CN101982173B - Danofloxacin mesylate-amoxicillin suspension injection applicable to livestock and poultry as well as preparation and application thereof - Google Patents
Danofloxacin mesylate-amoxicillin suspension injection applicable to livestock and poultry as well as preparation and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of preparation of medicines for the livestock and poultry, and particularly relates to a danofloxacin mesylate-amoxicillin suspension injection applicable to livestock and poultry as well as a preparation method and an application thereof. The danofloxacin mesylate content in the preparation is 2.5% (W/V), the amoxicillin content is 15% (W/V) and the balance is grease for injection, a suspending agent, an antioxidant and an emulsifier. The preparation method comprises the following steps: heating and stirring the grease for injection and the suspending agent to obtain gel; adding the micro powder of the raw medicines danofloxacin mesylate and amoxicillin, the antioxidant and the emulsifier into the gel; and dispersing and homogenizing by a high-speed dispersion machine and a colloid mill to prepare the suspension. The invention has simple preparation process; and the prepared suspension injection meets the pharmacopeia requirements, has obvious long-acting effect on the digestive tract and respiratory diseases after intramuscular injection, and is a novel veterinary drug preparation with long-acting effect on the digestive tract and respiratory diseases of the livestock and poultry.
Description
Technical field
The invention belongs to medicine for animal preparation and applied technical field, be specifically related to a kind of Danofloxacin mesylate-amoxicillin suspension injection and preparation and application of animal specific.
Background technology
Danofloxacin mesylate (Danofloxacin mesylate; DFM) belong to the third generation FQNS of animal specific; Has a broad antifungal spectrum all has excellent antibiotic active to escherichia coli, Salmonella, pasteurella multocida, pasteurella haemolytica, staphylococcus aureus, Actinobacillus pleuropneumoniae, mycoplasma hyopneumoniae, MG etc.The drug of first choice of the various infectious disease due to various sensitive bacterials of Chang Zuowei fowl poultry kind and the mycoplasma etc. clinically is like bovine pasteurellosis, pulmonis Bovis seu Bubali inflammation, porcine contagious pleuropneumonia, mycoplasmal pneumonia of swine, avian colibacillosis, fowl cholera, fowl chronic respiratory tract disease etc.The amoxicillin (Amoxicillin AMO) is Beta-lactam medicine, has a broad antifungal spectrum, and sterilizing power is strong, and main gram positive bacteria and gram negative bacteria are had stronger bactericidal action, but invalid to penicillin-fast staphylococcus aureus.Be mainly used in the systemic infectiones such as poultry respiratory system, urinary system, skin and soft tissue due to escherichia coli, Bacillus proteus, Salmonella, haemophilus, pasteurellosis bacillus, Brucella, staphylococcus and the streptococcus etc. clinically, like Bacillus pasteurii disease, staphylococcosis, streptococcicosis, pneumonia, mastitis, metritis, Hakuri, salmonellosis, septicemia etc.
Application number is that 200510016137.7 Chinese patent has been reported a kind of Danofloxacin mesylate solution for animals that contains clustered water; Application number is that 200810197943.2 Chinese patent has been reported a kind of Danofloxacin mesylate microsphere formulation for livestock and poultry and preparation method thereof; Application number is that 200910223852.6 Chinese patent has been reported a kind of danofloxacin mesylate liposome and preparation method thereof, and application number is that the United States Patent (USP) of US05811130 has been reported Danofloxacin mesylate injection and preparation method thereof.At present, approved uses and has at the preparation of market sale the normal injection of Danofloxacin mesylate soluble powder, solution and confession intramuscular injection.About the preparation of amoxicillin more research report is arranged both at home and abroad, approved uses and the preparation sold has the compound preparation of amoxicillin sheet, capsule, soluble powder, amoxicillin sodium for injection and amoxicillin with clavulanic acid.U.S. Pat 6756057B2; US6783773B1; US6878386B1; US7011849B2; US7217430B2; US7250176B1 and Chinese patent 96195022.6; 98113146.8; 97198977.X; 98804095.6; 200410083987.4; 200480016952.7; 200510039175.4; 200510034413.2; 200580019404.4; 200610081120.4; 200610045718.8; 200610033852.6; 200610152620.2; 200610118570.6; 200710306511.6; 200710087140.7; 200710042901.7; 200710189748.0; 200810212280.7; 200810212281.1; 200810030085.2; 200910308869.1 reported the compound preparation of amoxicillin and clavulanic acid (potassium).Application number is the compound preparation that 200310105248.6,200810134827.6,200910018012.6 Chinese patent has been reported amoxicillin and sulbactam (sodium).Application number is the oral formulations that 200510123859.2,200810055428.0 Chinese patent has been reported amoxicillin and (Pivaloyloxy)methyl penicillanate S,S-dioxide.Application number is that 200410069035.7,200510073169.0,200610066946.3,200810111818.5 Chinese patent has been reported amoxicillin-(hydrochloric acid) ambroxol compound preparation.Application number is that 200510073170.3 Chinese patent has been reported amoxicillin-Bisolvon compound preparation.The Chinese patent of the number of applying for a patent 200910114971.8 has been reported the suspension injection of amoxicillin and ofloxacin respectively.Mostly the amoxicillin compound preparation of above-mentioned patent report is tablet.Application number is the main component of the compound amoxicillin oil suspension injection reported of 200710189748.0 Chinese patent: the 100mL suspension injection contains soybean oil 100 ± 5mL, amoxicillin 15 ± 1.0g, clavulanate potassium 3.75 ± 0.3g or sulbactam sodium 7.5 ± 0.5g, phosphatidase 10 .05～0.07g, aluminium stearate 2.0 ± 0.2g, Arlacel-80 0.15～0.3g, benzyl alcohol 1 ± 0.1mL, vitamin E 0.15～0.3g; Its preparation technology processes gel with soybean oil and aluminium stearate earlier; Adopt the mortar abrasive method that amoxicillin, clavulanate potassium or sulbactam sodium, lecithin are dispersed in the gel carrier again; And then adding benzyl alcohol, Arlacel-80 and vitamin E, the back packing stirs.Application number is the amoxicillin reported of 200910114971.8 Chinese patent and the main component of ofloxacin suspension injection: the 1000mL suspension injection contains ofloxacin 10～40g, amoxicillin 20～60g, aluminium stearate 2～15g, vitamin E 0.1～1g, injection soybean oil; Its preparation technology is made into factice with soybean oil and aluminium stearate earlier; Then amoxicillin, ofloxacin, vitamin E and soybean oil-aluminium stearate factice is stirred, with high pressure homogenizer homogenizing, filtration, stirring packing.These two kinds of suspension injections that method is prepared, medicine disperses inhomogeneous, is prone to take place sedimentation, less stable.Do not see the research report of Danofloxacin mesylate and amoxicillin compound preparation both at home and abroad as yet.
Fowl bacterial infects and is generally the microbial mixed infection of various sensitivities clinically, presses for drug combination and treats.Danofloxacin mesylate, amoxicillin belong to FQNS and PCs respectively, and both antibacterial action mechanism are different, and the former mainly is through suppressing the DNA gyrase of antibacterial; And metabolism and the propagation of antibacterial have been destroyed; Can kill antibacterial rapidly, stable sterilization effect, the latter mainly is through suppressing the activity of bacteria cell wall mucopeptide enzyme; The formation of block cell wall; Thereby killing bacteria with the two combination can it duplicates in vegetative different phase blocking-up effectively, can significantly strengthen the antibacterial action of medicine.Therefore; The present invention is through prescription screening, preparation process research; Prepare safe and effective and make things convenient for the long-acting composite Danofloxacin mesylate-amoxicillin suspension injection of administration, the treatment of current very popular livestock and poultry alimentary tract and respiratory tract disease is had more practical significance.
Summary of the invention
The object of the present invention is to provide Danofloxacin mesylate-amoxicillin suspension injection that a kind of safe and efficient and medication is applicable to that easily poultry are used and preparation method thereof and application process.
The object of the invention is realized through following technical scheme:
A kind of Danofloxacin mesylate-amoxicillin suspension injection that is applicable to that poultry are used, the preparation composition is counted by mass/volume:
(1) the Danofloxacin mesylate crude drug 2.5%;
(2) the amoxicillin crude drug 15%;
(3) suspending agent 1～3%;
(4) antioxidant 0.008～0.024%;
(5) emulsifying agent 0.20～0.60%;
(6) by volume/stereometer replenishes the full dose of injection grease to suspension injection;
Wherein
Described suspending agent is aluminum monostearate or polyvinylpyrrolidone;
Described antioxidant is the mixture of Butylated hydroxyanisole and butylated hydroxytoluene, and by the mass/volume preparation as follows: Butylated hydroxyanisole is 0.004～0.012%, and butylated hydroxytoluene is 0.004～0.012%;
Said emulsifying agent is the mixture of lecithin and Arlacel-80, and wherein lecithin counts 0.05～0.15% by mass/volume, and Arlacel-80 is 0.15～0.45%;
As priority scheme:
Said injection grease is wherein a kind of in injection soybean oil or Oleum Gossypii semen or the glyceryl triacetate.
The concentration of suspending agent counts 2% by mass/volume in the dicyandiamide solution.
Butylated hydroxyanisole and butylated hydroxytoluene in the described antioxidant count 0.012% by mass/volume.
Described injection grease is the injection soybean oil.
The concentration of emulsifier lecithin counts 0.05% by mass/volume in the dicyandiamide solution, and the concentration of Arlacel-80 counts 0.45% by mass/volume.
The applicant provides the method for preparing of a kind of Danofloxacin mesylate-amoxicillin suspension injection, and the preparation composition is counted by mass/volume:
(1) the Danofloxacin mesylate crude drug 2.5%;
(2) the amoxicillin crude drug 15%;
(3) suspending agent 1～3%;
(4) antioxidant 0.008～0.024%;
(5) emulsifying agent 0.20～0.60%;
(6) by volume/stereometer replenishes the full dose of injection grease to suspension injection.
Preparation process of the present invention is:
(1) by formula ratio Danofloxacin mesylate crude drug and amoxicillin crude drug are obtained the drug powder of particle diameter less than 10 μ m with superfine grinding method respectively;
(2) in the injection grease, add suspending agent by formula ratio,, make it to form gel, put under the room temperature subsequent use in 150～160 ℃ of heating and stirring 40min;
(3) in the gel of step (2), add the Danofloxacin mesylate and the amoxicillin crude drug micropowder of step (1); Press formula ratio and add antioxidant and emulsifying agent; Under 8000r/min, disperse 20min with the high speed homogenization dispersion machine, obtain Danofloxacin mesylate-Amuxil;
(4) pour step (3) suspension into colloid mill and continue to stir, obtain Danofloxacin mesylate-amoxicillin suspension injection.
The present invention prepares Danofloxacin mesylate-amoxicillin suspension injection and can in the poultry pharmacy, apply.
The present invention prepares the application of Danofloxacin mesylate-amoxicillin suspension injection dosage, is the 0.1mL/kg body weight in the dosage intramuscular injection of treating bacterial respiratory tract of poultry sensitivity or digestive tract infection, and per 2 days once.
Utilization of the present invention delays to discharge the method with delayed absorption; Adopt injection Semen sojae atricolor wet goods vegetable oil or grease and other adjuvants to form gel in the preparation process; Form the gel skeleton thing with gel form, process the oil solution of medicine, Danofloxacin mesylate and amoxicillin micropowder are scattered in the gel rubber system; Thereby delay the diffusion and the stripping of medicine; With prolong drug effective acting time, improve therapeutic effect to various sensitive organism associated diseases, be the good medicine of current digestive tract of treatment and respiratory tract infectious disease.
Danofloxacin mesylate-amoxicillin the suspension injection that adopts the present invention to obtain is white oily suspension; The sharp aroma that PCs is arranged; Danofloxacin mesylate content is 2.5%; Amoxicillin content is 15%, and suspension Chinese medicine particle diameter all less than 15 μ m, meets the requirement of suspension injection.The said preparation method for preparing is simple, constant product quality, and have tangible long-acting slow-release effect; Both improve the therapeutic effect of medicine, reduced administration frequency again, and can effectively treat digestive tract and respiratory tract disease; Reduce the labour cost of large-scale farming, save the man power and material, can the long period keep excellent curative; Be a kind of livestock and poultry alimentary tract and respiratory tract disease to be had the novel veterinary drug preparation of long-acting, have high practical value.
Description of drawings
Fig. 1 for injection Danofloxacin mesylate injection, amoxicillin injection and Danofloxacin mesylate-amoxicillin suspension injection to the pig intramuscular injection after Danofloxacin mesylate concentration-time curve in the blood plasma.
Fig. 2: for injection Danofloxacin mesylate injection, amoxicillin injection and Danofloxacin mesylate-amoxicillin suspension injection to the pig intramuscular injection after amoxicillin concentration-time curve in the blood plasma.
The specific embodiment
Below in conjunction with specific embodiment the present invention is explained further details.These embodiment only are used to the present invention is described and do not limit the scope of requirement of the present invention protection.
Target of the present invention is to obtain a kind of Danofloxacin mesylate-amoxicillin suspension injection.The medicinal ingredient of this injection is Danofloxacin mesylate and amoxicillin; The dicyandiamide solution of medicine is mainly vegetable oil or grease; Form gel with suspending agent; Form the gel skeleton thing with gel form, Danofloxacin mesylate and amoxicillin micropowder are scattered in process suspension injection in the gel rubber system with long-acting.Wherein: gel rubber system accounts for 70～80% of preparation cumulative volume, and drug powder accounts for 20～30% of preparation cumulative volume.In the preparation process: medicine Danofloxacin mesylate and amoxicillin obtain the micropowder of particle diameter less than 10 μ m through micronizing; Antioxidant is selected from one or both the mixture in Butylated hydroxyanisole and the butylated hydroxytoluene, and the concentration of two kinds of antioxidants is 0.004～0.012% (W/V), is preferably 0.012% (W/V).
Dicyandiamide solution is processed transparent gel solvent system by injection grease and suspending agent.Wherein the injection grease is a kind of in injection soybean oil, Oleum Gossypii semen or the glyceryl triacetate, preferred injection soybean oil; Injection grease heating-up temperature is 150～160 ℃, and the time is 40min; Suspending agent is selected from a kind of in aluminum monostearate, the polyvinylpyrrolidone, and the concentration of suspending agent is 1～3% (W/V) in the dicyandiamide solution, is preferably 2% (W/V); Said emulsifying agent is selected from one or both the mixture in lecithin and the Arlacel-80; The concentration of lecithin is 0.05～0.15% (W/V) in the dicyandiamide solution; Be preferably 0.05% (W/V), the concentration of Arlacel-80 is 0.15～0.45% (W/V), is preferably 0.45% (W/V).
Among the present invention, gel rubber system, Danofloxacin mesylate and amoxicillin micropowder, antioxidant, emulsifying agent need in colloid mill, fully to mix just to obtain the long-acting suspension injection in Danofloxacin mesylate-amoxicillin.To do detailed introduction to the present invention through concrete embodiment below, optical microscope is used in the morphologic observation of test Chinese medicine particle diameter, and particle size determination is used the microscope micrometer, and the medicament contg of injection adopts HPLC-uv detection method.The preparation of suspension injection is all carried out in sterilizing room.
Suspension injection preparation technology is following in Danofloxacin mesylate of the present invention-amoxicillin:
(1) gets Danofloxacin mesylate and amoxicillin crude drug, pulverize with super micron mill between the sterile working and obtain particle diameter less than 10 microns Danofloxacin mesylate and amoxicillin crude drug micropowder.
(2) get in injection soybean oil 1000mL to the 1000mL beaker, add aluminum monostearate 10g, 150～160 ℃ of heated and stirred 40min process transparent gel, filter with 65 order yarns sieve, store subsequent use under the room temperature.
(3) get among the Danofloxacin mesylate crude drug micropowder 25g and amoxicillin micropowder 150g to 1000mL beaker of step (1) acquisition; The gel that adding few steps (2) makes fully soaks into medicine; Add Butylated hydroxyanisole 0.04g, butylated hydroxytoluene 0.04g, lecithin 0.5g and Arlacel-80 1.5g; Add gel that step (2) makes again to 1000mL, disperse 20min at 8000r/min with the high speed homogenization dispersion machine.
(4) drug system that step (3) is obtained is poured into and is continued to stir 4min in the colloid mill, after conventional treatment such as aseptic subpackaged, sterile sealing, promptly gets this routine Danofloxacin mesylate-amoxicillin suspension injection again.
With reference to " People's Republic of China's veterinary drug allusion quotation " 2005 editions, to the appearance character of this Danofloxacin mesylate-amoxicillin suspension injection, particle size; The settling volume ratio, dispersibility again, projects such as syringeability detect; Appearance character as a result, distributed test again, each item indexs such as cleansing pin test all meet the requirements; The settling volume ratio is near 1; The even particle size distribution of preparation, diameter of aspirin particle are all less than 15 μ m, and Danofloxacin mesylate and amoxicillin medicament contg are respectively 100.5% and 98.2% of dosage.
Suspension injection preparation technology is following in Danofloxacin mesylate of the present invention-amoxicillin:
(1) gets Danofloxacin mesylate and amoxicillin crude drug, obtain Danofloxacin mesylate and the amoxicillin crude drug micropowder of particle diameter with super micron mill through micronizing between the sterile working less than 10 μ m.
(2) get in injection soybean oil 1000mL to the 1000mL beaker, add aluminum monostearate 20g, 150～160 ℃ of heated and stirred 40min process transparent gel, filter with 65 order yarns sieve, store subsequent use under the room temperature.
(3) get Danofloxacin mesylate micropowder 25g that step (1) obtains and amoxicillin micropowder 150g in the 1000mL beaker; The gel that adding few steps (2) makes fully soaks into medicine; Add Butylated hydroxyanisole 0.12g, butylated hydroxytoluene 0.12g, lecithin 0.5g and Arlacel-80 3.0g; Add gel that step (2) makes again to 1000mL, high speed homogenization dispersion machine 8000r/min disperses 20min.
(4) drug system after step (3) is disperseed is poured into and is continued to stir 4min in the colloid mill, after conventional treatment such as aseptic subpackaged, sterile sealing, promptly gets this routine Danofloxacin mesylate-amoxicillin suspension injection again.
With reference to " People's Republic of China's veterinary drug allusion quotation " 2005 editions, to the appearance character of this Danofloxacin mesylate-amoxicillin suspension injection, particle size; The settling volume ratio, dispersibility again, projects such as syringeability detect; Appearance character as a result, distributed test again, each item indexs such as cleansing pin test all meet the requirements; The settling volume ratio is near 1; The even particle size distribution of preparation, diameter of aspirin particle are all less than 15 μ m, and Danofloxacin mesylate and amoxicillin medicament contg are respectively 101.6% and 98.6% of dosage.
The preparation technology of Danofloxacin mesylate of the present invention-amoxicillin suspension injection is following:
(1) gets Danofloxacin mesylate and amoxicillin crude drug, obtain Danofloxacin mesylate and the amoxicillin crude drug micropowder of particle diameter with super micron mill through micronizing between the sterile working less than 10 μ m.
Trial design in the experiment of table 1 bioavailability
Plasma sample is measured
Danofloxacin mesylate is measured: get plasma sample and thaw naturally in room temperature.Accurately draw blood plasma 0.5mL in centrifuge tube, add acetonitrile 2mL, vortex 5min with the centrifugal 10min of 10000r/min, gets supernatant in another centrifuge tube, and precipitate adds acetonitrile 1mL again and extracts, and merges the supernatant that extracts once more, and 50 ℃ of nitrogen dry up.Residue is crossed 0.22 μ m filter membrane with mobile phase 0.5mL dissolving, measures with HPLC.
The amoxicillin is measured: get plasma sample and thaw naturally in room temperature.Accurately draw blood plasma 0.5mL in centrifuge tube, add ultra-pure water 1mL, the whirlpool mixing adds 20% trichloroacetic acid solution 1mL; Vortex 2min with the centrifugal 10min of 10000r/min, gets supernatant in another centrifuge tube, adds 7% formalin 0.5mL; The whirlpool mixing heats 45min in 100 ℃ of boiling water baths, cools off 5min with frozen water immediately again, adds sodium chloride 0.5g; Ether 5mL, with 230 times/min vibration 20min, 60 ℃ of water-bath nitrogen dry up on agitator.Residue reaches 0.22 μ m filter membrane with mobile phase 0.5mL dissolving, measures with HPLC.
The drafting of working curve
Precision is drawn blank plasma 0.45mL, places the centrifuge tube of 10mL, adds an amount of Danofloxacin mesylate standard solution and makes Danofloxacin mesylate concentration be respectively 0.025,0.05,0.1,0.5,1 and 2.5 μ g/mL; Other gets blank plasma 0.45mL, adds an amount of amoxicillin standard solution, makes amoxicillin concentration be respectively 0.1,0.25,0.5,1,5 and 20 μ g/mL, leaves standstill 30min behind the whirlpool mixing 1min.After the plasma sample disposal methods, measure with the HPLC method.3 repetitions of each concentration; Repeat 3 days, the gained peak area is carried out match, the drawing curve with corresponding interpolation concentration; Draw regression equation: Danofloxacin mesylate y=103.16x-0.2771 (r=0.9999), amoxicillin y=24.441x+0.1262 (r=0.9999).
Detectability and quantitative limit
Get the standard solution that blank porcine blood plasma adds debita spissitudo; Prepare the lower sample of a series of concentration, detect by carrying out HPLC after the preparation of plasma sample method for preparing, each concentration repeats 5 times; Different time repetitive operation 5 times; Get and measure average S and N as a result for 25 times, the least concentration of sample is the detectability of method when reaching S/N=3, and the least concentration of sample was confirmed as the quantitative limit (LOQ) of method when the response rate and Variation Lines number average reached the quantitative analysis requirement.The detection of this method is limited to: Danofloxacin mesylate 0.02 μ g/mL, amoxicillin 0.05 μ g/mL; Quantitatively be limited to Danofloxacin mesylate 0.05 μ g/mL, amoxicillin 0.10 μ g/mL.
The response rate and precision test
Accurately draw blank plasma 0.45mL in the centrifuge tube of 10mL; Add an amount of titer; Make Danofloxacin mesylate concentration be respectively 0.05,0.5 and 5 μ g/mL, amoxicillin concentration is respectively 0.1,2 and 20 μ g/mL, leaves standstill 30min behind the whirlpool mixing 1min.Press the plasma sample sample treatment and handle, measure with HPLC.Each concentration sample determination 3 times repeats 3 days, respectively calculate recovery rate and in a few days, the coefficient of variation in the daytime.Average recovery rate when Danofloxacin mesylate interpolation concentration is 0.05～5 μ g/mL in the blood plasma is 89.4～100.6%, and RSD is 1.3～3.0%; Response rate when amoxicillin interpolation concentration is 0.10～20 μ g/mL is 89.0%～97.7%, and RSD is 6.1%～12.7%.Show that this method can satisfy pharmacokinetics test.
High-efficient liquid phase chromatogram condition
Danofloxacin mesylate is measured: chromatographic column is an Agilent SB-Aq chromatographic column, 250mm * 4.6mm, 5 μ m; Mobile phase: the 0.05mol/L phosphate sodium dihydrogen buffer solution (contains every 1000mL and contains ammonium acetate 2.3g, pH3.0): acetonitrile=80: 20 (V/V); Detect wavelength 282nm; Flow velocity: 1.0mL/min; Column temperature: 35 ℃; Sampling volume: 20 μ L.
The amoxicillin is measured: chromatographic column is an Agilent SB-Aq chromatographic column, 250mm * 4.6mm, 5 μ m; Mobile phase: 0.05mol/L phosphate sodium dihydrogen buffer solution (pH5.6): acetonitrile=80: 20 (v/v); Detect wavelength: excitation wavelength: 355nm, emission wavelength: 435nm; Flow velocity: 1.0mL/min; Column temperature: 35 ℃; Sampling volume: 80 μ L.
Date processing
With pharmacokinetics software WinNonlin (Version 5.2.1) blood drug level-time data is carried out match, calculate pharmacokinetic data available with non-compartment model, relatively the pharmacokinetic parameters of 3 kinds of different preparations is calculated relative bioavailability.
Result and analysis
Behind the agent of pig difference intramuscular injection Danofloxacin mesylate normal injection, amoxicillin normal injection agent and Danofloxacin mesylate-Amuxil; The concentration-time curve of Danofloxacin mesylate and amoxicillin is seen accompanying drawing 1 in the blood plasma, and pharmacokinetic parameters is seen table 2.The result shows, after Danofloxacin mesylate of the present invention-amoxicillin suspension injection intramuscular injection, and the elimination half-life (T of Danofloxacin mesylate
1/2 λ z) having prolonged 3 times than the agent of Danofloxacin mesylate normal injection, the elimination half-life of amoxicillin prolongs 14 times than amoxicillin normal injection agent.Therefore, Danofloxacin mesylate of the present invention-amoxicillin suspension injection has slow release effect preferably, and bioavailability is high.
The pharmacokinetic parameters of table 2 pig intramuscular injection Danofloxacin mesylate and the 3 kinds of different preparations in amoxicillin
Annotate: λ z in the table: terminal elimination rate constant; T
1/2 λ z: eliminate the half-life; T
Max: the time that reaches peak concentration; C
Max: peak concentration; AUC
All: area under the drug-time curve; Vz: apparent volume of distribution; CLz: elimination rate constant; MRT: mean residence time.
Embodiment 11 (application implementation example 2)
After selecting the conventional 7d of raising of 20 health pig, press 0.1mL/kg b.w. dosage intramuscular injection embodiment 2 preparations (Danofloxacin mesylate-amoxicillin suspension injection) 1 time, and the labelling injection site.Respectively at 1,3,7,21 after the administration, 42d randomly draws 4 pigs and butchers, and gets longissimus dorsi muscle, liver, kidney, stomach fat and injection site muscle, and is in-20 ℃ of storages, to be measured behind the homogenizing.
Tissue sample is handled
Danofloxacin mesylate: muscle, fat, liver, the kidney taking-up room temperature of-20 ℃ of preservations are thawed.Take by weighing 5g homogeneous structure in 50mL tool plug plastic centrifuge tube, add phosphate buffer (pH7.0) 10mL, the vortex mixing, the centrifugal 10min of 10000r/min gets supernatant to another test tube, repeats to extract once.Merge supernatant twice, add normal hexane 20mL, the vortex mixing, the centrifugal 10min of 10000r/min gets supernatant, and re-extract once merges supernatant twice, uses the SPE column purification.
HLB SPE post is used methanol, each 3mL activation of water successively, the sample upper prop, and (1: 4, the 3mL methanol-eluted fractions was used in v/v) drip washing then, collects eluent with the 3mL methanol.In 50 ℃ of water bath methods, be settled to 1mL with mobile phase.Cross 0.22 μ m microporous filter membrane, advance HPLC and detect.
Amoxicillin: take by weighing muscle and fatty sample 5g places 50mL glass centrifuge tube, add phosphate buffer 15mL, mixing; Then add normal hexane 5mL, mixing, the centrifugal 15min of 3000r/min; Discard the normal hexane layer, shift water layer to another clean glass centrifuge tube, repeat to extract 2 times with phosphate buffer 10mL, 5mL and normal hexane 5mL then; Abandon the normal hexane layer, merge 3 times water layer.Water intaking layer supernatant 3mL supplies SPE to purify.
Take by weighing kidney, liver sample 5g, place the 50mL centrifuge tube, add acetonitrile 20mL, mixing adds normal hexane 5mL again, and mixing leaves standstill 5min, and the centrifugal 15min of 3000r/min discards the normal hexane layer, shifts acetonitrile layer to another clean centrifuge tube A.Add phosphate buffer 10mL and normal hexane 5mL in the residue, mixing leaves standstill; The centrifugal 15min of 3000r/min abandons the normal hexane layer, and water layer is transferred among another clean glass centrifuge tube B; Repeat to extract once with phosphate buffer 10mL and normal hexane 5mL, abandon the normal hexane layer, merge 2 times extracting solution.From centrifuge tube A, get 2mL to centrifuge tube C, dry up in 45～50 ℃ of water-bath nitrogen.From centrifuge tube B, get solution 2mL to centrifuge tube C, fully mixing supplies SPE to purify.
The C18 solid-phase extraction column is used each the 3mL activation of methanol, water, sodium chloride solution and phosphate buffer successively, the sample upper prop, and water 1mL drip washing, acetonitrile 3mL eluting, 10mL glass centrifuge tube is collected eluent; 45～50 ℃ of water-bath nitrogen dry up, and with standard diluent 0.5mL dissolving, change the polypropylene centrifuge tube of 1.5mL over to, add anhydrous benzene formic anhydride solution 25 μ L; Mixing, 50 ℃ of water-bath 5min, water-bath is cooled to room temperature fast; Add derivatization reagent 250 μ L, mixing, 65 ℃ of water-bath 30min; Water-bath is cooled to room temperature fast, and 4 ℃ of centrifugal 10min of 15000r/min get supernatant and supply HPLC to detect.
The drafting of working curve
A collection of blank muscle, liver, kidney, fat are added an amount of Danofloxacin mesylate or amoxicillin titer respectively; Make that Danofloxacin mesylate concentration is respectively 20,100,400,1000 and 4000 μ g/kg in the tissue, amoxicillin concentration is respectively 25,250,1000,5000 and 15000 μ g/kg, after sample treatment; HPLC detects; Each concentration sample determination 5 times repeats 3 days, and the gained peak area is carried out match with corresponding interpolation concentration; The drawing curve is obtained working curve regression equation and the correlation coefficient of medicine in various edible tissues.The result shows that Danofloxacin mesylate and amoxicillin concentration and the peak area in four kinds of tissues has good linear relationship, and correlation coefficient (r) is all greater than 0.999.
Detectability and quantitative limit
Get the Danofloxacin mesylate and the amoxicillin titer of blank pig muscle, liver, kidney, fat adding debita spissitudo; Make the medicine that contains low concentration in the sample; After sample treatment, carrying out HPLC detects; Each concentration repeats 5 times, different time repetitive operation 5 times, gets 25 times and measures average S and N as a result; The least concentration of sample is the detectability (LOD) of this method when reaching S/N=3, and the least concentration of sample was the quantitative limit (LOQ) of method when the response rate and Variation Lines number average reached the quantitative analysis requirement.This method quantitatively is limited to Danofloxacin mesylate 20 μ g/kg, amoxicillin 25 μ g/kg to pig muscle, liver, kidney and fatty tissue Chinese medicine.
The response rate and precision
Add an amount of Danofloxacin mesylate and amoxicillin titer respectively at blank muscle, liver, kidney, fat; Make and organize Chinese medicine concentration to be respectively LOQ, MRL and 2MRL; After sample treatment, HPLC detects, each concentration sample determination 5 times; Repeat 3 days, respectively calculate recovery rate, the coefficient of variation (RSD) in a few days and in the daytime.The result shows that in adding concentration range, the response rate of Danofloxacin mesylate is 70%～110%, and the response rate of amoxicillin is 70%～95%, and RSD meets the requirement of residue of veterinary drug quantitative detecting method less than 10%.
Chromatographic condition
DFM: chromatographic column: Agilent SB-Aq chromatographic column, 250mm * 4.6mm, 5 μ m; Detect wavelength: 282nm; Mobile phase: phosphate sodium dihydrogen buffer solution (0.05mol/L, every 1000mL adds the 2.3g ammonium acetate, pH3.0): acetonitrile=82: 18 (v/v); Flow velocity: 1.0mL/min; Column temperature: 35 ℃; Sampling volume: 20 μ L.
AMO: chromatographic column: Agilent SB-C18 chromatographic column, 150mm * 4.6mm, 5 μ m; Detect wavelength: 325nm; Mobile phase: phosphate buffer (ADSP 4.97g, phosphate dihydrate disodium hydrogen 10.14g, Sodium Thio Sulphate(Anhydrous) 3.89g): second eyeball=65: 35 (v/v); Flow velocity: 1.0mL/min; Column temperature: 35 ℃; Sampling volume: 100 μ L.
Date processing
Danofloxacin mesylate and the amoxicillin MRL (MRL) in each tissue of pig according to European drug administration (EMEA) formulation; The method that adopts EMEA to recommend uses WT1.4 software to calculate the off-drug period of these two kinds of medicines in each tissue with bilateral 95% confidence limit.
Result and analysis
Behind pig intramuscular injection Danofloxacin mesylate-amoxicillin suspension injection, Danofloxacin mesylate and the amoxicillin mean concentration in each sampling time point and various tissue is seen table 3 and table 4, and the off-drug period after the drug withdrawal in the various tissues is seen table 5.
Table 3 Danofloxacin mesylate is residual quantity in the various edible tissues of pig
ND: do not detect
Table 4 amoxicillin is residual quantity in the various edible tissues of pig
ND: do not detect
Table 5 Danofloxacin mesylate and amoxicillin be residual eliminating equation, half-life and off-drug period in each tissue of pig
Claims (2)
1. Danofloxacin mesylate-amoxicillin suspension injection that is applicable to that poultry are used is characterized in that preparation is formed to count by mass/volume:
(1) the Danofloxacin mesylate crude drug 2.5%;
(2) the amoxicillin crude drug 15%;
(3) suspending agent 2%;
(4) antioxidant 0.008～0.024%;
(5) emulsifying agent 0.20～0.60%;
(6) by volume/stereometer replenishes the full dose of injection grease to suspension injection;
Wherein
Described suspending agent is aluminum monostearate or polyvinylpyrrolidone;
Described antioxidant is the mixture of Butylated hydroxyanisole and butylated hydroxytoluene, counts 0.012% by mass/volume;
Said emulsifying agent is the mixture of lecithin and Arlacel-80, and wherein lecithin counts 0.05% by mass/volume, and Arlacel-80 is 0.45%;
Said injection grease is the injection soybean oil.
2. the method for preparing of the described preparation of claim 1 is characterized in that according to the following step:
(1) by formula ratio Danofloxacin mesylate crude drug and amoxicillin crude drug are obtained the drug powder of particle diameter less than 10 μ m with superfine grinding method respectively;
(2) in the injection grease, add suspending agent by formula ratio,, make it to form gel, put under the room temperature subsequent use in 150～160 ℃ of heating and stirring 40min;
(3) in the gel of step (2), add the Danofloxacin mesylate and the amoxicillin crude drug micropowder of step (1); Press formula ratio and add antioxidant and emulsifying agent; Under 8000r/min, disperse 20min with the high speed homogenization dispersion machine, obtain Danofloxacin mesylate-Amuxil;
(4) pour step (3) suspension into colloid mill and continue to stir, obtain Danofloxacin mesylate-amoxicillin suspension injection.
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| CN102552160B (en) * | 2010-12-24 | 2013-07-17 | 华中农业大学 | Veterinary danofloxacin mesylate slow-release microsphere preparation as well as preparation method and application of veterinary danofloxacin mesylate slow-release microsphere preparation |
| CN102600075A (en) * | 2012-04-19 | 2012-07-25 | 烟台海研制药有限公司 | Danofloxacin mesylate suspension and preparation method thereof |
| CN103536531A (en) * | 2013-10-30 | 2014-01-29 | 王玉万 | Preparation method of long-acting injection containing danofloxacin mesylate |
| CN106727567A (en) * | 2016-11-25 | 2017-05-31 | 成都乾坤动物药业股份有限公司 | A kind of Amoxicillin Enrofloxacin oil suspension and preparation method thereof |
| CN110221008A (en) * | 2019-06-25 | 2019-09-10 | 华中农业大学 | A method of Danofloxacin mesylate in detection Swine plasma and alveolar fluid |
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| CN101406450A (en) * | 2007-10-12 | 2009-04-15 | 河南农业大学 | Technique for preparing compound amoxicillin oil suspension injection |
| CN101485658A (en) * | 2009-02-27 | 2009-07-22 | 江西新世纪民星动物保健品有限公司 | Method for preparing amoxicillin-ofloxacin suspension injection |
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| CN101406450A (en) * | 2007-10-12 | 2009-04-15 | 河南农业大学 | Technique for preparing compound amoxicillin oil suspension injection |
| CN101485658A (en) * | 2009-02-27 | 2009-07-22 | 江西新世纪民星动物保健品有限公司 | Method for preparing amoxicillin-ofloxacin suspension injection |
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