Antibacterial agent and pharmaceutical

Abstract

To provide a novel antibacterial agent and pharmaceutical.SOLUTION: An antibacterial agent contains pseudoiodinine or a salt thereof as an active ingredient, and has antibacterial activity against at least one bacterium selected from the group consisting of Yersinia, Vibrio, Escherichia, Aeromonas, Edwardsiella, Bacillus, and Streptococcus.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to antibacterial agents and medicines, and particularly to aquatic antibacterial agents for the treatment or prevention of diseases in fish and shellfish, etc.

Background Art

[0002] Conventionally, infectious diseases caused by bacteria have threatened our lives. Many antibacterial agents have been developed so far, but the emergence of resistant bacteria has often been a problem. As resistant bacteria, for example, methicillin-resistant Staphylococcus aureus (MRSA) and penicillin-resistant Streptococcus pneumoniae (PRSP) have been reported. Due to such problems of resistant bacteria, new antibacterial agents are demanded.

[0003] In addition, bacteria infect not only humans but also, for example, fish and shellfish, causing infectious diseases. For example, in the aquaculture of fish and shellfish, an environment of overcrowded breeding is likely to occur, so infectious diseases are likely to spread, and countermeasures against infectious diseases are an important issue in the aquaculture industry. Conventionally, as a measure against diseases of fish and shellfish (especially fish), administration of antibacterial agents has been carried out due to the wide range of application and the simplicity of use. For example, Patent Document 1 describes oral administration of antibacterial agents such as oxytetracycline and doxycycline to fish. However, also in this case, new antibacterial agents are demanded due to concerns about resistant bacteria.

[0004] By the way, bacteria of the genus Pseudomonas are known to produce a wide spectrum of heterocyclic antibiotics such as phenazine, quinoline, and pyrrole derivatives. For example, Non-Patent Document 1 reports P. fluorescens var. pseudoiodinum, which produces pseudoiodinine, a red pigment, as a strain of the genus Pseudomonas.

[0005] The structure of the above pseudoiodinine was identified as 3-Methoxy-7-methyl-7H-pyrazolo[4,3-e][1,2,4]triazine in Non-Patent Document 2. So far, the physiological activities of pyrazolo[4,3-e]as-triazine derivatives produced from similar bacteria have been studied, and it has been reported that they have a very weak antibacterial effect (Non-Patent Document 3). However, the physiological activity of pseudoiodinine itself is actually unknown.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a novel antibacterial agent and a medicine.

Means for Solving the Problems

[0009] The inventors of the present invention conducted intensive studies to solve the above problems, and surprisingly found that pseudoiodinine strongly inhibits the growth of various bacteria and the like. The present invention is based on such findings.

[0010] The antibacterial agent of the present invention contains pseudoiodinine or a salt thereof as an active ingredient.

[0011] The above antibacterial agent has antibacterial activity against at least one bacterium selected from the group consisting of Yersinia, Vibrio, Escherichia, Aeromonas, Edwardsiella, Bacillus, and Streptococcus.

[0012] The above antibacterial agent has antibacterial activity against at least one bacterium selected from the group consisting of Yersinia, Vibrio, Aeromonas, Edwardsiella, and Streptococcus.

[0013] It is characterized in that the minimum growth inhibitory concentration against the above bacteria is 1 mg / ml or less.

[0014] The above antibacterial agent is a fishery antibacterial agent for the treatment or prevention of diseases in fish and shellfish.

[0015] The medicament of the present invention contains pseudoiodinine or a salt thereof as an active ingredient.

Advantages of the Invention

[0016] Since pseudoiodinine strongly inhibits the growth of various bacteria and the like, it can be suitably used as an antibacterial agent.

[0017] In addition, as diseases of fish and shellfish, vibriosis, enteric redmouth disease, etc. are known, and since they strongly inhibit the growth of bacteria such as Vibrio and Aeromonas, which are the pathogens of these diseases, they can be particularly preferably used as aquaculture antibacterial agents for the treatment or prevention of diseases in fish and shellfish.

[0018] Pseudoiodinine can be preferably used as a medicine because it inhibits the growth of pathogens (e.g., Candida) of infectious diseases in mammals including humans.

Brief Description of the Drawings

[0019]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0020] The present inventors have found that pseudoiodinine strongly inhibits the growth of a wide range of bacteria. Therefore, pseudoiodinine is useful as an antibacterial agent. Here, the antibacterial agent broadly means a drug having a bactericidal action or a growth inhibitory action against bacteria.

[0021] In addition, pseudoiodinine is also useful as a medicine (especially an antibacterial drug). In the present invention, the medicine includes medicines used for humans, mammals (excluding humans; the same applies hereinafter), and fish and shellfish.

[0022] Pseudoiodinine is 3-Methoxy-7-methyl-7H-pyrazolo[4,3-e][1,2,4]triazine, a compound represented by the following formula (1).

[0023]

Chemical formula

[0024] Pseudoiodinine was discovered to be produced by Pseudomonas fluorescens var. pseudoiodinum in 1970 and reported as a red pigment (see Non-Patent Document 1). Subsequently, its structure was first determined in 1972 and reported as the compound represented by the above formula (2) (Chemische Berichte, 1972, 105, p. 1949-1955), but was corrected to the correct structure and became the above formula (1) according to the report of total synthesis in 2006 (see Non-Patent Document 2).

[0025] As the salt of pseudoiodinine, any pharmaceutically acceptable salt can be used without particular limitation. Examples of the salt include salts with inorganic acids, salts with organic acids, etc. Note that pseudoiodinine or its salt also includes the form of hydrates and the form of solvates with alcohols, etc.

[0026] In the present invention, the production method of pseudoiodinine is not particularly limited, and for example, it can be obtained by a method of chemically synthesizing from any starting material (synthesis method). For example, according to the above Non-Patent Document 2, pseudoiodinine can be synthesized by the following scheme.

[0027]

Chemical formula

[0028] The above method uses diethylaminomalonate hydrochloride as the starting material. After reacting this starting material with N,N-dimethylformamide dimethylacetal to form an amide compound, hydrazine is reacted to obtain dihydrotriazone (3). Then, dihydrotriazone (3) is reduced to triazone (4), and further chlorinated to obtain chlorotriazine (5). After reacting chlorotriazine (5) with Boc-hydrazine and then cyclizing, triazone (7) is obtained.

[0029] And by methylating triazone (7), Nostocine A (8) and Fluviol A (9) are obtained. Further, by methylating each compound, pseudoiodinine (1) is obtained.

[0030] In addition, pseudoiodinine may be obtained not only by the above synthesis method but also by a method (extraction method) of separating and collecting it from a culture of microorganisms that produce pseudoiodinine. When obtained by the extraction method, the antibacterial agent and medicine of the present invention may contain an extraction crude product containing pseudoiodinine. That is, in addition to pseudoiodinine, other products of the microorganism and components derived from the culture medium may be contained.

[0031] As shown in the examples described below, pseudoiodinine exhibits strong growth inhibitory activity against many prokaryotes such as Gram-negative bacteria and Gram-positive bacteria, and eukaryotes such as yeast. Examples of Gram-negative bacteria include, for example, Yersinia, Vibrio, Escherichia, Aeromonas, Edwardsiella, Pseudomonas, etc. Examples of Gram-positive bacteria include, for example, Bacillus, Streptococcus, etc. Examples of yeast include, for example, Saccharomyces, Candida, etc.

[0032] The antibacterial agent of the present invention preferably has antibacterial activity against at least one bacterium selected from the group consisting of Yersinia, Vibrio, Escherichia, Aeromonas, Edwardsiella, Bacillus, and Streptococcus. The MIC against the bacteria showing antibacterial activity is preferably 5 mg / mL or less, more preferably 1 mg / mL or less, even more preferably 500 μg / mL or less, and particularly preferably 100 μg / mL or less.

[0033] Since the antibacterial agent of the present invention strongly inhibits the growth of a wide range of bacteria, etc., it can be used by being incorporated into products ingested into the body or applied to the body surface of humans, mammals, and fish and shellfish, and other products for which it is desired to prevent or suppress the growth of bacteria. The antibacterial agent of the present invention can be used in various fields, for example, in the fields of pharmaceuticals, agricultural chemicals, livestock products, fisheries products, food and beverages, cosmetics, hygiene products, etc.

[0034] The above-mentioned pseudoiodinine exhibits growth inhibitory activity against, for example, pathogenic bacteria causing human infectious diseases (e.g., Candida). Pseudoiodinine is useful as a medicine for humans. Specifically, it can be used as a therapeutic or prophylactic agent for superficial mycosis such as tinea, vitiligo, cutaneous candidiasis, and deep mycosis such as aspergillosis and candidiasis.

[0035] For a medicine containing pseudoiodinine or a salt thereof as an active ingredient, the administration method, dosage form, dosage, etc. can be appropriately determined according to the purpose of use. The dosage form of the medicine is not particularly limited, and examples include external preparations such as solutions, lotions, emulsions, ointments, and creams, oral preparations such as tablets, solutions, and powders, and injections. The above-mentioned medicine is manufactured by a known production method generally used in the pharmaceutical field. Additives such as excipients, binders, disintegrants, lubricants, surfactants, sweeteners, suspending agents, emulsifiers, preservatives, colorants, fragrances, flavoring agents, thickeners, and stabilizers usually used in the pharmaceutical field can be appropriately formulated in the above-mentioned medicine as needed. For example, to prepare an external preparation, pseudoiodinine can be dissolved or dispersed in a base to obtain a desired dosage form.

[0036] In addition, the above-mentioned pseudoiodinine also exhibits strong growth inhibitory activity against pathogenic bacteria causing infectious diseases in fish and shellfish. Pseudoiodinine is useful as a therapeutic or prophylactic agent for diseases in fish and shellfish (e.g., antibacterial agents for aquatic products). Note that Ampicillin and erythromycin are known as antibacterial agents for aquatic products.

[0037] In the present invention, the fishery products are preferably cultured fishery products, fishery products bred in aquariums, ornamental fish, and tropical fish. For example, cultured fishery products include fish such as eel, ayu, yellowtail, trout, sea bream, carp, amberjack, tuna, salmon, horse mackerel, flounder, tilapia, pufferfish, hamachi, sea bass, mackerel, saury, catfish, and shark; crustaceans such as crabs and shrimp; cephalopods such as octopus; shellfish such as abalone; and sea cucumbers. Ornamental fish and tropical fish include goldfish, medaka, tetra, betta, and arowana.

[0038] Examples of fishery product diseases include Vibrio disease, columnaris disease, atypical Aeromonas salmonicida infection, Aeromonas hydrophila infection, Edwardsiella disease, red spot disease, Pseudomonas disease of ayu, redmouth disease, bacterial gill disease, columnaris disease (erosion of the fins, tail, and mouth), cold water disease, gliding bacteria disease, bacterial kidney disease, mycobacteriosis, nocardiosis, and streptococcal disease. These diseases are caused by percutaneous infection and are particularly problematic in fishery products bred in a closed environment.

[0039] When pseudoiodinine is used as an antibacterial agent for fisheries for the treatment or prevention of fishery product diseases, the dosage form for administration to fishery products is not particularly limited. For example, oral administration, injection, medicated bath (immersion), percutaneous administration, etc. may be mentioned. Pseudoiodinine is administered in a state suitable for the dosage form, and it may be administered as it is, or formulated into a predetermined dosage form for administration. When formulating, various additives used in the fisheries field can be appropriately blended.

[0040] In the case of oral administration, for example, pseudoiodinine alone or its formulation may be mixed with the feed of fishery products and administered. Other additives used in the fisheries field (for example, other antibacterial agents, etc.) may be added to the feed.

[0041] In the case of medicated bath (immersion), pseudoiodinine can be administered into the breeding water so as to reach the desired concentration. In the case of transdermal administration, for example, pseudoiodinine alone or its preparation may be applied to the epidermis of fish and shellfish, or microorganisms that produce pseudoiodinine may be made to colonize the epidermis of fish and shellfish.

[0042] The epidermis of fish and shellfish and the bacterial flora present therein function as a barrier that blocks the invasion of pathogenic bacteria and prevents bacterial infection. However, in an aquaculture environment or the like, skin damage may cause transdermal infection. In contrast, it can be said that performing medicated bath (immersion) or transdermal administration using an antibacterial agent for aquatic products makes it easier to effectively prevent transdermal infection. The epidermis of fish and shellfish includes the surfaces of the body, eyes, scales, fins, gills, mouth, etc. of fish, and the surfaces of the bodies of crustaceans such as shrimp.

Examples

[0043] [Test 1: Acquisition of pseudoiodinine] Pseudoiodinine was separated and collected by an extraction method. First, bacteria that produce pseudoiodinine were cultured, and the culture solution was centrifuged multiple times at 10,000×g for 10 minutes to remove the bacteria, and then sterilized with a sterilizing filter to obtain a culture supernatant.

[0044] Incidentally, it was found that this culture supernatant has antibacterial activity against Y. ruckeri, as shown in Fig. 1. Fig. 1 shows the results of performing the disk diffusion method after concentrating the culture supernatant. As the experimental method, a 0.5% soft agar medium containing the test bacterium was added to a 1.5% agar medium suitable for the test bacterium, a paper disk was placed, and as the test sample, a concentrate of the culture supernatant (obtained by concentrating 1 mL of the culture supernatant to 30 μL) was impregnated. As a positive control, 30 μL of a 200 μg / mL Ampicillin solution (Amp) was used, and as a negative control, a concentrate of NB2 medium (obtained by concentrating 1 mL of NB2 medium to 30 μL) was used. The plate was cultured overnight at a culture temperature suitable for the test bacterium, and the antibacterial activity was judged by the presence or absence of an inhibition zone. Incidentally, the test bacterium was suspended in the soft agar medium so that the final cell concentration was about Optical Density at 600 nm (OD 600 ) = 0.01.

[0045] Pseudoiodinine was purified from the culture supernatant obtained from the culture broth by the process shown in Fig. 2. First, the culture supernatant was extracted with hexane to remove lipophilic substances, and then extracted multiple times with ethyl acetate. The crude extract was roughly purified by silica gel chromatography using ethyl acetate and methanol, and then fractionally purified by high performance liquid chromatography (HPLC). It was subjected to the following HPLC conditions, and a peak with a retention time of about 6.1 minutes (reddish purple fraction) was fractionated.

[0046] <HPLC conditions> Analysis column: Reverse phase C18 column (particle size 5 μm, 4.6 mm × 150 mm) Column temperature: 40 °C Mobile phase: 50% by volume methanol Flow rate: 0.6 mL / min Detection wavelength: 220 nm

[0047] The obtained compound was subjected to mass spectrometry, 1 1H-NMR, and X-ray crystal structure analysis for structure determination, and was identified as pseudoiodinine (see the above formula (1)). Mass spectrometry, and 1The results of 1H-NMR are shown below. 1 For 1H-NMR, tetramethylsilane (TMS) was used as the internal standard, and deuterochloroform (CDCl3) was used as the solvent. The measurement results of the chemical shift were shown as δ values in ppm. The abbreviation s means singlet. LC / ESI-MS m / z: 166 [M+H] + 1 1H-NMR (CDCl3, 500 MHz) δ: 4.325 (3H, s), 4.444 (3H, s), 8.961 (1H, s)

[0048] For the crystallization of X-ray crystal structure analysis, the vapor diffusion method was adopted. Chloroform was used as the good solvent, and hexane was used as the poor solvent. As the procedure, first, about 0.8 mg of the purified sample was dissolved in about 300 μL of chloroform and put into a glass tube. An appropriate amount of hexane was put into a 50 mL sample bottle, the glass tube containing the sample was put in, the bottle was sealed, and it was left still at room temperature for several days while shielding from light. Using the resulting crystals, analysis was performed with a Rigaku XtaLAB P200 diffractometer to determine the structure.

[0049] [Test 2: Measurement of the MIC (minimum inhibitory concentration) of pseudoiodinine] MIC measurement was performed using pseudoiodinine. For the test, as the pathogenic bacteria of diseases in fishery products, Y. ruckeri (NVH3758), V. anguillarum, V. ordalii (NRIA90), E. coli (NBRC106373), A. hydrophila (ATCC700183), E. tarda (NRIA51), P. mosselii were used. As general bacteria, B. subtilis (isw1214), S. iniae (NRIA599) were used. As yeast, S. cerevisiae (Y2HGOLD) was used. Note that A. hydrophila (ATCC700183) is a strain resistant to Ampicillin.

[0050] The culture solution obtained by culturing these bacteria overnight was inoculated at 1 / 100 into 5 mL of a new medium. OD600 Adjust the culture medium to OD = 0.1 - 0.9 600 Adjust it to OD = 0.001, mix 98 μL of the bacterial solution and pseudoiodinine adjusted with a two-fold dilution series in a 96-well plate. Incubate statically at a predetermined temperature, and set the minimum concentration at which turbidity was not confirmed when the negative control of only the bacterial solution became turbid due to bacterial growth as the MIC. For the positive control, Ampicillin (final concentration 200 μg / mL) was used for bacteria and Hygromycin (final concentration 100 μg / mL) was used for yeast.

[0051] Regarding the culture media used, for V. anguillarum, V. ordalii, A. hydrophila, and E. tarda, Nutrient broth No.2 (NB2) medium (10 g / L peptone, 10 g / L beef extract, 5 g / L sodium chloride) was used; for S. iniae, Trypticase soy broth (TSB) medium (Becton and Dickinson (BD) Company, 211825) was used; for yeast, PYAD medium (20 g / L peptone, 20 g / L glucose, 10 g / L yeast extract, 40 mg / L adenine sulfate) was used; and for other bacteria, Mueller Hinton broth (MHB) medium (17.5 g / L casamino acids, 2 g / L beef extract, 1.5 g / L starch) was used. The results of the MIC measurement are shown in Table 1.

[0052]

Table 1

[0053] As shown in Table 1, it was confirmed that pseudoiodinine has antibacterial activity against many types of microorganisms. Also, pseudoiodinine is effective against Ampicillin-resistant strains and is useful as a novel antibacterial agent.

[0054] [Test 3: Antibacterial activity test of pseudoiodinine by the disk diffusion method] The disk diffusion method was performed in the same manner as the above-described procedure. B. subtilis (isw1214), Candida cylindracea (SL1B2), and S. cerevisiae (Y2HGOLD) were used in the test. The results are shown in Fig. 3.

[0055] As shown in Fig. 3, a inhibition circle was formed around the disk of pseudoiodinine, and it was confirmed that it has antibacterial activity.

[0056] [Test 4: Time-course change of antibacterial activity of pseudoiodinine] Next, in order to verify how pseudoiodinine inhibits the growth of pathogenic bacteria under aerobic conditions, the growth curves of pathogenic bacteria when pseudoiodinine was added were measured. Y. ruckeri, A. hydrophila, E. tarda, V. ordalii, and V. anguillarum were used as pathogenic bacteria in the test.

[0057] The above-mentioned pathogenic bacteria were pre-cultured for 24 hours. For pre-culture, Y. ruckeri was shake-cultured at 20 °C using NB2 medium and TSB medium. For A. hydrophila, E. tarda, V. ordalii, and V. anguillarum, they were shake-cultured at 28 °C using NB2 medium and TSB medium. 20 mL of a new medium was added to a 100 mL flask, and the pre-culture solution was inoculated so that OD 600 = 0.001. Pseudoiodinine was added so that its concentration became 8 μg / mL. Approximately 150 μL of the culture solution was sampled every 0, 12, 24, 36, and 48 hours from the start of the culture, and OD 600 was measured. The results are shown in Figs. 4 and 5.

[0058] As shown in Figs. 4 and 5, it was confirmed that when pseudoiodinine was added, the growth was significantly suppressed compared to the case where it was not added. Therefore, it was shown that pseudoiodinine has antibacterial activity even under aerobic conditions.

Industrial Applicability

[0059] Pseudoiodinine can be suitably used as an antibacterial agent or a medicine because it strongly inhibits the growth of a wide range of bacteria.

Claims

1. An antibacterial agent characterized by containing pseudiodinine or a salt thereof as an active ingredient.

2. The antibacterial agent according to Claim 1, which has antibacterial activity against at least one bacterium selected from the group consisting of Yersinia, Vibrio, Escherichia, Aeromonas, Edwardsiella, Bacillus, and Streptococcus.

3. The antibacterial agent according to Claim 1, which has antibacterial activity against at least one bacterium selected from the group consisting of Yersinia, Vibrio, Aeromonas, Edwardsiella, and Streptococcus.

4. The antibacterial agent according to Claim 2 or Claim 3, wherein the minimum growth inhibitory concentration against the bacterium is 1 mg / ml or less.

5. The antibacterial agent according to Claim 1 or Claim 2, which is an antibacterial agent for aquatic products for the treatment or prevention of diseases in fish and shellfish.

6. A pharmaceutical characterized by containing pseudiodinine or a salt thereof as an active ingredient.

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