Composition for treating streptococcosis in fish containing crystalline ceftiofur free acid

Abstract

A composition for treating streptococcal disease in fish is disclosed, which has significantly improved antibacterial activity against causative agents of streptococcal disease such as Streptococcus iniae and Streptococcus parauberis. The present invention provides a composition for treating streptococcal disease in fish comprising crystalline ceftiofur free acid as an active ingredient.

Description

Technology Field

[0001] The present invention relates to a composition for treating streptococcal disease in fish, and more specifically, to a composition for treating streptococcal disease in fish that has excellent antibacterial effects against causative bacteria causing streptococcal disease in fish. Background Technology

[0002] Major bacterial diseases occurring in domestically farmed fish include primarily streptococcosis, gliding bacteriosis, Edwardsiella disease, vibriosis, and intestinal leukocytosis; in particular, streptococcosis affects the flounder, a major domestic farmed species ( Paralichthys olivaceus Economic losses are continuously being reported due to frequent occurrences in ) and rockfish.

[0003] Streptococcus iniae, one of the bacteria that cause streptococcal infection Streptococcus iniae Fish infected with ) exhibit symptoms such as abnormal body color, cloudy eyes, hemorrhage, abdominal distension, and kidney enlargement, and in severe cases, it leads to death. An average of over 220 tons of aquatic antibiotics are used annually to treat these bacterial diseases, and among them, oxytetracycline, oxolinic acid, and erythromycin are widely used; however, there is still a need for the development of substances that exhibit excellent antibacterial effects against the causative agents of streptococcal disease.

[0004] Meanwhile, crystalline ceftiofur free acid (CCFA) is a long-acting formulation of ceftiofur, a third-generation cephalosporin antibiotic, and is an antibiotic that exhibits antibacterial effects against Gram-positive and Gram-negative bacteria and is used to prevent porcine respiratory disease (SRD). The problem to be solved

[0005] The present invention relates to Streptococcus iniae ( Streptococcus iniae ), Streptococcus parauberis( Streptococcus parauberisWe aim to provide a composition for treating streptococcal disease in fish that has significantly improved antibacterial activity against causative agents of streptococcal disease such as ). means of solving the problem

[0006] To solve the above problem, the present invention provides a composition for treating streptococcal disease in fish comprising crystalline ceftiofur free acid as an active ingredient.

[0007] In addition, the causative agent of the above-mentioned streptococcal infection is Streptococcus iniae ( Streptococcus iniae ) or Streptococcus parauberis( Streptococcus parauberis A composition for treating streptococcal disease in fish is provided, characterized by being ).

[0008] In addition, the above composition provides a composition for treating streptococcal disease in fish, characterized by further including a pharmaceutically acceptable excipient.

[0009] In addition, the present invention provides a composition for treating streptococcal disease in fish, characterized in that the excipient is one or more selected from the group consisting of butylhydroxytoluene, polysorbate, benzyl alcohol, migliol 840, ethanol (EtOH), cottonseed oil, benzyl alcohol, dimethyl sulfoxide (DMSO), medium-chain triglycerides, methyl parahydroxybenzoate, dicaprylate, caprylic / capric triglycerides, span 80, propylene glycol dicaprylate, and triethyl citrate.

[0010] In addition, the above excipient comprises butylhydroxytoluene, polysorbate, benzyl alcohol, and Migriol 840, thereby providing a composition for treating streptococcal disease in fish.

[0011] In addition, the above composition provides a composition for treating streptococcal disease in fish, characterized by comprising 0.01 to 1 weight part of butylhydroxytoluene, 0.05 to 5 weight parts of polysorbate, 5 to 100 weight parts of benzyl alcohol, and 500 to 1,200 weight parts of migriol 840, based on 100 weight parts of crystalline ceftiofer free acid.

[0012] In addition, a composition for treating streptococcal disease in fish is provided, characterized in that the minimum inhibitory concentration (MIC) of the crystalline ceftiofer free acid against the causative agent of the streptococcal disease is 0.031 μg / mL or less.

[0013] In addition, a composition for treating streptococcal disease in fish is provided, characterized in that the fish is a flounder.

[0014] In addition, the above composition is formulated as an injectable, and the dosage administered per single prescription is 5 to 50 mg of the ceftiofer free acid per 1 kg of body weight, thereby providing a composition for treating streptococcal disease in fish. Effects of the invention

[0015] The composition for treating streptococcal disease in fish according to the present invention comprises crystalline ceftiofur free acid as an active ingredient and Streptococcus iniae ( Streptococcus iniae ), Streptococcus parauberis( Streptococcus parauberis The antibacterial activity against causative agents of streptococcal disease, such as ), can be dramatically improved. Brief explanation of the drawing

[0016] Figure 1 is a spectrum showing the XRD analysis results of the crystalline ceftiofur free acid used in the example. Figure 2 is Streptococcus iniae This is a well photograph illustrating the determination of the MIC concentration of crystalline ceftiofer free acid. Specific details for implementing the invention

[0017] The present invention will be described in detail below through examples. Prior to this, terms and words used in this specification and claims should not be interpreted as being limited to their ordinary or dictionary meanings. Instead, based on the principle that the inventor may appropriately define the concepts of terms to best describe their invention, they should be interpreted in a meaning and concept consistent with the technical spirit of the present invention. Therefore, the configurations of the examples described in this specification are merely the most preferred embodiments of the present invention and do not represent all aspects of the technical spirit of the present invention; thus, it should be understood that various equivalents and modifications capable of replacing them may exist at the time of filing this application.

[0019] The inventors, in a composition for treating streptococcal disease in fish, adapt crystalline ceftiofur free acid (CCFA), conventionally used for the prevention of porcine respiratory disease (SRD), to Streptococcus iniae ( Streptococcus iniae ), Streptococcus parauberis( Streptococcus parauberis It was confirmed that when treated with causative bacteria of streptococcal disease such as ), the antibacterial effect against the causative bacteria is excellent, showing a dramatic effect in treating streptococcal disease in fish, leading to the present invention.

[0020] Accordingly, the present invention provides a composition for treating streptococcal disease in fish comprising crystalline ceftiofur free acid as an active ingredient.

[0021] According to an application example of the present invention, the composition is Streptococcus iniae ( Streptococcus iniae ) or Streptococcus parauberis( Streptococcus parauberis Suitable for treating streptococcal infections caused by ) and Streptococcus iniae ( Streptococcus iniae It is particularly suitable for treating streptococcal infections caused by )

[0022] Ceftiofur is a cephalosporin antibiotic represented by the following chemical formula 1. Like other cephalosporin antibiotics, it exhibits a bactericidal effect by interfering with peptidoglycan synthesis through binding to and inactivating penicillin-binding protein (PBP) present in the inner membrane of bacterial cell walls. In the present invention, it was confirmed that when used in the form of crystalline ceftiofur free acid to treat streptococcal disease in fish, it exhibits a significantly improved antibacterial effect compared to when used in the form of salts such as ceftiofur sodium.

[0023] [Chemical Formula 1]

[0024]

[0025] Ceftiofer free acid is classified into crystalline ceftiofer free acid and amorphous ceftiofer free acid, and the crystalline ceftiofer free acid in the present invention can be identified, for example, by an XRD spectrum having a sharp peak through XRD (X-ray Diffraction) analysis.

[0026] The composition for treating streptococcal disease in fish according to the present invention, even with the crystalline ceftiofer free acid alone (appropriately diluted), Streptococcus iniae, the causative agent of streptococcal disease ( Streptococcus iniae ), Streptococcus parauberry S ( Streptococcus parauberis In addition to exhibiting enhanced antibacterial effects against ) etc., it also exhibits enhanced antibacterial effects against causative agents of streptococcal disease in the form of a suspension containing crystalline ceftiofur free acid and additionally pharmaceutically acceptable excipients.

[0027] The above excipients are not specifically limited as long as they are pharmaceutically acceptable for the safety of fish and the effective delivery of the drug, and serve to improve the solubility, stability, bioavailability, osmotic pressure regulation, and suitability for application as an injectable of the active ingredient. Examples include butylhydroxytoluene, polysorbate, benzyl alcohol, migriol 840, ethanol (EtOH), cottonseed oil, benzyl alcohol, dimethyl sulfoxide (DMSO), medium-chain triglycerides, methyl parahydroxybenzoate, dicaprylate, caprylic / capric triglycerides, Span 80, propylene glycol dicaprylate, triethyl citrate, etc., which may be used alone or in combination. In this case, when used in the form of an injectable, water for injection or a buffer solution may be used.

[0028] These excipients help the active ingredients to function stably within the body without causing adverse effects on the physiology of fish. In the present invention, through empirical experiments on Streptococcus iniae and Streptococcus parauberis, which are causative agents of fish streptococcal disease, mixed with crystalline ceftiofer free acid, it was confirmed that an excellent antibacterial effect is achieved when butylhydroxytoluene, polysorbate, benzyl alcohol, and migriol 840 are included. In this regard, it is preferable that the content of the excipient components comprises 0.01 to 1 weight part of butylhydroxytoluene, 0.05 to 5 weight parts of polysorbate, 5 to 100 weight parts of benzyl alcohol, and 500 to 1,200 weight parts of migriol 840 per 100 weight parts of the crystalline ceftiofer free acid; more preferably, it may comprise 0.05 to 0.5 weight parts of butylhydroxytoluene, 0.1 to 2 weight parts of polysorbate, 10 to 60 weight parts of benzyl alcohol, and 600 to 1,100 weight parts of migriol 840 per 100 weight parts of the crystalline ceftiofer free acid; and even more preferably, 0.05 to It may contain 0.3 parts by weight, 0.1 to 1 part by weight of the polysorbate, 10 to 50 parts by weight of the benzyl alcohol, and 700 to 1,000 parts by weight of the migriol 840, and most preferably, with respect to 100 parts by weight of the crystalline ceftiofer free acid, it may contain 0.05 to 0.2 parts by weight of the butylhydroxytoluene, 0.2 to 1 part by weight of the polysorbate, 20 to 50 parts by weight of the benzyl alcohol, and 800 to 900 parts by weight of the migriol 840.

[0029] The composition for treating streptococcal disease in fish according to the present invention comprises crystalline ceftiofur free acid as an active ingredient, thereby treating Streptococcus iniae ( Streptococcus iniae ), Streptococcus parauberis( Streptococcus parauberis The antibacterial activity against causative agents of streptococcal disease, such as ) is significantly improved. Specifically, the minimum inhibitory concentration (MIC) of the crystalline ceftiofer free acid against the causative agents of streptococcal disease may be 0.031 μg / mL or less, and according to one embodiment, may be 0.0038 to 0.031 μg / mL.

[0030] Fish to which the composition for treating streptococcal disease in fish according to the present invention can be applied include Streptococcus iniae ( Streptococcus iniae ), Streptococcus parauberis( Streptococcus parauberis While not specifically limited to fish exhibiting streptococcal disease caused by causative agents such as ), flounder ( Paralichthys olivaceus It can be suitably applied to the treatment of streptococcal infection in ) and rockfish, and flounder( Paralichthys olivaceus It can be particularly suitable for the treatment of streptococcal infection.

[0031] The composition for treating streptococcal disease in fish according to the present invention is the above Streptococcus iniae ( Streptococcus iniae ), Streptococcus parauberis( Streptococcus parauberis To maximize the therapeutic effect of streptococcal disease caused by causative agents such as ) and ), it is desirable to formulate it as an injectable, and in this case, the dosage administered per single prescription is preferably 5 to 50 mg of crystalline ceftiofur free acid per 1 kg of body weight, more preferably 5 to 30 mg, and even more preferably 10 to 20 mg. Here, when administering the composition for treating streptococcal disease in fish according to the present invention, the injectable form can be administered by a conventional intramuscular injection method.

[0032] The present invention will be described in detail below with reference to examples. In this example, the efficacy of the composition for treating fish streptococcal disease according to the present invention was evaluated by investigating the antibacterial and bactericidal effects against the causative agents of fish streptococcal disease for each suspension containing crystalline ceftiofer free acid and an excipient.

[0034] Materials and Methods

[0035] 1. Test substance

[0036] (1) Crystalline Ceftiofur Free Acid

[0037] - Crystalline Ceftiofur Free Acid (CCFA) manufactured by QIRU SYNVA PHARMACEUTICAL, which exhibits a crystallinity XRD spectrum with sharp peaks when analyzed by XRD (see Figure 1).

[0038] - Appearance and characteristics: White powder

[0039] - Purity: Analytical standard (98.7%)

[0040] (2) Ceftiofur injectable suspension

[0041] - Composition: 1 mL of this product contains 100 mg of crystalline ceftiofur free acid, 0.1 mg of butylhydroxytoluene, 0.4 mg of polysorbate, 30 mg of benzyl alcohol, 840 mg of migriol, 860 mg of water for injection.

[0042] - Formulation and Appearance: Injectable, pale yellow or yellow suspension

[0044] 2. Test Method

[0045] (1) Test standards

[0046] MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) tests were conducted in accordance with CLSI VET04 (CLSI. Performance standards for antimicrobial susceptibility testing for bacteria isolated from aquatic animals. VET04-Ed3. 2020) and CLSI M26 (CLSI. Methods for determining bactericidal activity of antimicrobial agents. M26-A. 1999).

[0047] (2) Test strain

[0048] The test strains used in the MIC and MBC tests are shown in Table 1 below.

[0049] Bacterial species Strains Separation source Source Streptococcus iniae ( S.iniae ) S.iniae 1 KCTC3657 Pisces KCTC S.iniae 2 FP5228 Pisces National Institute of Fisheries Science S.iniae 3 BA1300015 Other unclassified (fish) Animal and Plant Quarantine Agency S.iniae 4 BA0000163 Other unclassified (fish) Animal and Plant Quarantine Agency S.iniae 5 BA0000164 Other unclassified (fish) Animal and Plant Quarantine Agency S.iniae 6 BA0000165 Other unclassified (fish) Animal and Plant Quarantine Agency S.iniae 7 BA0000166 Other unclassified (fish) Animal and Plant Quarantine Agency MIC reference strain S. aureus ATCC 29213, Streptococcus pneumoniae ATCC 49619(Ceftiofur)

[0050] (3) MIC and MBC Test 1) Preparation and Dilution of Test Substance

[0051] In the case of crystalline ceftiofur free acid, 20 mg was weighed and diluted with DMSO to make a Stock Solution (10 mg / mL), which was then further diluted with sterile distilled water for use. In the case of ceftiofur suspension, 1 mL was diluted with DMSO to make a Stock Solution (crystalline ceftiofur free acid: 10 mg / mL), which was then further diluted with 50% DMSO for use.

[0052] The concentration groups for the test substance were configured as shown in Table 2 below, and the same medium as the inoculum of the bacterial strain was used as the diluent (refer to Table 3 below for the medium and culture conditions of each bacterium). Specifically, a sterile 96-well microplate (F bottom, SPL) was prepared in advance, the bacterial strain name and the test substance name were written on it, and the medium was dispensed into all wells except the first well. 0.1 mL of the test substance was dispensed into each well of the highest concentration group, and 0.05 mL of this was taken and serially diluted with the broth dispensed into the wells of the next concentration group at an azeotropic ratio of 2.

[0053] test substance menstruum dilution solvent Concentration range (μg / mL) Crystalline ceftioper free acid DMSO Sterile distilled water 0.00095~500 Sectioper Suspension DMSO 50% DMSO 0.00095~500 Note) - Ceftiofer Suspension Concentration: Based on Crystalline Ceftiofer Free Acid Concentration

[0054] strain name culture medium MIC culture conditions agar MIC Broth Streptococcus iniae 5% blood agar CAMHB+LHB (2.5% v / v) 35±2℃, 5±2% CO2, 16~20h Staphylococcus aureus 5% blood agar CAMHB 35±2℃, non-CO2, 16~20h Streptococcus pneumoniae 5% blood agar CAMHB+LHB (2.5% v / v) 35±2℃, 5±2% CO2, 20~24h Note) - CAMHB: Cation-adjusted Mueller-Hinton broth - LHB: lysed horse blood

[0055] 2) Preparation of bacterial suspension: The test strain was plated onto 5% sheep blood agar and incubated at 35±2°C in a 5% CO2 incubator for 18 to 24 hours. Subsequently, 4 to 6 single colonies were taken, inoculated into liquid medium, and incubated at 35±2°C in a 5% CO2 incubator for 6 hours. The turbidity of the cultured bacteria was measured using MacFarland No. 0.5 (OD). 625 Adjusted to =0.08~0.132, the bacterial count is approximately 1.5×10 8 After adjusting to CFU / mL, dilute 100 times with the diluent to approximately 1.5 × 10⁻⁶ 6 A CFU / mL inoculum solution was prepared.

[0056] 3) Inoculation and culture of bacterial suspension

[0057] 1.5 × 10⁻⁶ in each well, excluding the negative control well (12th well) 60.05 mL of each bacterial suspension adjusted to CFU / mL was dispensed and inoculated, then incubated for 20 hours in a 5% CO2 incubator at 35±2℃. Additionally, 0.01 mL was taken from the growth-control well, diluted in 10 mL of sterile physiological saline, and 0.1 mL of this solution was inoculated onto solid media for plating, after which the initial inoculated bacterial count was measured. The bacterial count of the inoculated suspension used for the MIC test was confirmed to be 9.3×10⁶. 5 Up to 9.6×10 5 It was CFU / mL.

[0058] 4) Determining MIC

[0059] For the MIC concentration, after 20 hours, the presence or absence of bacterial growth was visually observed, 20 μL of resazurin solution (0.5 mg / mL) was dispensed into all wells, and after incubating for an additional 4 hours at 35±2℃ and 5% CO2, the color change (change from blue to pink) was observed, and the concentration at which no color change was observed was determined as the MIC concentration.

[0060] 5) MBC Decision

[0061] After conducting the MIC test, 0.01 mL aliquots were taken three times from the MIC concentration well and from wells 5 to 6 with concentrations higher than the MIC concentration, inoculated onto solid media without the test substance, and cultured for 24 hours in a 5% CO2 incubator at 35±2℃. The concentration at which 99.9% of the initial inoculated bacteria were reduced was determined as the MBC concentration.

[0062] (4) Validation of MIC test

[0063] For QC (Quality Control) of MIC testing S. aureus ATCC 29213 (Ceftiofur QC MIC range: 0.25~1.0 μg / mL), S. pneumoniaeThe MIC values ​​of Ceftiofur for ATCC 49619 (Ceftiofur QC MIC range: 0.12~0.5 μg / mL) were 0.488 μg / mL and 0.244 μg / mL, respectively, which fall within the QC MIC range suggested by CLSI, so all MIC results were valid.

[0065] test

[0066] Streptococcus iniae The MIC and MBC test results for crystalline ceftiofur free acid (CCFA) and ceftiofur suspension are shown in Table 4 below (unit: μg / mL), and for example Streptococcus iniae Figure 2 shows a well photograph illustrating the determination of the MIC concentration of crystalline ceftiofer free acid.

[0067] division Ceftiofur Crystalline Free acid (CCFA) Sectioper Suspension MIC MBC MIC MBC S.iniae 1 KCTC3657 0.015 0.015 0.015 0.015 S.iniae 2 FP5228 0.031 0.031 0.031 0.031 S.iniae 3 BA1300015 0.0038 0.0038 0.031 0.0038 S.iniae 4 BA0000163 0.031 0.031 0.031 0.031 S.iniae 5 BA0000164 0.031 0.031 0.031 0.031 S.iniae 6 BA0000165 0.031 0.031 0.031 0.031 S.iniae 7 BA0000166 0.031 0.031 0.031 0.031 MIC 50 0.031 - 0.031 - MIC 90 0.031 - 0.031 - MBC 50 - 0.031 - 0.031 MBC 90 - 0.031 - 0.031 MBC 90 / MIC 90 1 1 Note) - Ceftiofer Suspension Concentration: Based on Crystalline Ceftiofer Free Acid Concentration

[0068] Referring to Table 4 and Figure 2, Streptococcus iniae The MIC ranges for crystalline ceftiofur free acid (CFAA) and ceftiofur suspension were both 0.0038 to 0.031 μg / mL, and MIC 50 Value and MIC 90 It can be confirmed that the value is the same at 0.031 μg / mL. Also Streptococcus iniae The bactericidal effects of crystalline ceftiofur free acid (CFAA) and ceftiofur suspension on [the bacteria] were equal to the MIC concentrations of each strain, and the minimum bactericidal concentration (MBC) 90 It can be confirmed that ) is 0.031 μg / mL.

[0069] Meanwhile, examining prior literature confirming the therapeutic effect of substances in the form of ceftiofur sodium on streptococcal infections, isolated from Jeju flounder farms Streptococcus spp. ( Streptococcus iniae 1 strain, Streptococcus parauberisThe MIC concentration range of ceftiofer for 28 strains was 0.03 to 4 μg / mL (Ye Ji Kim, Lyu Jin Jun, Da Won Lee, Young Juhn Lee, Ye Jin Ko, Yeong Eun Oh, Soo Ji Woo, Myoung Sug Kim, Seung Min Kim and Joon Bum Jeong. Antibiotic Susceptibility of Bacterial Pathogens That Infect Olive Flounder (Paralichthys olivaceus) Cultivated in Korea. Int. J. Environ. Res. Public Health. 2022. 1, 8110), Streptococcus paraueris The MIC range of ceftiofur for 21 strains was 0.0039 to 1 μg / mL (Naila Boby, Muhammad Aleem Abbas, Eon-Bee Lee, and Seung-Chun Park. Pharmacodynamics of Ceftiofur Selected by Genomic and Proteomic Approaches of Streptococcus parauberis Isolated from the Flounder, Paralichthys olivaceus. International Journal of Genomics. 2020. Article ID 4850290, 17 pages). Referring to this, the composition having crystalline ceftiofur free acid as an active ingredient according to the present invention is compared to when ceftiofur is used in salt form Streptococcus iniae It is confirmed that it has an excellent effect in treating streptococcal disease in fish, with an antibacterial effect of about 8 to 128 times.

[0071] The preferred embodiments of the present invention described above are disclosed to solve technical problems, and those skilled in the art to which the present invention belongs may make various modifications, changes, and additions within the spirit and scope of the present invention, and such modifications and changes should be considered to fall within the scope of the following claims.

Claims

Claim 1 A composition for treating streptococcal disease in fish comprising crystalline ceftiofur free acid as an active ingredient, wherein the causative agent of the streptococcal disease is Streptococcus iniae ( Streptococcus iniae ) and the above fish is a flounder ( Paralichthys olivaceus A composition for treating streptococcal disease in fish, characterized in that the minimum inhibitory concentration (MIC) of the crystalline ceftiofur free acid against the causative agent of the streptococcal disease is 0.0038 to 0.031 μg / mL. Claim 2 delete Claim 3 A composition for treating streptococcal disease in fish according to claim 1, characterized in that the composition further comprises a pharmaceutically acceptable excipient. Claim 4 A composition for treating streptococcal disease in fish according to claim 3, wherein the excipient is one or more selected from the group consisting of butylhydroxytoluene, polysorbate, benzyl alcohol, migliol 840, ethanol (EtOH), cottonseed oil, benzyl alcohol, dimethyl sulfoxide (DMSO), medium-chain triglycerides, methyl parahydroxybenzoate, dicaprylate, caprylic / capric triglycerides, span 80, propylene glycol dicaprylate, and triethyl citrate. Claim 5 A composition for treating streptococcal disease in fish according to claim 3, characterized in that the excipient comprises butylhydroxytoluene, polysorbate, benzyl alcohol, and Migriol 840. Claim 6 A composition for treating streptococcal disease in fish according to claim 5, characterized in that the composition comprises 0.01 to 1 weight part of butylhydroxytoluene, 0.05 to 5 weight parts of polysorbate, 5 to 100 weight parts of benzyl alcohol, and 500 to 1,200 weight parts of migriol 840, based on 100 weight parts of crystalline ceftiofer free acid. Claim 7 delete Claim 8 delete Claim 9 A composition for treating streptococcal disease in fish, characterized in that, in any one of claims 1 and 3 to 6, the composition is formulated as an injectable and the dosage administered per single prescription is 5 to 50 mg of the ceftiofur free acid per 1 kg of body weight.

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