JPH0645543B2 - Preventive and therapeutic agents for fish diseases - Google Patents

Description

Detailed Description of the Invention Technical Field This invention relates to a preventive and therapeutic agent for fish diseases (fish diseases), and more specifically to a compound represented by the following general formula (I): (wherein R represents an alkanoyl group; a benzoyl group which may be substituted with one or more substituents selected from lower alkyl and halogen; a phenylacetyl group; or a cyclohexanecarbonyl group) as an active ingredient.

BACKGROUND ART In recent years, the cultivation of fish (both saltwater and freshwater) has become popular, and this has led to the outbreak of various diseases. To prevent and treat these fish diseases, for example, in the case of pseudotuberculosis in yellowtail, antibacterial substances such as ampicillin and oxolinic acid are commonly used. However, the frequent use of these antibacterial substances has also led to the problem of an increase in pathogenic bacteria that have acquired resistance to the antibacterial substances used.

Therefore, there is a demand for new antibacterial agents that are free from cross-resistance with existing drugs and are effective against resistant bacteria as preventive and therapeutic agents for fish diseases.

In order to solve this problem, the inventors have developed bicozamycin [WS-
The same substance as 4545 substance (see JP 48-29158)
However, there were some difficulties with intestinal absorption.

On the other hand, an ester of bicozamycin (the same substance as the acylated WS-4545 substance (see Japanese Patent Application Laid-Open No. 1973-39497)) synthesized to improve the oral absorption of bicozamycin improved its oral absorption (see The Journal of Antibiotics, Vol. 25, No. 10, pp. 576-581). However, when administered to fish, particularly yellowtail, it is often mixed with live feed (minced fish). This could lead to the esterases present in the minced fish breaking down the bicozamycin ester back into bicozamycin, which could defeat the purpose of improving oral absorption when administered to yellowtail. (In fact, as will be described later, the ester of bacampicin, an ester of ampicillin (one of the existing translations), is broken down in the minced fish before being administered to fish.) For this reason, there was some hesitation in using the ester of bicozamycin in fish.

Under these circumstances, the present inventors have attempted an experiment in which bicozamycin esters were administered to fish. Among these esters, a specific bicozamycin ester [i.e., the above-mentioned compound (I)] was actually mixed into minced fish and administered to the fish.
Contrary to the above expectations, the inventors discovered that bicozamycin was hardly decomposed in minced fish, was well absorbed by fish, and maintained at a high concentration in the blood of administered fish for a long period of time. Based on this discovery, the inventors continued their research and completed the present invention.

DISCLOSURE OF THE INVENTION The present invention has been provided to solve the above problems and is a preventive and therapeutic agent for fish diseases, which contains the above compound (I) as an active ingredient.

The compound (I) used in the present invention is known as described above. Bicozamycin, which is a raw material for producing the compound (I), is grown in Streptomyces sapporonensis ATCC2 as described in the above-mentioned Japanese Patent Publication No. 48-29158.
1532 is known as an antibiotic produced by the bacteria.

The deposit status of the producing strain is as follows:

Depositary: American Type Culture Collection Address: 12301 Park Lawn Drive, Rockville, Maryland, USA Date of Deposition: April 21, 1970 Accession Number: ATCC 21532 Preferred examples of the alkanoyl group in Compound (I) include acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, octanoyl, palmitoyl, etc., and examples of the lower alkyl group include methyl,
Examples of the halogen include chlorine, bromine, and iodine.

Representative examples of the compounds (I) used in the present invention are as follows:

Examples of fish and diseases to which the preventive and therapeutic agent of the present invention is usually applied include pseudotuberculosis (Pasteurella piscicida) in yellowtail (Yellowtail) and horse mackerel, Vibriosis (Vibrio angularum) in yellowtail, sweetfish and sea bream, Edwardsiella infection (Edwardsiella tarda) in eel, flounder and sea bream, and Hessow's disease (Aeromonas salmonicida) in rainbow trout.

The preventive and therapeutic agent for fish diseases of this invention can be prepared by diluting Compound (I) with or without dilution with a solid, semi-solid or liquid carrier or diluent in the form of a powder, dust, fine granules, granules, trickle, tablet, liquid, pellet or syrup, or by adding Compound (I) directly or in the form of any of the above-mentioned various dosage forms to fish feed including compound feed.

Specific examples of the carrier include live bait such as minced fish (for example, minced mackerel, sardines, sand lance, giant eel, Pacific saury, pollock, squid, etc.), fish meal, soybean meal, yeast, wheat flour,
Examples of commonly used feed include compound feed containing vitamins as the main ingredient, lactose, sucrose, glucose, starch, talc, and acid clay.

Test example 1 (change in blood concentration in yellowtail): The drug was mixed with minced mackerel and given to yellowtail at a dose of 50 mg/
The dose was 1 kg and administered once.
Blood was collected from the heart at 6, 48, and 72 hours.

The amount of bicozamycin in the blood was measured by a microbiological assay using E. coli BS-10 as the test bacterium.

The results are shown in the following table.

Test Example 2 (Stability in Various Enzyme Solutions 1) Each drug was dissolved in water to a concentration of 2000 μg/ml.

0.1 ml of this solution is added to 0.9 ml of each enzyme solution.

The methods for preparing the various enzyme solutions are as follows.

Ion-exchanged water: used as an enzyme-free control.

Yellowtail plasma: Blood was collected from the heart of live farmed yellowtail using a heparinized syringe, and the collected blood was centrifuged at 3000 rpm for 15 minutes to separate the plasma.

Yellowtail liver homogenate: The liver of live farmed yellowtail was removed, crushed, and homogenized.

Yellowtail intestinal tract homogenate: The intestines of live farmed yellowtail were removed, crushed, and then homogenized.

Porcine esterase: Commercially available esterase (Sigma) was diluted 50 times with water and used.

Mackerel mince: Fresh mackerel was purchased, the whole fish was crushed, and minced in a meat grinder. Two times the amount of water was added to this, and
The mixture was homogenized and centrifuged at 3000 rpm for 15 minutes, and the supernatant was used as the enzyme solution.

Minced sardines: Prepared in the same manner as minced mackerel.

Minced saury: Prepared in the same manner as minced mackerel.

The mixture of the drug solution and enzyme solution is reacted at 25°C for 2 hours.

To stop the enzymatic reaction, 1.0 ml of methanol is added.

After gentle stirring, the mixture is centrifuged at 6400 rpm for 5 minutes, the supernatant is soaked into a paper disc, and the concentrations of ampicillin, cefteram piboxyl parent compound, and bicozamycin are determined by a microbiological assay using E. coli BS-10 as the test strain.

The results are shown in the table below. The values in the table indicate the ratio of bacampicillin to ampyrin, and cefteram piboxyl to its parent compound (Japanese Journal of Chemotherapy, Vol. 34, S-2, P44-6).
0, 1986) and for the compounds used in this invention, the respective conversion rates (%) to bicozamycin are shown.

Test Example 3 (Stability in Various Enzyme Solutions 2): Each drug was dissolved in water to a concentration of 1000 μg/ml.

0.04 ml of this drug solution is added to 0.36 ml of each enzyme solution.

The methods for preparing the various enzyme solutions are as follows.

Ion-exchanged water: used as an enzyme-free control.

Porcine esterase: Commercially available esterase (Sigma) was diluted 50 times with water and used.

Minced raw bait: Fresh mackerel and sardines were purchased, and equal amounts of whole fish were crushed and mixed together, then minced in a meat grinder. Two volumes of water were added to this, and the mixture was homogenized and centrifuged at 3000 rpm for 15 minutes. The supernatant was used as the enzyme solution.

Yellowtail serum: Blood was collected from the heart of a live farmed yellowtail using a syringe. The collected blood was left at room temperature for 1 hour and then heated at 3000 rpm.
The serum was separated by centrifugation at RT for 15 min.

Rainbow trout plasma: Blood was collected from the heart of live farmed rainbow trout using a heparinized syringe, and the collected blood was centrifuged at 3000 rpm for 15 minutes to separate the plasma.

The mixture of the drug solution and enzyme solution is reacted at 30°C for 3 hours.

To stop the enzymatic reaction, 0.4 ml of methanol is added.

After gentle stirring, the mixture is centrifuged at 6400 rpm for 5 minutes, the supernatant is soaked into a paper disc, and the bicozamycin concentration is determined by a microbiological assay using E. coli BS-10 as the test strain.

The results are shown in the table below, and the values in the table indicate the conversion rate (%) of the compound of the present invention to bicozamycin.

Test Example 4 (Comparison with existing drugs) Yellowtail weighing approximately 200g infected with pseudotuberculosis in the field were placed in one group.
The fish were divided into 7 groups, with group I being the non-medicated control group, group II being the group treated with 20 mg/kg ampicillin, and group III being the group treated with 30 mg/kg oxolinic acid.
Group IV was administered 20 mg/kg of Compound 1, Group V was administered 40 mg/kg of Compound 1, and Group VI was administered 20 mg/kg of Compound 1.
Group VII was administered 20 mg/kg, and Group VII was administered 40 mg/kg of Compound 2. After measuring the total weight of each group and fasting for one day, a prescribed amount of the drug (10% lactose formulation) was mixed with yellowtail formula feed and mixed with minced giant melon, and administered once daily for five consecutive days. Dead fish were collected every morning and the number recorded. All dead fish were dissected to confirm that they had died from pseudotuberculosis.

Water temperature was measured and recorded every morning.

The results are shown in the following table.

Test Example 5 (Comparison with bicozamycin) Yellowtail weighing approximately 200 g and infected with pseudotuberculosis in the field were placed in a group of 3
The fish were divided into four groups of 00 to 320 fish, with Group I being the non-medicated control group and Group II
Group III was administered 20 mg/kg of bicozamycin, Group II was administered 20 mg/kg of Compound 1, and Group IV was administered 20 mg/kg of Compound 2. The total weight of each group was measured and the animals were fasted for one day, after which the animals were weighed.
A prescribed amount of the drug (10% lactose preparation) was mixed with yellowtail compound feed and mixed with minced giant lance.
The drugs were administered for 3 days in a row. Dead fish were collected every morning.
The number of dead fish was recorded, and all dead fish were dissected to confirm that they had died from pseudotuberculosis.

Water temperature was measured and recorded every morning.

The results are shown in the following table.

Example 1 6.7 parts by weight of Compound 1 (5 parts by weight in terms of bicozamycin potency) and 93.3 parts by weight of lactose were mixed to form a powder.

By mixing this 100g daily dose with 200kg of minced live bait made from giant eel and administering it to a total of 1 ton of yellowtail, each with an average fish weight of 50g, for five days, the purpose of preventing and treating pseudotuberculosis in yellowtail can be achieved.

Example 2 6.7 parts by weight of Compound 1 (5 parts by weight in terms of bicozamycin potency) and 93.3 parts by weight of lactose are mixed to form a powder.

By administering a daily dose of moist pellets containing 300g of this powder (cylindrical pellets made by mixing raw minced sardines and powdered feed mainly consisting of fish meal in a 6:4 ratio and then processing them in a pelleting machine) for five days to a total of 3 tonnes of yellowtail, consisting of approximately 15,000 fish with an average weight of 200g, the goal of preventing and treating pseudotuberculosis in yellowtail can be achieved.

INDUSTRIAL APPLICABILITY This invention is configured as described above and is useful for preventing and treating fish diseases.
By using it as a therapeutic agent, particularly for treating fish diseases, it can increase fish yields.

Claims (14)

[Claims] [Claim 1] General formula (wherein R represents an alkanoyl group; a benzoyl group which may be substituted with one or more substituents selected from lower alkyl and halogen; a phenylacetyl group or a cyclohexanecarbonyl group) and a live bait. [Claim 2] General formula (wherein R represents an alkanoyl group; a benzoyl group which may be substituted with one or more substituents selected from lower alkyl and halogen; a phenylacetyl group or a cyclohexanecarbonyl group) and a live bait. [Claim 3] General formula (wherein R represents an alkanoyl group; a benzoyl group which may be substituted with one or more substituents selected from lower alkyl and halogen; a phenylacetyl group or a cyclohexanecarbonyl group) and a live bait. 4. The preventive and therapeutic agent for fish diseases according to claim 1, wherein the fish disease is pseudotuberculosis of yellowtail. 5. The method for preventing and treating fish diseases according to claim 2, wherein the live bait is minced fish. 6. The method for preventing and treating a fish disease according to claim 5, wherein the fish disease is pseudotuberculosis of yellowtail. 7. The agent for preventing and treating fish diseases according to claim 1 or 4, wherein R is a benzoyl group or a cyclohexanecarbonyl group. 8. The method for preventing or treating a fish disease according to claim 2, 5 or 6, wherein R is a benzoyl group or a cyclohexanecarbonyl group. 9. The method for preventing and treating fish diseases according to claim 2, wherein the feed is a mixture of minced fish and compound feed. 10. The method for preventing and treating a fish disease according to claim 9, wherein the fish disease is pseudotuberculosis of yellowtail. 11. The method for preventing and treating fish diseases according to claim 10, wherein R is a benzoyl group or a cyclohexanecarbonyl group. 12. A method for preventing and treating fish diseases according to claim 2, wherein the feed is moist pellets made from a mixture of minced fish and compound feed. 13. The fish feed according to claim 3, wherein the feed is moist pellets made from a mixture of minced fish and compound feed. 14. The fish feed according to claim 13, wherein R is a benzoyl group or a cyclohexanecarbonyl group.