JP4732702B2 - Anti-chlamydia agent, preventive agent, relapse preventive agent and therapeutic agent for diseases involving chlamydia - Google Patents

Description

  The present invention relates to an anti-chlamydia agent, and a preventive agent, a recurrence preventive agent and a therapeutic agent for a disease involving chlamydia.

  The genus Chlamydia is an obligate intracellular parasitic bacterium that can only grow in living cells. There are four known species of Chlamydia, Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pneumoniae and Chlamydia pecorum. Chlamydia trachomatis is further divided into 15 serotypes, A, B, Ba, C, D, E, F, G, H, I, J, K, L1, L2, and L3.

  Chlamydia trachomatis is the etiological agent of eye infections and urogenital infections, and it causes infections in the eyes and respiratory tract of newborns through birth canal infections, and is the main pathogenic agent of sexually transmitted diseases (STD). is there. In addition, serotypes A to C are trachoma, B and D to K are urethritis, epididymis, prostatitis, cervicitis, tubitis, pelvic inflammatory disease (PID), perihepatitis (Fitz-Hugh-Curtis syndrome), fallopian tube infertility, ectopic pregnancy, inclusion body conjunctivitis, L1 to L3 cause genopathic lymphogranulomatosis (LGV). Parrot disease Chlamydia is a pathogen common to humans and animals, and is a pathogen of parrot disease that causes pneumonia by infecting humans from birds. Chlamydia pneumonia is known as a pathogen of respiratory infections that infect humans from person to person. Chlamydia pecolum is known as a pathogen that causes diarrhea and miscarriage in ruminants (such as cattle or sheep).

  Briefly explaining the life cycle of Chlamydia (1 cycle 48-72 hours), Chlamydia has an elementary body (EB) that propagates from solid to solid or from host cells to cells and non-infectious and dividing. There is a period of reticulate body (RB) which is a form of the growth phase. When EBs (spheres with a diameter of about 0.3 μm) are invaded and infected in the cells of the host, the EBs turn into RBs (spheres with a diameter of about 0.5 μm) within the cells, and after further dividing and proliferating, It then matures and is released again as an EB and either infects other hosts or metastasizes to other cells in the same host.

  Tetracycline antibiotics such as tetracycline, doxycycline, and minocycline are most recommended as conventional anti-chlamydia agents that suppress infection and growth after infection in humans of the genus Chlamydia. Next, the antibacterial activity of macrolide antibiotics such as erythromycin and rifampicin is excellent as an anti-chlamydia agent.

  The mechanism of action of antibiotics such as tetracyclines on Chlamydia utilizes the specificity of Tetracycline for ribosomes in host cells and ribosomes in Chlamydia cells. In other words, the host animal ribosome has a protein precipitation constant of 80S, which is composed of 40S and 60S subunits, whereas the chlamydial intracellular ribosome has a protein precipitation constant of 70S. Yes, this is composed of 30S and 50S subunits. According to the difference in the structure of ribosomes, tetracycline antibiotics are said to act on ribosomes in chlamydial cells of smaller subunits and inhibit their protein synthesis.

  In recent years, it has become clear that Chlamydia pneumonia is involved in the development of arteriosclerosis. In other words, Chlamydia pneumonia is confirmed at a high probability of about 50 to 60% in the arteriosclerosis site of atherosclerosis patients, and Chlamydia pneumonia specific antibodies are at a high rate of about 79% in atherosclerosis patients. Chlamydia pneumonia is involved in at least the onset or worsening of atherosclerosis due to the high infection rate in cases of coronary restenosis after PTCA (percutaneous coronary angioplasty). It is thought that. The mechanism of the pathogenesis of arteriosclerosis has not been clarified, but the migration of Chlamydia pneumonia infected with airway epithelial cells and alveolar macrophages to peripheral blood mononuclear cells is thought to play an important role. . Macrolides and quinolone drugs are widely used to sterilize Chlamydia pneumonia. However, in vitro studies by the inventor have already reported that Chlamydia pneumonia infected with monocyte / macrophage cells is extremely resistant to these drugs (see below, non-patent Reference 1). It is also known that Chlamydia pneumonia that has migrated to peripheral blood cells changes its form into a persistent infection type and its sensitivity to drugs decreases.

  By the way, it is known that 2-aminophenoxazine-3-one derivatives and 3-aminophenoxazin-2-one derivatives, which are phenoxazine derivatives, exhibit strong antitumor activity against cancer cells (for example, , See Patent Document 1 and Patent Document 2). Further, 2-aminophenoxazin-3-one is a Gram-positive bacterium, Mycobacterium phlei, Mycobacterium tuberculosis BCG, Mycobacterium tuberculosis H-2 (Mycobacterium tuberculosis H-2). It is known to be effective against Penicillium chrysogenum, Monilia formosa, and the fungus Schizosaccharomyces actosporas (the following, non-patented) Reference 2). However, 2-aminophenoxazin-3-one is found in Escherichia coli, Staphylococcus aureus 209P, Micrococcus flavus, Bacillus subtilis, Bacillus argi (Bacillus argi). ), Pseudomonas pyocyaneus, the fungus Aspergillus niger, the fungus Candida albicans, Toruta utilis, the yeast Saccharomyces ceromies Saccharomyces pastorianus yeast, Saccharomyces fragilis yeast, Kleockera apicuiata, Torulaspora dellviickii, Torulaspora dellviickii Endomyces magnusii, yeasts Schizosaccharomyces pompe, Pichia menfranaefaciens, Hansemula anomala, and yeast Schizosaccharomyces monolus (Schizosaccharomyces monolshurocus), Phototorula glutinis, Torulaspora fermentali, and yeast Schizosaccharomyces salsus (Schizosaccharomyces salsus) have no antibacterial activity (see Non-Patent Document 2 below) reference). Thus, 2-aminophenoxazin-3-one is known to have antibacterial activity against some gram-positive bacteria, fungi, and yeast, but is known for antibacterial activity against other bacteria. Not.

JP 02-193984 A (4th page, lower left column, lines 2-7) Patent No. 3290172 (page 5, left column, lines 32-40) Yamaguchi et al., Antimicrobial Agents and Chemotherapy, 47, 1972-1975, 2003 Kentaro Anzai et al., The New Antibiotics, Questiomycins A and B, The Journal of Antibiotics, Ser. A. Vo. XIII, No. 2, 125-132, 1960

  An object of the present invention is to provide an antibacterial and antibacterial agent that can be used for prevention and treatment of chlamydia infection.

  The present invention relates to an anti-chlamydia agent comprising 2-aminophenoxazin-3-one or a pharmacologically acceptable salt or ester thereof as an active ingredient, and a prophylactic agent, a recurrence preventing agent or a Therapeutic agents (hereinafter collectively referred to as “agents of the present invention”) are provided.

  2-Aminophenoxazin-3-one and their pharmacologically acceptable salts and esters have an antibacterial effect on chlamydia. Therefore, 2-aminophenoxazin-3-one and pharmacologically acceptable salts and esters thereof are effective as an anti-chlamydia agent and a prophylactic agent, relapse preventive agent or therapeutic agent for diseases associated with chlamydia. .

  2-Aminophenoxazin-3-one is represented by the following formula.

  The agent of the present invention contains 2-aminophenoxazin-3-one or a pharmacologically acceptable salt or ester thereof as an active ingredient. 2-Aminophenoxazin-3-one has an antibacterial effect on chlamydia (see Examples 1-4 below). The 2-aminophenoxazin-3-one derivative (compound a in Test Example 1 below) having a substituent (methyl group) at positions 4a and 7 of 2-aminophenoxazin-3-one is less than chlamydia. Does not show antibacterial effect. In addition, the 3-aminophenoxazin-2-one derivative (compound b of Test Example 1 below) does not show antibacterial effect against chlamydia.

  As used herein, the term “anti-chlamydia agent” refers to a drug that antibacterials or disinfects chlamydia. Furthermore, the “prophylactic agent, relapse preventive agent or therapeutic agent for a disease associated with chlamydia” as used in the present invention is an antibacterial or antibacterial agent for chlamydia, which is used for the purpose of preventing chlamydia-derived diseases. Used as a prophylactic agent when used for the purpose of preventing recurrence of the disease once cured, or as a therapeutic agent when used for the purpose of treating the disease by antibacterial or removing chlamydia To do. The “relapse prevention agent or treatment agent” can also be used for the purpose of preventing and treating relapse at the same time.

  The agent of the present invention has an antibacterial effect against chlamydia. “Chlamydia” includes, but is not limited to, Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydia pecorum.

  The inhibitory action of 2-aminophenoxazin-3-one on the growth of chlamydia in infected cells was measured by measuring MIC (Minimum Inhibitory Concentration) in the presence of cycloheximide (see Example 1 below). Measurement of the degree of growth of chlamydia in HEp-2 cells using the inclusion body count (IFU) method in the absence of cycloheximide (see Example 2 below), in the absence of cycloheximide Measurement of Chlamydia growth in THP-1 cells using the inclusion body count (IFU) method (see Example 3 below) or real-time-RT-PCR in the absence of cycloheximide Although it can be determined by measuring the degree of Chlamydia proliferation in the HEp-2 cells (see Example 4 below), it is not limited to these methods.

In Example 1 below, 2-aminophenoxazin-3-one was shown to inhibit the growth of Chlamydia pneumonia in the presence of cycloheximide which inhibits host cell protein synthesis (MIC was 32 μg). / ml or 160 μM). As shown in Test Example 1 below, compounds a and b, which are 2-aminophenoxazin-3-one derivatives, have no inhibitory action under these experimental conditions, and therefore are judged to have no inhibitory action on Chlamydia growth. It was done.
In Examples 2 and 3 below, 2-aminophenoxazin-3-one (final concentration 1 μM or 10 μM) is chlamydia in the absence of cycloheximide, ie, under conditions where protein synthesis of infected cells is not inhibited. Proliferation of Chlamydia pneumonia was significantly suppressed in the infected cells (HEp-2 cells and THP-1 cells).
In Example 4, 2-aminophenoxazin-3-one was confirmed to suppress the growth of chlamydia even at the RNA level in the absence of cycloheximide in HEp-2 cells infected with chlamydia. Therefore, it is clear that 2-aminophenoxazin-3-one has a remarkable inhibitory effect on the growth of chlamydia in infected cells in the absence of cycloheximide.
It has been clarified that chlamydia infected with cells are extremely resistant to conventional macrolides and quinolones. In contrast, 2-aminophenoxazin-3-one inhibits the growth of chlamydia in infected cells at low concentrations in the absence of cyclohexamide. This effect is not found in conventional macrolides and quinolone drugs.
Thus, although the mechanism of action of chlamydia antibacterial by the agent of the present invention is not clear, it is thought to suppress the proliferation of chlamydia in infected cells through some mechanism in the cells infected with chlamydia. Therefore, 2-aminophenoxazin-3-one is thought to be effective in the prevention, treatment and recurrence of diseases associated with chlamydia by suppressing the growth of chlamydia by a mechanism different from that of macrolides and quinolones. It is done.

  The agent of the present invention can be used for prevention, relapse prevention, or treatment of diseases associated with chlamydia. In the case of Chlamydia trachomatis, diseases associated with Chlamydia are chronic keratoconjunctivitis, nongonococcal urethritis, inclusion body conjunctivitis, cervicitis, tubalitis, perihepatitis, neonatal Chlamydia pneumonia, neonatal Chlamydia ophthalmitis Inguinal (sexually pathogenic) lymphogranulomatosis, infertility, ectopic pregnancy, miscarriage. In the case of parrot disease chlamydia, examples of the disease include parrot disease, meningitis, conjunctivitis, and pneumonia. In the case of Chlamydia pneumonia, examples of the disease include asthma, pneumonia, bronchitis, otitis media, arteriosclerosis, and atherosclerosis (Atherosclerosis). In the case of Chlamydia pecolum, examples of the disease include diarrhea and miscarriage in ruminants (such as cattle or sheep).

  As a synthesis method of 2-aminophenoxazin-3-one, a method of reacting 2-amino-5-methylphenol in the presence of human hemoglobin at 37 ° C. for 3 days (Akio Tomoda et al., Phenoxazinone Synthesis by Human Hemoglobin, Biochimica et Biophysica Acta, 1117, 306-314, 1992), a method of reacting 2-amino-5-methylphenol in the presence of bovine hemoglobin at 37 ° C. for 5 days (Akio Tomoda et al. , An Improved Method for the Rapid Preparation of 2-Amino-4,4a-dihydro-4a, 7-dimethyl-3H-phenoxazine-3-one, a Novel Antitumor Agent, Bioorganic & chemistry Letters, Vol. 11, 1057-1058 Page, 2001), but is manufactured according to a chemical synthesis method using potassium ferricyanide as a catalyst (Japanese Patent No. 3290172), but is not limited thereto.

  Salts that can be used in the agent of the present invention include, for example, inorganic salts such as alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as magnesium salts; metal salts such as copper salts and zinc salts; , Diethanolamine salts, alkanolamine salts such as 2-amino-2-ethyl-1,3-propanediol salt and triethanolamine salt; heterocyclic amine salts such as morpholine salt, piperazine salt and piperidine salt; ammonium salt and arginine salt And basic amino acid salts such as lysine salts and histidine salts. Here, the basic amino acid may be D-form, L-form, or a mixture thereof.

  Examples of the ester that can be used in the agent of the present invention include formic acid ester, acetic acid ester, and propionic acid ester.

  The agent of the present invention may contain a pharmaceutically acceptable carrier or additive. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, Others such as gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, etc. And artificial cell structures such as liposomes. The additive to be used is selected appropriately or in combination from the above according to the dosage form of the present invention.

  The agent of the present invention can be used in the form of a general pharmaceutical preparation. In the preparation, diluents such as fillers, extenders, binders, moisturizers, disintegrants, surfactants, lubricants and the like that are usually used, or excipients can be blended. Various forms of the agent of the present invention can be selected according to the purpose of treatment, and representative examples thereof include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, and liquids. And injections such as suspensions.

  The carrier to be formed into a tablet form can include various pharmaceutically acceptable carriers or additives. Examples of such carriers and additives include excipients such as lactose, sucrose, glucose, sodium chloride, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid; water, ethanol, propanol, simple syrup , Glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc .; dry starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene Dissolving agents such as sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose; disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil; quaternary ammonium base, lauryl sulfate Absorption promoters such as thorium, humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; lubricants such as purified talc, stearate, boric acid powder and polyethylene glycol Can be used. Further, the tablets can be made into tablets with ordinary coatings as necessary, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multilayer tablets.

  When forming into the form of a pill, those conventionally known in this field can be widely used as the carrier. Examples include excipients such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, kaolin and talc; binders such as gum arabic powder, tragacanth powder, gelatin and ethanol; and disintegrants such as laminaran and agar it can.

  Capsules are usually prepared by mixing an active ingredient compound with the various carriers exemplified above and filling them into hard gelatin capsules, soft capsules and the like according to a conventional method.

  In molding into a suppository form, conventionally known carriers can be widely used. Examples thereof include polyethylene glycol, cacao butter, higher alcohols, higher alcohol esters, gelatin, semi-synthetic glycerides and the like.

  When prepared as an injection, solutions, emulsions and suspensions are preferably sterilized in advance and are isotonic with blood. In molding into these forms, any conventionally used diluent in this field can be used. For example, water, ethanol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be used. In this case, a sufficient amount of sodium chloride, glucose or glycerin may be contained in the pharmaceutical preparation to prepare an isotonic solution, and a normal solubilizing agent, buffering agent, soothing agent, etc. may be added. May be.

  In addition, by using 2-aminophenoxazin-3-one in combination with known antibiotics (tetracycline antibiotics such as tetracycline, doxycycline and minocycline, and macrolide antibiotics such as erythromycin), antibacterial activity against chlamydia The effect can be enhanced.

  It is more preferable that the antibiotic used in combination is sensitive to chlamydia. For example, tetracycline antibiotics such as tetracycline, doxycycline, minocycline, macrolide antibiotics such as rithromycin, rifampicin, clindamycin, penicillin-G, ampicillin, and the like can be used. The amount of antibiotic added is not particularly limited.

  The agent of the present invention can be administered orally. In this case, a solid preparation such as a tablet, granule, powder, pill or the like, or a liquid preparation such as a liquid or syrup may be used. In particular, granules and powders can be made into unit dosage forms as capsules, and in the case of liquid preparations, they may be dried products that are redissolved when used.

  Among these agents, oral solid preparations usually contain additives such as binders, excipients, lubricants, disintegrating agents, wetting agents and the like that are generally used in pharmaceutical compositions. Oral liquid preparations usually contain additives such as stabilizers, buffering agents, corrigents, preservatives, fragrances, coloring agents and the like that are generally used in the formulation.

  The method of using the agent of the present invention is not particularly limited, and it is used in a method according to various preparation forms, age of patients or users, sex and other conditions, degree of disease, and the like. For example, in the case of tablets, pills, solutions, suspensions, emulsions, granules and capsules, they are administered orally. In the case of an injection, it is administered intravenously alone or mixed with a normal replacement fluid such as glucose or amino acid, and further administered alone intramuscularly, intradermally, subcutaneously or intraperitoneally as necessary. In the case of a suppository, it is administered intrarectally.

  The amount of the agent of the present invention is appropriately selected depending on the usage, the age of the intended user or patient, gender and other conditions, the degree of the disease, etc. About 0.1 to 2000 mg per day, preferably about 0.5 to 1800 mg, particularly preferably about 1 to 1500 mg, and can be administered 1 to 4 times a day, for example, on an empty stomach.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by these Examples.

[Example]
[Production example]
After adding 100 mL of 50 mM ortho-aminophenol (manufactured by Tokyo Chemical Industry Co., Ltd.) solution (neutralized to pH 7.0 with 0.1 N sodium hydroxide aqueous solution) to 1 L of bovine hemoglobin solution and stirring thoroughly And left at 37 ° C. for 6 days. Six days later, 5 L of 100% methanol was added to the reaction solution to denature hemoglobin and protein. The denatured hemoglobin and protein were centrifuged with a centrifuge (SCR-20BS type, Hitachi, Ltd.) (10,000 × g, 5 minutes) to obtain a supernatant. After collecting the supernatant, water and methanol were evaporated and concentrated using a rotary evaporator (NE type, manufactured by Tokyo Rika Kikai Co., Ltd.). To the remaining powder, 300 ml of 100% methanol was added and dissolved. This lysate was subjected to separation and purification by attaching it to a column (inner diameter 7 cm × height 30 cm) packed with Sephadex LH20 resin (manufactured by Amarsham Pharmacia Japan) equilibrated by pre-swelling with 50% ethanol. . Elution was performed with 50% ethanol (volume / volume) and fractionated at regular intervals. The fraction (reddish brown) part that appeared third was collected. This fraction was again concentrated by an evaporator to obtain a powder. 50 ml of 100% methanol was added to the powder and completely dissolved. The lysate was applied to a column (inner diameter 4 cm × height 50 cm) packed with Sephadex LH20 resin previously swollen with 50% ethanol and equilibrated for purification. Elution was performed with 50% ethanol (volume / volume) and fractionated at regular intervals. The reddish brown fraction was collected, and this fraction was again concentrated by an evaporator to obtain about 3.5 g of a reddish brown powder. By comparing the data values of UV, IR, ¹ H-NMR (270 MHz), ¹³ C-NMR (50.3 MHz) of this powder with those of the standard 2-aminophenoxazin-3-one, The powder was identified as 2-aminophenoxazin-3-one.

MIC (Minimum Inhibitory Concentration) of 2-aminophenoxazin-3-one against Chlamydia pneumonia was measured.
(1) Test compound 2-Aminophenoxazin-3-one obtained in the above production example was used.
(2) Dilution of test compound
A stock solution (0.75 mg / ml) containing 15 mg of 2-aminophenoxazin-3-one in 20 ml of 99% ethanol (special grade, manufactured by Wako Pure Chemical Industries, Ltd.) was prepared.
As a control, at the time of cell culture after infection, the same amount of ethanol without 2-aminophenoxazin-3-one was used so that the final dilution was the same as that of the test compound.
(3) Preparation of Chlamydia pneumonia for inoculation
Chlamydia pneumoniae TW183 ATCC (Japan name: Chlamydia pneumonia, source: Department of Infectious Diseases, Kyorin University School of Medicine) (Mycoplasma free) was used. The ingesting strain was grown in Heb-2 cells, released from the cells by sonication, and collected by centrifugation. The collected cells were resuspended in sucrose phosphate buffer (pH 7.4: 0.2 M Sucrose, 0.004 M KH ₂ PO ₄ , 0.007 MK ₂ HPO ₄ ). Adjust the bacterial count to 5x10 ⁷ IFUs (IFU is almost the same as the bacterial count) / ml, dispense 1 ml each into a 2 ml freezing vial, deep freezer (model number Ultra Flow MDF-392, Sanyo Electric And frozen at -80 ° C.
(4) Cell line
HEp-2 cells (Japanese name: human pharyngeal squamous cell carcinoma-derived cells, available from Kyorin University School of Medicine Department of Infectious Diseases)
The cell line was subcultured in D-MEM medium (Sigma, St. Louis, Missouri, USA) supplemented with 10% inactivated bovine infant serum (FCS) and adjusted to a cell number of 1 × 10 ⁷ cells / ml. 1 ml each was dispensed into 2 ml freezing vials, and stored frozen at −80 ° C. in a deep freezer (model number Ultra Flow MDF-392, manufactured by Sanyo Electric Co., Ltd.).

(5) Experimental method
Each well of a 24-well plate (Iwaki Glass Co., Ltd.) was subjected to monolayer culture of HEp-2 cells and infected with Chlamydia pneumonia at the stage where HEp-2 cell proliferation spread across the entire plate surface. After infection, cycloheximide 1 μg / ml and 2-aminophenoxazin-3-one (0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 or 32 μg at final concentrations / ml: 32 μg / ml corresponds to a concentration of 160 μM) and cultured for 3 days. After the culture, 70% alcohol was added to the 24-well plate to fix HEp-2 cells, and the inclusion bodies formed in each well were stained with FITC-anti-chlamydia antibody. Measure the number of inclusion bodies stained per hole with a fluorescence microscope (Olympus BX50, manufactured by Olympus Corporation) and calculate the infectivity value as IFU (Inclusion forming units) from that number. Was determined as the MIC of the 2-aminophenoxazin-3-one drug.

(6) Results
The minimum inhibitory concentration (MIC) at which 2-aminophenoxazin-3-one completely suppressed chlamydia growth in HEp-2 cells was 32 μg / ml.

Inclusion body count (IFU) method was used to measure the degree of growth of Chlamydia pneumonia in HEp-2 cells in the absence of cycloheximide.
(1) The test compound, dilution of the test compound, and preparation of Chlamydia pneumonia for inoculation were the same as in Example 1 except that cycloheximide was not added.

(2) Experimental method Infection of Chlamydia pneumonia into HEp-2 cells was carried out at a multiplicity of infection (MOI) = 10 (10 Chlamydia pneumonia was exposed per HEp-2 cell). It was. Specifically, after preparing HEp-2 cells at a rate of 1x10 ⁵ cells / well in a 24-well plate, add 1 ml of 1x10 ⁶ IFU containing Chlamydia pneumonia and centrifuge at 400 xg for 1 hour at room temperature. The cells were adsorbed and infected on the cells. After washing with D-MEM medium containing 10% FCS three times, 2-aminophenoxazin-3-one was added to a final concentration of 1 or 10 μM in the absence of cycloheximide, respectively. did. After culturing in D-MEM medium supplemented with 10% FCS for 72 hours, the number of inclusion bodies formed (IFU) method (FEMS Microbiology Letters, 216, 229-234, 2002; Infection and Immunity, 69, No. 7753-7759 (2001), the number of bacteria was quantitatively measured. As a control, instead of 2-aminophenoxazin-3-one, only the culture solution used for drug dilution was added and cultured for 72 hours (control). The 2-aminophenoxazin-3-one stock solution is also dissolved in ethanol. Therefore, in order to confirm that ethanol had no effect on the cells, ethanol was added to the final dilution factor at the time of addition of the test compound and cultured for 72 hours (control (ethanol)).

(3) Results
FIG. 1 shows the number of Chlamydia pneumonia bacteria (IFU) after 72 hours of culture after infection at each concentration of 2-aminophenoxazin-3-one (final concentration 1 or 10 μM) in HEp-2 cells. Table 1 specifically shows the number of bacteria and the degree of proliferation in FIG.

  As a result of quantifying the number of intracellular bacteria by IFU method, the growth of Chlamydia pneumonia in HEp-2 cells was performed by adding 2-aminophenoxazin-3-one (final concentration 10 μM) in the absence of cycloheximide. After 72 hours, it was almost completely suppressed (p <0.05) (FIG. 1).

Inclusion body formation count (IFU) method was used to measure the degree of proliferation (increase in the number of bacteria) of Chlamydia pneumonia in THP-1 cells in the absence of cycloheximide.
(1) Experimental method Example 2 was used except that THP-1 cells (human monocyte / macrophage cell line, available from Kyorin University School of Medicine) were used instead of HEp-2 cells in Example 2. A similar test was conducted. The cell line was subcultured in RPMI1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% inactivated calf infant serum (FCS), adjusted to 1 × 10 ⁶ cells / ml, and 2 ml freezing vial 1 ml each was dispensed and then frozen and stored at −80 ° C. in a deep freezer (model number Ultra Flow MDF-392, manufactured by Sanyo Electric Co., Ltd.).

(2) Results
FIG. 2 shows the number of Chlamydia pneumonia bacteria (IFU) after incubation for 72 hours after infection at each concentration of 2-aminophenoxazin-3-one (final concentration 1 or 10 μM) in THP-1 cells. Table 2 specifically shows the number of bacteria and the degree of proliferation shown in FIG.

  As a result of quantifying the number of intracellular bacteria by the IFU method, the growth of Chlamydia pneumonia in THP-1 cells was carried out in the absence of cycloheximide, and 2-aminophenoxazin-3-one was added (final concentration 1 or 10 μM). ) Was almost completely suppressed after 72 hours of culture (p <0.05) (FIG. 2).

Using real-time-RT-PCR method, the growth inhibitory effect of Chlamydia pneumonia in HEp-2 cells in the absence of cycloheximide was tested.
(1) The test compound, dilution of the test compound, and preparation of Chlamydia pneumonia for inoculation are the same as in Example 1.

(2) Experimental method Real-time RT-PCR was performed using primers specific to Chlamydia pneumonia 16S rRNA (Berger M, Schroder B, Daeschlein G et al., Chlamydia pneumoniae DNA in Non-coronary Atherosclerotic Plaques and Circulating Leukocytes J Lab Clin Med, 2000, 136, 194-200). According to the experimental method of Example 2, cells were infected with Chlamydia pneumonia HEp-2 cells at a multiplicity of infection MOI = 10. Specifically, after adding HEp-2 cells to a 24-well plate at a rate of 1x10 ⁵ cells / well, add 1 ml of a solution containing 1x10 ⁶ IFU of Chlamydia pneumonia and centrifuge at 400 xg for 1 hour at room temperature. The cells were adsorbed and infected on the cells. After washing with 10% FCS-added D-MEM medium by centrifugation three times, 2-aminophenoxazin-3-one was added to a final concentration of 1 or 10 μM in the absence of cycloheximide, respectively. . After 72 hours of culturing in D-MEM medium supplemented with 10% FCS, total RNA was extracted from infected HEp-2 cells using RNeasy Mini Kit (Qiagen). This operation was repeated a plurality of times, and the total extraction amount was 2 to 6 μg / 40 μl extract. 40 μl of this extract was treated with DNase (DNA removal kit; Ambion, Austin, Texas, USA) to decompose contaminating DNA.
The complete removal of DNA from the RNA-containing extract is due to the amplification of chlamydial target products from DNA by real-time PCR (without RT) using RNA solution prior to reverse transcription (RT) treatment. It was confirmed that there was no. 1 μg of RNA in the extract was obtained from a commercial RT reaction mixture (20 μl; Reverse Transcription System; Promega, Madison, Wis., USA) and avian myeloblastosis virus reverse transcriptase with random primers. It was converted to cDNA by action.
The quality of the extracted RNA and the efficiency of cDNA synthesis in the RT reaction are uniform between samples. Real-time RT-PCR targeting the G3PDH gene, which is always expressed in cells, is performed. It was confirmed by. Real-time RT-PCR was performed using a GeneAmp 5700 sequence detection system (sequence detection system: PE Biosystems, Foster City, Calif., USA).
Chlamydia can be detected by using specific primers (sense, 5-GGA CCT TAG CTG GAC TTG ACATGT-3; antisense, 5-CCA TGC AGC ACC TGT GTA TCT G-3) The SYBY green method (QuantiTect SYBR green PCR kit, Qiagen) was used.
Amplification of the target product by PCR was performed under the conditions of 95 ° C. for 10 minutes, 1 cycle, and 50 cycles of 95 ° C. for 15 seconds, 60 ° C. for 1 minute, and 72 ° C. for 20 seconds. Each time PCR was performed to confirm whether the PCR product was specifically amplified, a melting temperature (Tm) assay was performed. The Tm value of the chlamydia target product of the primer used in this experiment was 76.9 ° C. Samples in which amplification was observed at other Tm values were excluded as nonspecific amplification reactions taking place.
Moreover, about the amplification product in the sample determined to be positive, it was confirmed that the target product was specifically amplified by directly determining the oligonucleotide sequence.
A calibration curve for Real-time PCR was prepared by diluting DNA extracted from a Chlamydia cryopreservation solution with a known number of bacteria. The results are shown in FIG. The numerical value on the vertical axis in FIG. 3 indicates the number of Chlamydia pneumonia 16S rRNA cDNA copies amplified in the PCR tube.

(3) Results
As a result of examining by real-time-RT-PCR method, the growth inhibitory effect of Chlamydia pneumonia in HEp-2 cells was significantly (p <0.05) in both 1 and 10 μM addition systems ( FIG. 3).

(4) Discussion From the results of Example 1, 2-aminophenoxazin-3-one suppresses the growth of Chlamydia pneumonia in the presence of cycloheximide that suppresses host cell DNA protein synthesis (MIC: 32 μg / ml). From the results of Examples 2 to 4, 2-aminophenoxazin-3-one remarkably suppresses the growth of Chlamydia pneumonia in the absence of cycloheximide (1 μg / ml). This shows that 2-aminophenoxazin-3-one suppresses Chlamydia pneumoniae growth at a lower concentration in the absence than in the presence of cycloheximide. This suggests that 2-aminophenoxazin-3-one has an effect on some mechanism of the host cell and consequently suppresses the growth of Chlamydia pneumonia.

  100 g of 2-aminophenoxazin-3-one and 900 g of lactose were mixed well to prepare a powder containing 10 mg of 2-aminophenoxazin-3-one in 1 g (10-fold powder).

  2-Aminophenoxazin-3-one 500 g and lactose 500 g were mixed well to prepare a powder containing 2 mg of 2-aminophenoxazin-3-one in 1 g (double powder).

  2-aminophenoxazin-3-one 100 g, lactose 50 g, 6% HPC lactose 40 g, potato starch 6 g and talc stearate 4 g are mixed well and tableted, and 2-aminophenoxazin-3-one in one tablet Tablets containing 100 mg were made.

  2-aminophenoxazin-3-one 100 g, lactose 90 g, potato starch 6 g and calcium stearate 4 g are mixed well and filled into a hard capsule, and each capsule contains 100 mg of 2-aminophenoxazin-3-one The agent was made.

[Test Example 1]
Using the same method as in Example 1, the MIC (minimum growth inhibitory concentration) of the following test compounds was measured.
(1) Test compound 1) 2-aminophenoxazin-3-one derivative 2-amino-4,4a-dihydro-4a, 7-dimethyl-3H-phenoxazin-3-one (hereinafter referred to as compound a) is Akio Tomoda et al., An Improved Method for the Rapid Preparation of 2-Amino-4,4a-dihydro-4a, 7-dimethyl-3H-phenoxazine-3-one, a Novel Antitumor Agent, Bioorganic & chemistry Letters, Volume 11. , Pages 1057-1058, 2001.

2) 3-Aminophenoxazin-2-one derivative 3-Amino-1,4a-dihydro-4a, 8-dimethyl-2H-phenoxazin-2-one (hereinafter referred to as compound b) was obtained by Akio Tomoda et al. It was produced according to the method described in Oxidative Condensation of 2-Amino-4-Methylphenol to Dihydrophenoxazinone Compound by Human Hemoglobin, J. Biochem., 110, 1004-1007, 1991.

(2) Results As a result, compounds a and b had no effect on Chlamydia growth at a concentration of 32 μg / ml. Thus, compounds a and b are not effective against chlamydia.

  2-Aminophenoxazin-3-one and their pharmacologically acceptable salts and esters are effective against chlamydia. Therefore, the agent of the present invention has industrial applicability.

Results of suppression of Chlamydia pneumoniae growth by 2-aminophenoxazin-3-one in HEp-2 cells by IFU method Results of suppression of Chlamydia pneumoniae growth in THP-1 cells by 2-aminophenoxazin-3-one by IFU method Results of Chlamydia pneumoniae growth inhibition effect of 2-aminophenoxazin-3-one in HEp-2 cells by real-time RT-PCR

Claims (8)

An anti-chlamydia agent comprising 2-aminophenoxazin-3-one or a pharmacologically acceptable salt thereof as an active ingredient. The anti-chlamydia agent according to claim 1, wherein the chlamydia is Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pneumoniae or Chlamydia pecorum. A prophylactic, relapse preventive or therapeutic agent for a disease involving chlamydia comprising 2-aminophenoxazin-3-one or a pharmacologically acceptable salt thereof as an active ingredient , wherein the disease is chronic horn Conjunctivitis, non-gonococcal urethritis, inclusion body conjunctivitis, cervicitis, tubal inflammation, perihelitis, neonatal chlamydia pneumonia, neonatal chlamydia ophthalmitis, inguinal (genderous) lymphogranulomatosis, infertility, ectopic pregnancy , Miscarriage, trachoma, epididymis, prostatitis, intramedullary inflammatory disease, parrot disease, meningitis, conjunctivitis, pneumonia, asthma, bronchitis, otitis media, arteriosclerosis, atherosclerosis, or ruminant The said agent which is diarrhea or miscarriage .   The disease associated with Chlamydia according to claim 3, wherein the Chlamydia is Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pneumoniae or Chlamydia pecorum. Preventive agent, relapse preventive agent or therapeutic agent. Chronic keratoconjunctivitis caused by Chlamydia trachomatis, nongonococcal urethritis, inclusion body conjunctivitis, cervicitis, tubalitis, perihepatitis, neonatal chlamydia pneumonia, neonatal chlamydial ophthalmitis, inguinal (sexually pathogenic) 4. The preventive agent or the recurrence preventive agent for a disease associated with chlamydia according to claim 3, which is a lymphogranulomatosis, infertility, ectopic pregnancy , miscarriage , trachoma, epididymis, prostatitis or intramedullary inflammatory disease Or a therapeutic agent.   The preventive agent, relapse preventive agent or therapeutic agent for a disease involving chlamydia according to claim 3, wherein the disease is parrot disease, meningitis, conjunctivitis or pneumonia caused by parrot disease chlamydia.   The preventive agent, relapse preventive agent or therapeutic agent for a disease associated with chlamydia according to claim 3, wherein the disease is asthma, pneumonia, bronchitis, otitis media, arteriosclerosis or atherosclerosis caused by Chlamydia pneumonia .   The preventive agent, relapse preventive agent, or therapeutic agent for a disease associated with chlamydia according to claim 3, wherein the disease is ruminant diarrhea or miscarriage caused by chlamydia pecolum.

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