to a CH ₂ Cl ₂ solution (40.80 mL) of EM936 (1.718 g, 2.047 mmol) under N ₂ atmosphere, and the mixture was stirred for 1.5 hours. After stirring, saturated Na ₂ S ₂ O ₃ solution (300.0 mL) was added, extracted with CHCl ₃ , washed with saturated Na ₂ S ₂ O ₃ solution, saturated NaHCO ₃ solution and brine, and the organic layer was washed with Na ₂ SO ₄ . After drying, it was filtered and concentrated to obtain a crude product (1.700 g). The obtained crude product was separated and purified by flash column chromatography (CHCl ₃ : MeOH: NH ₄ OH aq = 50: 1: 0.1) to obtain a white powder EM937 (1.668 g, 97%). .

EM937;
HR-MS m / z: 8386.4012 [M + H] +, Calcd for C 45 H 60 NO 14: 838.4014 [M + H]

To EM937 (1.461 g, 1.745 mmol) were added Pd (OH) ₂ (292.2 mg) and EtOH (34.90 mL) under N ₂ atmosphere, and the mixture was stirred at room temperature for 3 hours under H ₂ atmosphere. After stirring, Pd (OH) ₂ (292.2 mg) was added under N ₂ atmosphere and stirred at room temperature under H ₂ atmosphere for 2.5 hours. After further stirring, Pd (OH) ₂ (146.1 mg) was added under N ₂ atmosphere, and the mixture was stirred at room temperature for 1 hour under H ₂ atmosphere. Filtration and concentration gave a crude product (1.302 g). The obtained crude product was separated and purified by flash column chromatography (CHCl ₃ : MeOH: NH ₄ OH aq = 50: 1: 0.1-30: 1: 0.1), and white powder EM938 (967. 3 mg, 97%).

EM938;
HR-MS m / z: 570.3307 [M + H] +, Calcd for C 29 H 48 NO 10: 570.3278 [M + H]

To a solution of EM938 (303.4 mg, 0.533 mmol) in CHCl ₃ (5.330 mL) was added i-Pr ₂ NEt (928.4 μL, 5.330 mmol), p-ClBnBr (1.095 g, 5.330 mmol), The mixture was stirred at room temperature for 2 hours under N ₂ atmosphere. After stirring, saturated Na ₂ S ₂ O ₃ solution (50.00 mL) was added, extracted with CHCl ₃ , washed with saturated Na ₂ S ₂ O ₃ solution, saturated NH ₄ Cl solution and brine, and the organic layer was washed with Na ₂ SO _4. After being dried over, it was filtered and concentrated to obtain a crude product (350.1 mg). The obtained crude product was separated and purified by flash column chromatography (CHCl ₃ : MeOH: NH ₄ OH aq = 100: 1: 0.1) to obtain white powder EM939 (342.5 mg, 93%). .

EM939;
HR-MS m / z: 694.3353 [M + H] +, Calcd for C 36 H 53 NO 10 Cl: 694.3358 [M + H]

[Example 1]
The influence of EM900 and EM905 on the cell proliferation of fibroblasts isolated and cultured from human nasal polyps was examined. Fibroblasts have a certain amount of ATP, and the number of fibroblasts (ie, cell field proliferative ability) was evaluated from the amount of ATP.

<Preparation of fibroblasts>
Fibroblasts were isolated and cultured from nasal polyps extracted at the time of surgery. Cut the nostrils to 1 mm ³ or less, once with PBS (phosphate saline), 10% FBS (fetal bovine serum), penicillin (100 U / mL), streptomycin (100 μg / mL), amphotericin B (2. 5 mg / mL) Washed twice with added Dulbecco's Modified Eagle Medium (D'MEM) (manufactured by SIGMA), then put several sections into a beaker and 0.1% collagenase type 2 Hanks' Balanced Salt Solution (HBSS) (manufactured by Worthington) was stirred for 3 hours using a stirrer in 30 mL (manufactured by GIBCO). Next, 10 mL of the culture solution was added to the cells separated by a centrifuge (3000 rpm, 5 minutes) and suspended, and suspended. The same operation was repeated 4 times, and cell culture was started in a 75 cm ² flask (manufactured by IWAKI) at 37 ° C. and 5% by volume CO ₂ . After several weeks of culture, the 75 cm ² flask was almost full of fibroblasts. Subsequently, the passage was changed by trypsin treatment, and fibroblasts of the third passage were used for the experiment.

<Measurement of cell proliferation activity>
In each well of a 24-well plate (manufactured by FALCON), 1 mL of a culture solution was placed at a concentration of 5 × 10 ⁴ cells / mL, and cultured at 37 ° C. under 5% CO ₂ for 24 hours (n = 4). After culturing, each drug [EM900, EM905, EM914, EM939, clarithromycin (CAM), erythromycin (EM), ampicillin (AMPC)] dissolved in DMSO (dimethyl sulfoxide) was added to each concentration (25 μM, 50 μM, 100 μM) was added to the culture solution. To the control, only a certain amount of DMSO was added to the culture solution. After culturing for 48 hours, ATPlite Assay (manufactured by PerkinElmer) was performed on the cells in each well, and the change in the number of fibroblasts was evaluated from the amount of ATP. The amount of ATP in each well was subjected to Dunnett's multiple comparison test after analysis of variance of 1-way ANOVA.

  FIG. 1 shows the calculation results of the relative cell number (%) of each drug-treated group, assuming that the number of control cells (ATP amount) is 100%. 1A shows the results when the drug concentration is 25 μM, FIG. 1B shows the results when the drug concentration is 50 μM, and FIG. 1C shows the results when the drug concentration is 100 μM. At each drug concentration of 25 μM, EM905 significantly decreased ATP (fibroblast count) compared to vehicle (p <0.0001), but other drugs (EM900, EM914, EM939, CAM, EM, AMPC) did not decrease the amount of ATP (FIG. 1 (a)). At each drug concentration of 50 μM, EM900 (p <0.01) and EM905 (p <0.0001) significantly decreased ATP compared to vehicle (FIG. 1 (b)). At each drug concentration of 100 μM, EM900 (p <0.0001), EM905 (p <0.0001), and CAM (p <0.05) significantly decreased ATP compared to vehicle. (FIG. 1 (c)). That is, EM900 and EM905 were confirmed to have a stronger cell growth inhibitory effect than the known macrolide antibiotic CAM.

[Example 2]
The effect of EM900 and EM905 on the apoptosis of human nasal fin-derived fibroblasts prepared in the same manner as in Example 1 was examined.

In each well of a 24-well plate (manufactured by FALCON), 1 mL of a culture solution was placed at a concentration of 5 × 10 ⁴ cells / mL, and cultured at 37 ° C. under 5% CO ₂ for 24 hours (n = 4). After culturing, each drug [EM900, EM905, EM914, EM939, clarithromycin (CAM), erythromycin (EM), ampicillin (AMPC)] dissolved in DMSO (dimethyl sulfoxide) was added to each concentration (25 μM, 50 μM, 100 μM) was added to the culture solution. To the control, only a certain amount of DMSO was added to the culture solution. After culturing for 48 hours, the cells in each well were subjected to Caspase-Glo (registered trademark) 3/7 Assay (manufactured by Promega) to evaluate the caspase-3 / 7 activity of fibroblasts. The caspase activity value of each well was subjected to Dunnett's multiple comparison test after analysis of variance of 1-way ANOVA.

  FIG. 2 shows the calculation result of the relative caspase activity value (%) of each drug-treated group, assuming that the control caspase activity value is 100%. 2A shows the results when the drug concentration is 25 μM, FIG. 2B shows the results when the drug concentration is 50 μM, and FIG. 2C shows the results when the drug concentration is 100 μM. At a concentration of 25 μM for each drug, EM905 significantly increased caspase-3 / 7 activity (apoptosis induction) compared to vehicle (p <0.0001), but other drugs (EM900, EM914, EM939, CAM, EM, AMPC) did not induce (FIG. 2 (a)). At each drug concentration of 50 μM, EM905 (p <0.0001) significantly increased caspase-3 / 7 activity compared to vehicle (FIG. 2 (b)). At a concentration of 100 μM for each drug, EM900 (p <0.001) and EM905 (p <0.0001) significantly decreased the increase in Caspase-3 / 7 activity compared to vehicle (FIG. 2 ( c)). That is, EM900 and EM905 were confirmed to have apoptosis-inducing activity against human nasal fin-derived fibroblasts.

Claims (3)

Following formula (1)

Or a pharmacologically acceptable salt thereof, or the following formula (2):

A therapeutic agent for chronic rhinosinusitis, comprising one or more selected from the group consisting of a compound represented by the formula: or a pharmacologically acceptable salt thereof as an active ingredient.   The therapeutic agent for chronic rhinosinusitis according to claim 1, which has an inhibitory effect on the proliferation of nasal or sinus fibroblasts.   The therapeutic agent for chronic rhinosinusitis according to claim 1, which has an effect of inducing apoptosis of nasal cavity or sinus fibroblasts.

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