KR20200038435A - Antibiotics composition for animals - Google Patents

Abstract

The present invention relates to a complex antibiotic composition for animals. More specifically, the present invention relates to a complex antibiotic composition for animals in which ceftiofur, neomycin, and dexamethasone are complexly prescribed with a weight ratio of 1 to 5 : 1 to 5 : 0.01 to 0.05, preferably 1 to 3 : 1 to 3 : 0.01 to 0.03, and especially a weight ratio of 1 : 1 : 0.01, thereby having an excellent effect on prevention and treatment of mastitis in animals compared to a single prescription.

Description

Compound antibiotic composition for animals {ANTIBIOTICS COMPOSITION FOR ANIMALS}

The present invention relates to a composite antibiotic composition for animals, more specifically, Ceftiofur, Neomycin and Dexamethasone in a weight ratio of 1-5: 1-5: 0.01-0.05. It relates to a composite antibiotic composition for animals having an excellent effect on the prevention and treatment of mastitis in animals compared to when prescribed alone.

Particularly, the present invention is a single prescription by combining Ceftioper, Neomycin and Dexamethasone in a weight ratio of 1-3: 1-3: 0.01-0.03, particularly 1: 1: 0.01. Compared to a composite antibiotic composition for animals having an excellent effect on the prevention and treatment of mastitis in animals.

In recent years, the productivity of milk has decreased due to the occurrence of mastitis in cows by penicillin-resistant bacteria, streptococcus, pseudomonas, and E. coli in cow farms, and the demand for safe therapeutic agents is increasing in farms.

As the causative agent of mastitis such as cattle, cattle, pigs, and goats, about 140 microorganisms are known to be involved in a wide variety of ways, and many types of antibacterial drugs have been introduced.

Ceftiofur is a third-generation cephalosporin antibiotic, and the lineage is {6R- [6α, 7β (Z)]} -7- {[(2-amino-4-thiazolyl) (methoxyi Mino) Acetyl] amino} -3- {[(2-furanyl carbonyl) -thio] methyl} -8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2 -It is a carboxylic acid and exists in monosodium salt or monohydrochloride form. Cefthioper, like other beta-lactam antibiotics (penicillin and cephalosporin), is a bactericidal antibiotic that exhibits antibacterial activity by inhibiting the cell wall synthesis of bacteria. Betalactam antibiotics inhibit the third stage of bacterial cell membrane formation by binding to one or more Penicillin Binding Proteins (PBPs) present on the sensitive bacterial cell wall. Therefore, the antibacterial activity of betalactam antibiotics depends on the binding affinity with these PBPs. For example, the affinity of Staphylococcus aureus with PBPs is generally greater in the first generation cephalosporins than in the third generation cephalosporins. However, the affinity of Enterobacteriaceae bacteria for PBPs is generally greater for the third generation cephalosporins. In addition, the antimicrobial activity of individual antibiotics varies depending on the stability to beta-lactam produced by gram-positive and negative bacteria and the permeability of the drug to the gram-negative bacteria. Ceftiofer, like other first-generation cephalosporins, has a very high antibacterial activity against most Gram-positive bacteria and a high degree of antibacterial activity against some Gram-negative bacteria. Recently, some E. coli strains have been reported to be resistant to cefthioper, but are currently formulated only as an infusion solution (oil suspension). (See EP 36812 (1981), USP 4464367 (1984) and Am. J. Vet.Res. 48, 1050-3 (1987), ibid. 53, 2201, (1992))

In addition, aminoglycoside antibiotics include amikacin, arbecacin, gentamicin, kanamycin, neomycin, netylmycin, paromomycin, streptomycin, tobramycin, etc. Neomycin is a drug that has been widely used since ancient times, and its line name is (2R, 3S, 4R, 5R, 6R) -5-amino-2- (aminomethyl) -6-[(1R, 2R, 3S, 4R , 6S) -4,6-diamino-2-[(2S, 3R, 4S, 5R) -4-[(2R, 3R, 4R, 5S, 6S) -3-amino-6- (aminomethyl)- 4,5-dihydroxyoxan-2-yl] oxy-3-hydrooxy-5- (hydroxymethyl) oxolan-2-yl] oxy-3-hydroxy cyclohexyl] oxyoxane-3,4- As the diol, neomycin A, neomycin B, neomycin B hydrochloride, neomycin B sulfate, and the like are known. It is a bactericidal antimicrobial agent that specifically inhibits the formation of DNA supercoils by inhibiting DNA gyrase (topoisomerase II), which is used only for animals and essential for DNA replication and transcription. Escherichia coli (E. coli), Pseudomonas Ah Labor (Pseudomonas aeruginosa), keulrep City in the Ella Rouge (Klebsiella spp.) Enterococcus batteo (Enterobacter), Campanile days bakteo (Campylobacter), when Gela (Shigella), salmonella (Salmonella), to remain on Pseudomonas (Aeromonas), Haemophilus (Haemophilus), Proteus (Proteus), Yersinia (Yersinia), Serratia marcescens (Serratia), Vibrio species (Vibrio species) superior to exhibit a strong antimicrobial activity on pathogenic bacteria, such as It can be said to have a therapeutic and preventive effect. Neomycin is a unique mechanism of action as described above, and is characterized by not causing cross-resistance with other types of antibacterial agents. (See USP 2799620, 2848365, 3108996, 3005815, 3022228 and J. Antibiot. 40, 1464, (1997))

Dexamethasone is a well-known synthetic corticosteroid that exhibits anti-inflammatory activity. It can be administered in several physical forms for use in the treatment of various inflammatory situations. The system name of dexamethasone is (8S, 9R, 10S, 11S, 13S, 14S, 16R, 17R) -9-fluoro-11,17-dihydroxy-17- (2-hydroxyacetyl) -10,13, As 16-trimethyl-6,7,8,11,12,14,15,16-oxahydroxycyclopenta [a] phenanthrene-3-one, 21-acetate, 21-phosphate disodium as its salt Salts and 17, 21-dipropionate and the like are known. (See DE 1113690, GB 869511 and N. Engl. J. Med. 311. 549 (1984))

As described above, cefthioper, neomycin and dexamethasone are antibiotics that are frequently used as single agents in the veterinary clinical field, but veterinary for these three types of complex preparations considering microorganisms or resistant bacteria that are not antimicrobial by itself alone Pharmacological studies have not been conducted at home and abroad.

EP 36812; USP 2796620, 2848365, 3005815, 3022228, 3108996; DE 1113690; GB 869511

Am. J. Vet. Res. 48, 1050-3 (1987) ibid 53, 2201 (1992), J. Antibiot. 40, 1464 (1987) N. Engl. J. Med. 3ll. 549 (1984)

Accordingly, the present inventors have developed research efforts to obtain the optimal composition for composite antibiotics for animals, and developed a composite antibiotic composition for animals of cefthioper, neomycin and dexamethasone, which have excellent inhibitory effects on animal pathogenic microorganisms, and used alone. The present invention has been completed by confirming that it has an excellent synergistic effect in the prevention and treatment of mastitis in animals compared to cases and conventional combination preparations.

Therefore, the present invention is excellent in the inhibitory effect of pathogenic microorganisms causing mastitis in animals, cefthioper, neomycin and dexamethasone are 1-5: 1-5: 0.01-0.05, preferably 1-3: 1-3: 0.01. It is an object to provide a composite antibiotic composition for animals contained by mixing in a weight ratio of -0.03, especially 1: 1: 0.01.

The present invention is a mixture of cefthioper, neomycin and dexamethasone in a weight ratio of 1-5: 1-5: 0.01-0.05, preferably 1-3: 1-3: 0.01-0.03, especially 1: 1: 0.01. It provides a composite antibiotic composition for animals. The present invention will be described in more detail as follows.

The present invention is a combination of cefthioper, neomycin and dexamethasone in a weight ratio of 1-5: 1-5: 0.01-0.05, preferably 1-3: 1-3: 0.01-0.03, especially 1: 1: 0.01. It relates to a composite antibiotic composition for animals having an excellent effect on the prevention and treatment of mastitis in animals compared to a single prescription and a conventional combination formulation.

The present invention relates to a new pharmaceutical composition for animals with excellent antimicrobial activity against mastitis-causing bacteria in animals by optimizing the mixing ratio of cefthioper, neomycin and dexamethasone, and in the present invention, cefthioper, neomycin and In order to characterize the interaction between dexamethasone and to investigate the correlation between mixing ratios, the antibiotic multi-resistant microorganisms, ie, gram positive and negative bacteria isolated from chickens, pigs, and cows affected by various bacterial diseases The antimicrobial effect of pthioper, neomycin and dexamethasone combination preparations was measured by testing at the minimum inhibition concentration (MIC).

In the present invention, the mixing ratio of cefthioper, neomycin and dexamethasone is 1-5: 1-5: 0.01-0.05, preferably 1-3: 1-3: 0.01-0.03, particularly 1: 1: 0.01. desirable. If the mixing ratio is less than 1: 1: 0.01, although it is economical, the antibacterial effect is reduced, and the antibacterial effect of the desired bar is difficult to obtain, and when it is 5: 5: 0.05 or more, it is uneconomical due to the high price of cefthioper. Preferred weights for each formulation include 100-500 mg of ceftioper, 100-500 mg of neomycin and 1-5 mg of dexamethasone, preferably 100-300 mg of ceftioper, 100-300 mg of neomycin, and dexamethasone 1 Contains -3 mg. Further, in the present invention, the aminoglycoside antibiotics amikacin, arbecacin, gentamicin, kanamycin, netylmycin, paromomycin, streptomycin, or tobramycin, which are the same aminoglycoside antibiotics as neomycin, are mixed with cefthioper. It was confirmed that the antimicrobial effect was superior to mixing neomycin with ceftiofer than to do it.

In addition to this, dexamethasone was added to further broaden the antibacterial range.

The composite antibiotic composition of the present invention is Cefthioper, Neo mixed in a weight ratio of 1-5: 1-5: 0.01-0.05, preferably 1-3: 1-3: 0.01-0.03, particularly 1: 1: 0.01 It contains mycin and dexamethasone as active ingredients, and additives, powders, animal injections, orally administered liquids with one or more carriers according to a method that can be easily carried out by a person skilled in the art to which the present invention pertains And ointments, and the preferred formulations in the present invention are ointments.

On the other hand, animal additives and powders containing the composite antibiotic of the present invention, one or more selected from saccharides, starches, powder, jade powder, malt powder, calcium carbonate or sodium hydrogen carbonate, such as commonly used vitamins, glucose and lactose It is prepared by containing excipients or enzymes in an amount used. The enzyme is bromelain or allantoin.

In addition, when formulating the complex antibiotic of the present invention as an ointment, it may further include an antioxidant, a pigment, a moisturizing agent, a solvent, and the like. One or more selected from BH (BHT, butyl-hydroxytoluene), vitamin A, C, E or derivatives thereof as the antioxidant, and one or more selected from allantoin, glycerin, petrolatum, or wax as the moisturizing agent, as the solvent One or more selected from aluminum monostearate, olive oil, soybean oil, or coconut oil may be used.

In the administration of the composite antibiotic prepared in the present invention, the additive is mixed and administered in the feed, and is formulated and administered as a powder, an animal injection, an oral administration liquid, and an ointment.

In the case of additives as a composite antibiotic mixed at a ratio determined in the present invention, it is administered at 200 g to 10 kg per ton of feed (usually 500 g to 2 kg), and in particular, when administered as an ointment, 0.5 to 2 cows per minute, usually 1 It is preferably administered as a dog. One ointment contains approximately 200 to 1000 mg of antibiotics in total.

The present invention is prepared by mixing ceftioper, neomycin and dexamethasone in the above ratio as described above, thereby suppressing the appearance of antibiotic-resistant strains, having a broad antibacterial range, and administering alone by reducing the dose when used in combination. It can reduce toxicity than poetry, and can be useful for the prevention and treatment of mastitis by acting more effectively when a complex infection disease occurs.

As described above, the present invention can suppress the appearance of antibiotic-resistant strains by preparing cefthioper, neomycin and dexamethasone by mixing at the above ratio, has a broad antibacterial range, and decreases the dose when administered in combination, Toxicity can be reduced more than when administered alone, and it can be useful for the prevention and treatment of mastitis by acting more effectively when a complex infection disease occurs.

1 and 2 are diagrams showing changes in the number of somatic cells after administration of the present invention ointment, cefthioper ointment, and neomycin ointment to cows with non-organic mastitis.
3 is a view showing the clinical index, such as papillary lesions and crude sediment index.
Figure 4 is a change in the number of bacteria in the milk of cows with non-organic mastitis.

The composite antibiotic composition for animals according to the present invention prevents or treats mastitis in livestock animals, characterized in that cefthioper, neomycin and dexamethasone are mixed in a weight ratio of 1-5: 1-5: 0.01-0.05. As a composite antibiotic composition for animals, it is preferably a composition in which cefthiopi, neomycin and dexamethasone are mixed in a weight ratio of 1-3: 1-3: 0.01, particularly 1: 1: 0.01.

The composition formulation may be formulated as a powder, a water-receiving agent or an ointment agent, and an ointment agent is preferable in consideration of efficacy, stability of the formulation, and a method of administration.

In addition, the present invention provides a prophylactic and therapeutic agent for mastitis in livestock animals comprising the composite antibiotic composition as a medicinal active ingredient.

Since the clinical trial of cow's non-organic mastitis of the drug preparation of the present invention was performed, the ointment of the present invention has a higher antibacterial effect than the single administration of each component alone, resulting in a decrease in somatic cell count and a decrease in the total number of bacteria in the milk. It has been identified as an effective agent for the treatment of breast cow mastitis.

Hereinafter, the present invention will be described in detail with reference to the following examples, but the scope of the present invention is not limited by these examples.

[Example]

Example 1: Comparison of antibacterial effects by strain (○○ Biomedical Research Institute, Asan City, Chungnam)

1) MIC measurement

E. coli , Staphylococcus spp ( S. aureus , S. epidermidis ), S treptococcus uberis , Pseudomonas aeruginosa , Klebsiella pneumoniae , E nterococcus faecalis , Bacillus cereus, etc. At 5 weeks), the minimum growth inhibitory concentration (MIC) of mastitis-causing bacteria collected from the field was measured using ceftioper, neomycin and dexamethasone.

2) Materials and devices

① antibiotics

Cefthioper hydrochloride and neomycin sulfate were weighed at 40 mg and 45 mg (30 mg as neomycin), respectively, and diluted with sterile distilled water to dilute the active ingredient to a concentration of 1 mg / mL.

② MIC and MBC test: conducted according to CLSI VET01-A4 (2013) and CLSI M26-A (1999).

③ Badge

After spreading each test strain on 5% sheep blood agar, incubate for 18 ~ 24 hours in a 37 ℃ (S.uberis is 37 ℃, 5% CO ₂ ) incubator, take 4 ~ 6 single colonies, and inoculate the liquid medium. Incubate for 2-6 hours in a 37 ° C incubator. The turbidity of the cultured bacteria is MacFarland No. Adjust the number of bacteria to be about 1.5 Х10 ⁸ CFU / mL according to 0.5, and then dilute it 100 times with diluent to use as the inoculation bacterial solution of about 1.5 Х10 ⁶ CFU / mL

④ Experimental strain: 7 strains of 42 strains collected from the field (standard strain for each strain 1 week, outdoor strain 5 weeks)

Escherichia coli

Staphylococcus epidermidis

Staphylococcus aureus

Streptococcus uberis

Pseudomonas aeruginosa

Klebsiella nuoniae

Enterococcus faecalis

Bacillus cereus

3) Fungus inoculation and culture

After inoculation by injecting 0.05 mL of the culture solution adjusted to 1.5 Х10 ⁶ CFU / mL into each well except the negative control well (12th well) and incubating for 18 to 24 hours in a 37 ° C incubator. In addition, the inoculum solution was diluted 10-fold up to 10 ^-5 with sterile physiological saline, and 10 μL per dilution was repeated 3 times to inoculate the solid medium to measure the initial inoculation bacteria. As a result of confirming the number of inoculation bacteria in the inoculum solution used in the MIC test, it was 1.5 to 6.510 ⁵ CFU / mL.

4) MIC decision

After 18 to 24 hours, the presence or absence of the growth of the bacteria was visually observed to determine the concentration at which the bacteria did not grow as the MIC concentration.

* 5) MIC & FIC (FRACTORY INHIBITION CONCENTRATION) results

∑ FIC 0.5: Synergism (rising)

0.5 <∑ FIC = 0.75: Partial synersim (partial rise)

0.75 <∑ FIC 4: Indifference

∑ FIC> 4: antagonism

As shown in Table 2, the FIC value for E.coli at each antibiotic concentration of ceftioper and neomycin was observed to be an additive or synergistic effect from 1.008 to 1.500, and the combination preparation of the present invention was administered to a single administration group. In comparison, the antibacterial activity against E. coli was confirmed to be partially synergistic or additive.

As a result of investigating the antibacterial effects of cefthioper, neomasin and the combination preparation of the present invention with respect to five strains of E.coli outdoor strain and one week of standard strain, the MIC range of cefthioper was 0.122 to 0.244 μg / mL, and the MIC _{The 50} and MIC ₉₀ values were 0.244 μg / mL, and the antimicrobial effect was very excellent at a concentration of about 8 times lower than the 2 μg / mL, which is the cefthioper MIC susceptibility standard for E.coli , the causative agent of anti-inflammatory. In the case of neomycin, the MIC was 1.953 to 250 μg / mL, and both the MIC ₅₀ and MIC ₉₀ were 250 μg / mL, and the antibacterial effect was minimal. The MICs of the combination preparations of the present invention were all 0.488 μg / mL, and when converted to the amounts of the active ingredients cefthioper and neomycin, the antimicrobial effect was excellent at 0.006 + 0.006 μg / mL.

In the concentrations of the antibiotics of ceftiofer and neomycin, FIC values for S. aureus were 1.008 to 1.500, respectively.

Staphylococcus spp. As a result of investigating the antibacterial effects of cefthioper, neomasin, and the combination preparation of the present invention against 6 strains of outdoor isolates (4 weeks of S. aureus , 2 weeks of S. epidermidis ) and 1 week of standard strains, it was excellent in all disclosed antibiotics. Antibacterial effect appeared. The Cefthioper MIC ₅₀ and MIC ₉₀ values for the outdoor isolate strains were 0.244 μg / mL and 1.953 μg / mL, respectively, which were below 2 μg / mL, which is the Cefthioper's MIC susceptibility standard for S. aureus , a bovine mastitis causative agent. The antibacterial effect was confirmed.

MIC ₅₀ and Neomycin The value of MIC ₉₀ was 0.977 μg / mL (Cefthioper + Neomycin: 0.012 + 0.012 μg / mL), and the antibacterial effect was remarkable.

)은 각각 3.906 μg/mL, 2.0 μg/mL, 1.953 μg/mL으로 MIC 농도보다 2 배 높은 농도에서 살균효과가 나타났다.Minimum sterilization concentration (MBC) of cefthioper, neomycin and the combination preparation of the present invention for Staphylococcus spp

) Was 3.906 μg / mL, 2.0 μg / mL, and 1.953 μg / mL, respectively, and the sterilizing effect was found at a concentration twice as high as the MIC concentration.

Partial synergy and additive activity were observed with FIC values for S.uberis at the concentrations of antibiotics of cefthioper and neomycin of 1.008 ~ 1.500, respectively.

As a result of investigating antibacterial effects of cefthioper, neomasin , and the combination preparation of the present invention for 5 strains of S.uberis outdoor strain and 1 strain of standard strain, the antimicrobial effect was excellent in all the disclosed antibiotics. That is, the MIC ₅₀ and MIC ₉₀ values of cefthioper for the outdoor isolate strain were 0.0024 μg / mL and 0.0049 μg / mL, respectively, and neomycin was 0.488 μg / mL, and the MIC ₅₀ and MIC ₉₀ values of the combination preparation of the present invention Silver has an excellent antibacterial effect with 0.004 μg / mL (Cefthioper + Neomycin: 0.000048 + 0.000048 μg / mL).

The minimum sterilization concentration (MBC ₉₀ ) of cefthioper , neomycin and the combination preparation of the present invention for S. uberis is 0.908 μg / mL, 1.953 μg / mL, 0.015 μg / mL (ceftioper + neomycin: 0.0002), respectively. + 0.0002 μg / mL), showing a sterilizing effect at a concentration 2 to 4 times higher than the MIC concentration.

Partial synergy and additive activity were observed with FIC values for Pseudomonas aeruginosa at the concentrations of antibiotics of cefthioper and neomycin of 0.625 to 1.125, respectively.

As a result of investigating antibacterial effects of cefthioper, neomasin, and the combination preparation of the present invention for 5 weeks of Pseudomonas aeruginosa and 5 strains of standard strains, antibacterial effects were recognized in all the disclosed antibiotics. Cefthioper's MIC ₅₀ and MIC ₉₀ values for outdoor isolate strains were 15.625 μg / mL and 31.250 μg / mL, respectively, and Neomycin's MIC ₅₀ and MIC ₉₀ 62.5 μg / mL and 125.0 μg / mL had insufficient antibacterial effects. Did. The MIC ₅₀ and MIC ₉₀ values of the combination preparation of the present invention were both 0.488 μg / mL, and the antibacterial effect was excellent. The antimicrobial effect was confirmed to be 15.625 μg / mL (Cefthioper + Neomycin: 0.195 + 0.195 μg / mL) for both MIC of Cefthioper and the combination preparation of the present invention.

)는 각 공시 항생제의 MIC 농도와 같거나 2 배 높은 농도인 62.5 μg/mL, 125 μg/mL, 31.250 μg/mL (세프티오퍼+네오마이신: 0.391 + 0.391 μg/mL) 이었다. Cefthioper , Pseudomonas aeruginosa , Antimycin , Sterilizing Effect of the Composite Formulation of the Invention (MBC

) Were 62.5 μg / mL, 125 μg / mL, 31.250 μg / mL (Cefthioper + Neomycin: 0.391 + 0.391 μg / mL), which are the same or twice as high as the MIC concentration of each test antibiotic.

At each antibiotic concentration of cefthioper and neomycin, FIC values for Klebsiella pneumoniae were 1.008 to 1.500, respectively, and additive activity was observed.

As a result of investigating the antibacterial effects of cefthioper, neomasin, and the combination preparation of the present invention for Klebsiella pneumoniae 5 strains and 1 strain of standard strains, the antimicrobial effect of both cefthioper MIC ₅₀ and MIC _{90 was} 0.488 μg / mL Was excellent. Both the MIC of Cefthioper and the combination preparation of the present invention had an excellent antibacterial effect with 0.488 μg / mL (0.006 + 0.006 μg / mL).

The minimum sterilization concentrations (MBC ₉₀ ) of cefthioper, neomycin and the combination preparation of the present invention for Klebsiella pneumoniae are 0.907 μg / mL, 0.488 μg / mL, and 0.488 μg / mL (Cefthioper + Neomycin: 0.006 +, respectively). 0.0069 μg / mL).

Partial synergy and additive activity were observed with FIC values for Enterococcus faecalis at the concentrations of antibiotics of ceftioper and neomycin of 0.507 to 1.125, respectively.

As a result of investigating the antibacterial effects of cefthioper, neomycin, and the combination preparation of the present invention for 5 weeks of Enterococcus faecalis outdoor strain and 1 week of standard strain, the MIC range of cefthioper was 0.244 to 125 μg / mL, MIC ₅₀ And MIC ₉₀ values were 1.953 μg / mL and 125 μg / mL, respectively. Neomycin had a MIC range of 0.015 to 250 μg / mL, and MIC ₅₀ and MIC ₉₀ values of 1.953 μg / mL and 125 μg / mL, respectively. Neomycin's MIC range was 0.015 to 250 μg / mL, and the MIC ₅₀ and MIC ₉₀ values were 31.250 μg / mL and 250 μg / mL, respectively.

The MIC ₅₀ and MIC ₉₀ values of the combination preparation of the present invention were 7.813 μg / mL (ceftioper + neomycin: 0.098 + 0.098 μg / mL).

The bactericidal effect of ceftioper, neomycin, and the combination preparation of the present invention on Enterococcus faecalis is 125 μg / mL, 500 μg / mL, 31.250 μg / mL (three taxes 1 to 4 times higher than the MIC concentration of each disclosed antibiotic) Pthioper + Neomycin: 0.391 + 0.391).

At each antibiotic concentration of ceftiofer and neomycin, FIC values for Bacillus cereus were 1.008 to 1.500, respectively, and additive activity was observed.

As a result of investigating the antibacterial effects of cefthioper, neomycin, and the combination preparation of the present invention for Bacillus cereus outdoor isolate strain 5 weeks and standard strain 1 week, both MIC ₅₀ and MIC ₉₀ values of cefthioper were 7.813 μg / mL. While the antibacterial effect was weak, neomycin was 0.488 μg / mL, and the MIC ₅₀ and MIC ₉₀ values of the combination preparation of the present invention were 0.488 μg / mL (Cefthioper + Neomycin: 0.006 + 0.006 μg / mL). The effect was excellent.

The bactericidal effect of each disclosed antibiotic on Bacillus cereus was that of cefthioper , neomycin, and the combined preparation of the present invention was at a concentration 1 to 2 times higher than the MIC concentration of each disclosed antibiotic, and the minimum sterilization of each antibiotic Concentrations (MIC ₉₀ ) were 15.625 μg / mL, 0.488 μg / mL, and 0.977 μg / mL (Cefthioper + Neomycin: 0.012 + 0.012 μg / mL), respectively.

[Preparation example]

Formulation Example 1: Preparation of non-organic ointment

Ointments were prepared from the compositions listed in Table 9 below.

1. After moisturizing agent and solvent are weighed, dry heat sterilization is performed at 160 ° C for 1 hour, mixed, and then put into a preparation tank to cool to 50 ° C.

2. Separately, cefthioper, neomycin, dexamethasone, and pigment were put in a plastic container to mix sufficiently.

3. The raw material of cefthioper, neomycin, dexamethasone, pigment, and antioxidant mixed in a small amount of sterile solvent was added and stirred to disperse homogeneously.

4. The solution of 3 was added to the solution of 1, mixed for 30 minutes or more, and the operation was terminated.

5. Appearance: Injection, grayish blue uniform ointment, Storage condition: Room temperature

6. Usage and Dose: After disinfecting the breast and nipple cleanly, inject 1 syringe (8 g) in 1 minute per day for 3-5 days.

Formulation Example 2: Preparation of non-organic ointment

Ointment was carried out in the same manner as in Formulation Example 1 with the composition shown in Table 10 below.

Formulation Example 3: Preparation of non-organic ointment

Ointment was carried out in the same manner as in Preparation Example 1 above with the composition shown in Table 11.

Formulation Example 4: Preparation of non-organic ointment

Ointment was carried out in the same manner as in Formulation Example 1 with the composition shown in Table 12 below.

Formulation Example 5: Preparation of non-organic ointment

Ointment was carried out in the same manner as in Preparation Example 1 with the composition shown in Table 13 below.

Formulation Example 6: Preparation of non-organic ointment

Ointment was carried out in the same manner as in Preparation Example 1 with the composition shown in Table 14 below.

Formulation Example 7: Preparation of non-organic ointment

Ointment was carried out in the same manner as in Preparation Example 1 above with the composition shown in Table 15 below.

Formulation Example 8: Preparation of non-organic ointment

Ointment was carried out in the same manner as in Preparation Example 1 above with the composition shown in Table 16 below.

Formulation Examples 9-13: Formulation examples of powders and additives

Manufacturing method: The above-mentioned prescription ingredients are mixed to prepare according to the powder manufacturing method.

Formulation Examples 14-19: Formulation examples of water-soluble agents

Preparation method: The active ingredient is prepared by dissolving or suspending it in a solvent.

Comparative formulation example 1: cefthioper ointment

Composition (this syringe 1, in 8 g)

    Cefthioper hydrochloride --------- 100 mg (titer)

    Butylhydroxytoluene --------- suitable amount

Formulation and Properties: Infusion in powder

Usage and dosage: After disinfecting the breast and nipple cleanly, inject 1 syringe (8 g) in 1 minute per day for 3-5 days.

Comparative Formulation Example 2: Neomycin Ointment

Composition (this syringe 1, in 8 g)

    Neomycin Sulfate --------- 100 mg (titer)

    Dexamethasone disodium phosphate ---------- 1.3 mg (1 mg as dexamethasone)

    Butylhydroxytoluene --------- suitable amount

Formulation and Properties: Infusion in powder

Usage and dosage: After disinfecting the breast and nipple cleanly, inject 1 syringe (8 g) in 1 minute per day for 3-5 days.

[Clinical Experimental Example]

Clinical efficacy test

In order to confirm the therapeutic effect of the formulation example 1 ointment and the comparative formulation examples 1 and 2 ointment for non-organic mastitis, the individual syringes with a somatic cell count of 2 Х 10 ⁵ cell / mL or more are selected and the syringe of the present invention is administered in a fraction. Changes in somatic cell number, hematology, number of bacteria in crude oil, nipples, and milk were confirmed for 14 days after injection.

Somatic cell number change

Somatic cells of the administration group before administration of the test substance to cows suffering from non-organ mastitis (Day 0), ointment of formulation of the present invention (Formulation Example 1), cefthioper ointment (Comparative Preparation Example 1), and neomycin ointment (Comparative Preparation Example 2) The number was 1040.3 ± 557.6 Х 10 ³ cell / mL, 1021.2 ± 246.0Х10 ³ cell / mL, and 1276.7 ± 692.8 Х 10 ³ cell / mL, respectively, and the number of somatic cells was 676.3 ± 298.9Х10 ³ cell / on the third day after the administration of the test substance. mL, 710.2 ± 250.1 Х 10 ³ cell / mL, reduced to 851.5 ± 293.5 Х 10 ³ cell / mL. On the 6th day after the start of administration, the number of somatic cells in each administration group decreased by about 45-50% compared to before administration, respectively, 429.0 ± 223.2Х10 ³ cell / mL, 507.2 ± 160.0 Х 10 ³ cell / mL, 612.0 ± 172.5 Х 10 ³ cell / mL Was. The number of somatic cells on the 10th day after the start of drug administration was reduced by 70% compared to before the administration, and the number of somatic cells in each group on the 14th day after the start of the administration was decreased by about 80% to 90% compared to before the administration, so that the number of somatic cells was normal in most subjects. It was confirmed to recover. (See Table 19, Figures 1 and 2)

3) Hematological changes

10⁹/L, 24.2 ± 4.4

10⁹/L, 22.6 ± 4.5

10⁹/L 이었으나, 각 공시물질 투여 개시 후 3일째의 총백혈구수가 각각 14.6 ± 2.7

10⁹/L, 17.1 ± 2.1

10⁹/L, 18.3 ± 4.0

10⁹/L으로 감소하였으며, 본발명 연고와 세프티오퍼 연고 투여군의 경우 총 백혈구수는 투여 전 보다 유의하게 감소하였다. The present invention ointment, cefthioper ointment, and neomycin ointment were administered to cows with non-organic mastitis, and changes in total leukocytes were observed in hematological examination before and after administration. The total number of white blood cells before administration of the present invention ointment, cefthioper ointment, and neomycin ointment to each test group was 21.9 ± 5.2, respectively.

10 ⁹ / L, 24.2 ± 4.4

10 ⁹ / L, 22.6 ± 4.5

10 ⁹ / L, but the total number of white blood cells on the third day after the start of administration of each test substance was 14.6 ± 2.7, respectively.

10 ⁹ / L, 17.1 ± 2.1

10 ⁹ / L, 18.3 ± 4.0

It decreased to 10 ⁹ / L, and the total leukocyte count in the administration group of the present invention ointment and cefthioper ointment decreased significantly before administration.

From the 6th day after the start of administration to the 14th day after the start of administration, the total leukocyte count of all test groups was significantly decreased than the total leukocyte count before administration. In addition, the total leukocyte count of the ointment administration group of the present invention on the 14th day after the start of administration was significantly lower than that of the neomycin administration group (Table 20).

4) Nipple lesion and crude sediment index

After administering the present invention ointment, cefthioper ointment, and neomasin ointment to mastitis-expressing cows, the nipple and crude oil conditions were confirmed until the end of the experiment (FIG. 3).

In the individual in which the papillary induration or redness of the ointment administration group of the present invention was observed, nipple infringement or redness was not observed from the 4th day after the start of drug administration, and nipple infringement or redness was not observed in all the individuals from the 7th day after the start of administration. A small amount in the crude oil of the ointment administration group of the present invention was not observed from day 4, and was not observed in all subjects from day 7.

In the case of the cefthioper ointment administration group, no papillary induration or redness was observed from the 4th day after the start of drug administration, and no papillary induration or redness was observed in all subjects from the 9th day after the start of the administration. A small amount in the crude oil of the cefthioper ointment-administered group was not observed from day 3 to day 4, and was not observed in all subjects from day 9. In the case of the neomycin ointment administration group, no papillary induration or redness was observed from the 5th day after the start of drug administration, and from the 9th day after the start of administration, no papillary induration or redness was observed in all subjects. In some subjects in the neomycin ointment administration group, small amounts of sediment or small fragments in crude oil were not observed from day 4, and were not observed in all subjects from day 10.

5) Measuring the number of bacteria in crude oil

The present invention ointment, cefthioper ointment, and neomycin ointment 1 syringe each were injected for 5 days in a small fraction of non-organic mastitis, and the total number of bacteria in the crude oil was examined for 14 days from the start of drug administration (Table 21, FIG. 4). .

The total number of bacteria in crude oil before injection of the present invention ointment, cefthioper, neomycin ointment into the breast is 12.1 ± 14.3 Х 10 ⁵ CFU / mL, 18.1 ± 14.3 Х 10 ⁵ CFU / mL, 15.2 ± 19.0 Х 10 ⁵ CFU, respectively / mL, but the total number of bacteria in crude oil on the third day after the start of drug administration is 1.1 ± 1.1 Х 10 ⁴ CFU / mL, 2.1 ± 2.3 Х 10 ⁵ CFU / mL, 2.1 ± 2.7 Х 10 ⁵ CFU / mL, the ointment administration group of the present invention In total, the number of bacteria decreased significantly compared to the ceftioper and neomycin groups.

In the case of the ointment administration group of the present invention, bacteria were not detected in 2 heads on the 6th day after the start of drug administration, and bacteria were detected in other individuals except the total number of 633 CFU / mL bacteria was detected in 1 head on the 10th day after the start of the administration. On the 14th day after the start of drug administration, no bacteria were detected in all subjects.

In the cefthioper group, the total number of bacteria was 3.2 ± 5.2 Х 10 ⁴ CFU / mL and 972 ± 1067 CFU / mL in ⁴ heads on the 6th and 10th days after the start of drug administration. A total number of bacteria of 200 ± 298 CFU / mL was detected.

In the neomycin-administered group, the total number of bacteria of 7.7 ± 15.9 Х 10 ⁴ CFU / mL was detected in 5 heads on the 6th day after the start of drug administration, and the total number of bacteria in 4 heads was 4.7 ± 7.9 Х 10 ³ CFU / in the 10th day after the start of administration. It was mL, and on the 14th day after the start of administration, 356 ± 446 CFU / mL was detected in 3 heads.

6) Conclusion

In order to confirm the efficacy of the ointment of the present invention against non-organic mastitis, the syringe of the present invention was injected for 5 days in a fraction where the number of somatic cells was confirmed to be over 2Х10 ⁵ cells / mL, and somatic cell number, hematology and changes in the number of bacteria in the crude oil were observed. As a result, the number of somatic cells on the 3rd day after the administration of the ointment of the present invention was reduced to about 27% than before the administration of the present invention to cows suffering from non-organic mastitis, and the number of somatic cells on the 6th, 10th and 14th days after administration was 53%, respectively, before administration. The decrease was 75% and 87%, and it was confirmed on the 14th day after the initiation of administration that the somatic cell number was restored to the normal range in most subjects. In addition, the total number of white blood cells was 21.9 ± 5.2 Х 10 ⁹ / L before administration, but the total number of white blood cells decreased to 14.6 ± 2.7 Х 10 ⁹ / L from the third day after the start of administration, and the total number of white blood cells was 10.7 ± 1.8 Х on the 14th day after the start of administration. It decreased to 10 ⁹ / L, and was significantly lower than the neomycin administration group. From the 4th day after the administration of the ointment of the present invention, papillary induration or redness was not observed in some animals, and from the 7th day after the initiation of administration, papillary induration or redness was not observed in all individuals, and small amounts of sediment or small fragments in crude oil were also observed from the 7th day. Not observed in all subjects. In addition, the total number of bacteria in the crude oil before administration of the ointment of the present invention was 12.1 ± 14.3 Х 10 ⁵ CFU / mL, but the total number of bacteria in the crude oil was 1.1 ± 1.1 Х 10 ⁴ CFU / mL on the third day after the drug administration was initiated. It was significantly decreased than the neomycin administration group. In the case of the ointment administration group of the present invention, bacteria were not detected in 2 heads on the 6th day after the start of drug administration, and bacteria were detected in other individuals except the total number of 633 CFU / mL bacteria was detected in 1 head on the 10th day after the start of the administration. On the 14th day after the start of drug administration, no bacteria were detected in all subjects.

In conclusion, the present invention ointment for non-organic mastitis is evaluated as an effective drug for the treatment of non-organic mastitis because the antibacterial effect is more effective than the administration of ceftioper and neomycin alone, resulting in a decrease in the number of somatic cells and a decrease in the total number of bacteria in crude oil. Became.

[Stability evaluation of composition]

In order to confirm the stability according to the storage of the prepared composition, after obtaining the formulation example 1, formulation example 2 and formulation example 6 of the present invention, the prepared injection composition temperature: 25 ± 2 ℃, relative humidity 60 ± 5% Stored for 24 months under conditions, the content of cefthioper, neomycin and dexamethasone over time was measured by high performance liquid chromatography (HPLC). The HPLC is YL9101 vaccum degasser, YL9110 Quaternary pump, YL9131 Column compartment, YL9120 uv / vis detecter, YL9150 Autosampler System (Younglin equipment), Column: Cosmosil C ₁₈ , 5 μm (Nacalai tesque), Mobile phase: Septhioper (solution) A + MeOH + THF-1000: 200: 110, Solution A-50 mM Ammonium acetate + 10 mM Tetrabutylammonium bromide → to DW (distilled water) 1 L), Neomycin 0.1% Trifloracetic acid solution + ACN (95: 5), Dexamethasone (Mobile phase A: water + methanol + Buffer (3.5 + 3.5 + 3), mobile phase B-methanol + Buffer (7: 3), Buffer: 7 g of ammonium acetate is dissolved in water to make 1 L, and adjusted to pH 4.0 with acetic acid. ). Wavelength: cefthioper (290 nm), neomycin (refractive index detector, ELSD), dexamethasone (254 nm), flow rate: cefthioper 1.2 ml / min, neomycin and dexamethasone 1.0 ml / min, injection volume: 20 μl Dexamethasone was analyzed by Gradient system, and the analysis conditions are as follows (Table 22). Table 23, 24 and 25 show changes in the content of cefthioper, neomycin and dexamethasone over time.

Dexamethasone Gradient system conditions

[Equation 1] Content retention rate (%) = {(A1 / A0) × 100}

In the formula 1, A0 is based on the initial content of each component in the composite antibiotic composition for animals, A is 0, 3, 6, 9 in the conditions of 25 ± 2 ℃ and relative humidity of 60 ± 5% of the composite antibiotic composition for animals , The content of each component measured after leaving for 12, 18 and 24 months.

[Toxicity test]

Toxicity test of oral administration to the following composite antibiotic composition for animals was carried out as follows.

Complex antibiotic composition / in 8 g

Cefthioper 100 mg

Neomycin 100 mg

Dexamethasone 1 mg

Experimental animals were prepared with SD (Spraque Dawley) 6-7 week old rats, and the number of animals was purified and quarantined for 5 days using 54 animals (27 female, 27 male), and 7-8 at the start of administration. 50 animals (25 females, 25 males) were given to the animals of the age of age, and the animals were freely fed with feed and drinking water during the purification and quarantine period, and the general condition was observed to provide only healthy animals.

Test results of toxicity and toxic intensity by single oral exposure, dead animals and clinical symptoms, weight change, feed intake and negative intake for 14 days after single oral administration in doses of 250, 500, 1,000 and 2,000 mg / kg bw in SD rat male and female A change in intake was observed, and as a result of visual examination of the rats that survived after the end of the observation period, no abnormality was observed.

In conclusion, the semi-lethal dose (LD50) in single oral administration was more than 2,000 mg / kg in acute oral administration of the combination preparation of the present invention in rats, and as a result, this product was a safe substance without toxicity by oral exposure. It was confirmed as.

The composite antibiotic composition for animals according to the present invention has excellent efficacy in treating and preventing mastitis in animals, and is a very stable agent.

Claims (8)

Prevention of mastitis in ointment-type animals, characterized in that cefthioper or a salt thereof, neomycin or a salt thereof, and dexamethasone or a salt thereof are mixed in a weight ratio of 1-3: 1-3: 0.01-0.03. Therapeutic composite antibiotic composition. According to claim 1,
Cefthioper, neomycin and dexamethasone, characterized in that made by mixing in a weight ratio of 1: 1: 0.01, composite antibiotic composition. According to claim 1,
Cefthioper, neomycin and dexamethasone 100 ~ 300 mg: 100 ~ 300 mg: characterized in that consisting of a mixture of 1-3 mg, composite antibiotic composition. According to claim 1,
A composite antibiotic composition wherein the animal is a cow. Cefthioper or a salt thereof, neomycin or a salt thereof, and dexamethasone or a salt thereof as a drug active ingredient are mixed in a weight ratio of 1-3: 1-3: 0.01-0.03, to which one excipient on paper is added. Characterized in, the ointment-type method of manufacturing a composite antibiotic composition for preventing or treating mastitis in animals. The method of claim 5,
A method for preparing a composite antibiotic composition, wherein ceftioper, neomycin, and dexamethasone are mixed and contained in a weight ratio of 1: 1: 0.01 of the animal. The method of claim 5,
Method of preparing a composite antibiotic composition, wherein ceftioper, neomycin and dexamethasone are mixed with 100 to 300 mg: 100 to 300 mg: 1 to 3 mg. The method of claim 5,
Method for preparing a composite antibiotic composition wherein the animal is a cow.

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