CN102772359A - Enrofloxacin injection and preparation method thereof - Google Patents
Enrofloxacin injection and preparation method thereof Download PDFInfo
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- CN102772359A CN102772359A CN201210274300XA CN201210274300A CN102772359A CN 102772359 A CN102772359 A CN 102772359A CN 201210274300X A CN201210274300X A CN 201210274300XA CN 201210274300 A CN201210274300 A CN 201210274300A CN 102772359 A CN102772359 A CN 102772359A
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- zhusheye
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Abstract
The invention relates to an enrofloxacin injection. The enrofloxacin injection comprises, by weight, 21-25 parts of enrofloxacin, 5-15 parts of lactic acid, 0.1-0.5 part of sodium hydrogensulfite and 59.5-73.9 parts of water for injection. The enrofloxacin injection is faintly acid and can be compatible with most of acidic drugs; the concentration of the enrofloxacin is high and reaches 21-25%, so that the single administration dosage can be reduced, and stimulation and harm on animals are reduced; and besides, potential of hydrogen (pH) of the injection is 5.0-6.9, the range of the pH of the injection is identical to that of organism muscular tissue, the injection is small in stimulation, safe in clinical usage, obvious in curative effects and controllable in quality, curative effects of the enrofloxacin injection are better than those of existing products, and the whole preparation process is simple, practicable and applicable to modern mass production.
Description
Technical field
The invention belongs to the chemicals field, especially a kind of ENNUOSHAXING ZHUSHEYE and preparation method thereof.
Background technology
(enrofloxacin ERFX) is third generation QNS to enrofloxacin, is the animal specific antimicrobial drug, is widely used in the prevention and the treatment of various animal infectious diseases.Enrofloxacin all has good action to escherichia coli, Salmonella, Cray Bai Shi bacillus, pasteurellosis bacillus, pleuropneumonia actinomycetes, erysipelas bacillus, Bacillus proteus, clayey Serratieae, suppurative corynebacterium, deteriorated blood Bo Teshi bacillus, staphylococcus aureus, mycoplasma, chlamydia etc.Enrofloxacin can be used for sensitive organism and the digestive system due to the mycoplasma, respiratory system, urinary system and the skin soft-tissue infection property disease of pig, cattle, fowl, dog, cat.
Enrofloxacin is on 6 of quinolones structure, to have introduced fluorine atom, has strengthened the affinity of medicine and antibacterial target position and has enlarged antimicrobial spectrum; Cyclopropyl on 1 can promote the tissue permeability of medicine.Enrofloxacin metabolism in animal body is that ciprofloxacin is brought into play antibacterial activity.Identical with the antibiotic mechanism of other Du-6859as, enrofloxacin is a target with DNA (DNA) enzyme of antibacterial.The distrand DNA distortion of antibacterial becomes loop shape or helical form (being called superhelix), makes DNA form supercoiled enzyme and is called the DNA gyrase.Medicine suppresses the cutting and the linkage function of this enzyme through combining with DNA of bacteria gyrase subunit A, and chromosome is caused irreversible lesion, stops bacterial cell division and brings into play antibacterial action.Simultaneously, it has the penetration of height to bacteria cell wall, and destroys the after birth of antibacterial.Therefore, enrofloxacin is to reach bacteriolyze, antibacterial purpose through dual antibacterial action.A large amount of experiment confirms, enrofloxacin generally is better than ciprofloxacin, norfloxacin and Tri-Biocin to the curative effect of disease that antibacterial, mycoplasma etc. causes.
Enrofloxacin pharmacokinetics in vivo is characterized as: for oral administration and the intramuscular injection post-absorption is rapid; The peak value of blood Chinese medicine concentration and remedy,tissue's substrate concentration all is higher than most of pathogen and mycoplasma MIC several times to tens times, and the elimination half-life is long, the interior distribution of body is extensive, apparent volume of distribution is big.Enrofloxacin can be assembled in phagocyte, is 2～8 times of blood drug level, discharges rapidly from inflammation part.A lot of researchs show that the drug level of enrofloxacin in serum often is lower than the concentration in other tissue.Because the special medicine mechanical characteristics of this medicine makes the special-purpose enrofloxacin of poultry become the first-selected antibacterials of the infection of treatment deep tissue, pulmonary infection (various pneumonia) and pyoderma.Particularly it can reach high concentration at lung and urogenital tract, the respiratory tract disease, urogenital infections and the mycoplasma that therefore are widely used in treating animal.
Have many superioritys on the drug effect of enrofloxacin pathogen and the characteristics of pharmacokinetics in animal body; Have that antibacterial activity is strong, has a broad antifungal spectrum, oral absorption is good, bioavailability is high, tissue distribution is wide, long half time, be difficult for producing drug resistance, to the fastbacteria of other antimicrobial drugs effectively and characteristics such as untoward reaction is less relatively, the while toxic and side effects is less.Based on the plurality of advantages of enrofloxacin, it has very high using value at veterinary clinic.
At present mostly clinical case is mixed infection, needs combination drug, and ENNUOSHAXING ZHUSHEYE is basic formulations or neutral preparation at present, can't Combined application with a lot of acidic drugs, be unfavorable for the clinical medicine compatibility, and restricted the ENNUOSHAXING ZHUSHEYE clinical application range; At present,,, increased administration number of times, improved cost because the concentration of enrofloxacin is lower though also there is acid ENNUOSHAXING ZHUSHEYE, the waste resource, and also method for preparing is also comparatively complicated.
Through retrieval, find the following patent publication us relevant with application of the present invention:
1, a kind of ENNUOSHAXING ZHUSHEYE and preparation method thereof (CN201010179923.X), the pH of injection of the present invention are neutral, comprise 10～20 parts enrofloxacin, also comprise 0.1～0.5 part of antioxidant, 0.1～0.5 part of chelating agen.The pH value of the ENNUOSHAXING ZHUSHEYE that the present invention processes is for neutral; Nonirritant when animal injection is used like this; And make in the injection contents of enrofloxacin bring up to 20%, and having increased the therapeutic effect of single-dose greatly, the ENNUOSHAXING ZHUSHEYE character of processing simultaneously is stable; Long-term storage does not have crystallization and separates out, and is good, the non-stimulated injection of a kind of therapeutic effect.
2, a kind of acid ENNUOSHAXING ZHUSHEYE and preparation method thereof (CN201010582402.9) the present invention relates to acid ENNUOSHAXING ZHUSHEYE and preparation method thereof.The component of this injection comprises enrofloxacin, lincomycin hydrochloride, acetic acid and benzyl alcohol; Wherein, Enrofloxacin 5g, an amount of 1.25g of acetic acid, benzyl alcohol 1ml, lincomycin hydrochloride 0.5%-5% and water for injection 100ml, aforementioned each component deal can change according to the clear and definite ratio of above-mentioned material and solution.This injection preparation may further comprise the steps: get the 5g enrofloxacin and add 80-90ml water for injection, stir; Be heated to 50～60 ℃, put coldly, add 1.25g acetic acid and 1ml benzyl alcohol; After stirring; Add hydrochloric acid woods Mycosporin 0.5-5g, dissolve during stirring, add to 100ml with water for injection at last; Fill, autoclaving promptly get.
Through contrast, application of the present invention has the different of essence with above-mentioned patent publication us.
Summary of the invention
The objective of the invention is to overcome the weak point of prior art, a kind of weakly acidic high concentration ENNUOSHAXING ZHUSHEYE and preparation method thereof is provided.
The objective of the invention is to realize through following technical scheme:
A kind of ENNUOSHAXING ZHUSHEYE, its composition and parts by weight are following:
21～25 parts of enrofloxacins;
5～15 parts of lactic acid;
0.1～0.5 part of sodium sulfite;
59.5～73.9 parts of waters for injection.
And, described ENNUOSHAXING ZHUSHEYE, its composition and parts by weight are following:
21 parts of enrofloxacins;
9 parts of lactic acid;
0.2 part of sodium sulfite;
69.8 parts of waters for injection.
And, described ENNUOSHAXING ZHUSHEYE, its composition and parts by weight are following:
25 parts of enrofloxacins;
15 parts of lactic acid;
0.5 part of sodium sulfite;
59.5 parts of waters for injection.
And the pH of said ENNUOSHAXING ZHUSHEYE is 5.0～6.9.
And the usage of said ENNUOSHAXING ZHUSHEYE and consumption are: according to 2.5mg/kg b.w intramuscular injection, and 1d administration 1～2 time, continuous use 2～3d.
The method for preparing of above-mentioned ENNUOSHAXING ZHUSHEYE, step is following:
⑴ take by weighing and be used for dissolved water for injection;
⑵ add sodium sulfite by weight in step ⑴ solution, be stirred to dissolving fully;
⑶ add enrofloxacin by weight in step ⑵ solution, stir;
⑷ add lactic acid by weight in step ⑶ solution, be stirred to the solution clarification;
⑸ add remaining water for injection to full dose in step ⑷ solution, filter, sterilize, and promptly gets ENNUOSHAXING ZHUSHEYE.
Advantage of the present invention and good effect are:
1, ENNUOSHAXING ZHUSHEYE of the present invention is a faintly acid, can use with most of acidic drug compatibilities, has enlarged the clinical application range of ENNUOSHAXING ZHUSHEYE widely.
2, the concentration of enrofloxacin is high in the prepared ENNUOSHAXING ZHUSHEYE of the present invention, has reached 21～25%, can reduce single-dose dosage, has reduced stimulation and injury to animal; And the pH of this injection is 5.0～6.9, and is identical with body muscular tissue pH scope, little, clinical safe in utilization, the determined curative effect, quality controllable of zest.
3, method for preparing scientific formulation of the present invention, each component raw material are easy to get, and with low cost, and curative effect is superior to existing product, and the whole preparation process simple possible is suitable for modern large-scale production.
Description of drawings
Fig. 1 is the chromatogram (0.2 μ g/mL) of enrofloxacin reference substance of the present invention;
Fig. 2 is a blank plasma chromatogram of the present invention;
Fig. 3 adds medicine chromatogram (0.5 μ g/mL) for blank plasma of the present invention;
Fig. 4 surveys the plasma sample chromatogram for the present invention;
Fig. 5 curve chart during for the medicine of pig single dose intramuscular injection ENNUOSHAXING ZHUSHEYE of the present invention.
The specific embodiment
Through specific embodiment the present invention is made further detailed description below, following examples are descriptive, are not determinate, can not limit protection scope of the present invention with this.
Need to prove: like no specified otherwise, employed method is conventional method in the present patent application; Employed each component is the commercially available prod.
A kind of ENNUOSHAXING ZHUSHEYE comprises principal agent, cosolvent, antioxidant and water for injection, wherein:
The constitutive material medicine and the parts by weight of principal agent are:
Enrofloxacin 21～25;
Cosolvent constitutive material and parts by weight are:
Antioxidant constitutive material and parts by weight are:
Sodium sulfite 0.1～0.5;
The parts by weight of water for injection are: 59.5～73.9.
The pH of above-mentioned ENNUOSHAXING ZHUSHEYE is 5.0～6.9.
The usage of above-mentioned ENNUOSHAXING ZHUSHEYE and consumption are: according to 2.5mg/kg b.w intramuscular injection, and 1d administration 1～2 time, continuous use 2～3d.
The method for preparing of above-mentioned ENNUOSHAXING ZHUSHEYE, step is following:
⑴ take by weighing the water for injection of dissolving with various medicaments;
⑵ add antioxidant, is stirred to dissolving fully;
⑶ add principal agent, stirs;
⑷ add cosolvent, is stirred to the solution clarification;
⑸ add remaining water for injection to full dose, and filtration, packing, sterilization promptly get ENNUOSHAXING ZHUSHEYE.
A kind of ENNUOSHAXING ZHUSHEYE, its composition and parts by weight are following:
Enrofloxacin 21g;
Lactic acid 5g;
Sodium sulfite 0.1g;
Water for injection 73.9g.
The method for preparing of above-mentioned ENNUOSHAXING ZHUSHEYE, step is following:
⑴ take by weighing the 50g injection water;
⑵ add sodium sulfite 0.1g in step ⑴ solution, be stirred to dissolving fully;
⑶ add enrofloxacin 21g in step ⑵ solution, stir;
⑷ add lactic acid 5g in step ⑶ solution, be stirred to the solution clarification;
⑸ add residue water for injection to 100g in step ⑷ solution, filtration, packing, sterilization promptly get ENNUOSHAXING ZHUSHEYE.
Embodiment 2
A kind of ENNUOSHAXING ZHUSHEYE, its composition and parts by weight are following:
Enrofloxacin 22g;
Lactic acid 8g;
Sodium sulfite 0.2g;
Water for injection 69.8g.
The method for preparing of above-mentioned ENNUOSHAXING ZHUSHEYE, step is following:
⑴ take by weighing the 50g injection water;
⑵ add sodium sulfite 0.2g in step ⑴ solution, be stirred to dissolving fully;
⑶ add enrofloxacin 22g in step ⑵ solution, stir;
⑷ add lactic acid 8g in step ⑶ solution, be stirred to the solution clarification;
⑸ add residue water for injection to 100g in step ⑷ solution, filtration, packing, sterilization promptly get ENNUOSHAXING ZHUSHEYE.
A kind of ENNUOSHAXING ZHUSHEYE, its composition and parts by weight are following:
Enrofloxacin 23g;
Lactic acid 10g;
Sodium sulfite 0.2g;
Water for injection 66.8g.
The method for preparing of above-mentioned ENNUOSHAXING ZHUSHEYE, step is following:
⑴ take by weighing the 50g injection water;
⑵ add sodium sulfite 0.2g in step ⑴ solution, be stirred to dissolving fully;
⑶ add enrofloxacin 23g in step ⑵ solution, stir;
⑷ add lactic acid 10g in step ⑶ solution, be stirred to the solution clarification;
⑸ add residue water for injection to 100g in step ⑷ solution, filtration, packing, sterilization promptly get ENNUOSHAXING ZHUSHEYE.
Embodiment 4
A kind of ENNUOSHAXING ZHUSHEYE, its composition and parts by weight are following:
Enrofloxacin 24g;
Lactic acid 12g;
Sodium sulfite 0.4g;
Water for injection 63.6g.
The method for preparing of above-mentioned ENNUOSHAXING ZHUSHEYE, step is following:
⑴ take by weighing the 50g injection water;
⑵ add sodium sulfite 0.4g in step ⑴ solution, be stirred to dissolving fully;
⑶ add enrofloxacin 24g in step ⑵ solution, stir;
⑷ add lactic acid 12g in step ⑶ solution, be stirred to the solution clarification;
⑸ add residue water for injection to 100g in step ⑷ solution, filtration, packing, sterilization promptly get ENNUOSHAXING ZHUSHEYE.
A kind of ENNUOSHAXING ZHUSHEYE, its composition and parts by weight are following:
Enrofloxacin 25g;
Lactic acid 15g;
Sodium sulfite 0.5g;
Water for injection 59.5g.
The method for preparing of above-mentioned ENNUOSHAXING ZHUSHEYE, step is following:
⑴ take by weighing 50g water for injection;
⑵ add sodium sulfite 0.5g in step ⑴ solution, be stirred to dissolving fully;
⑶ add enrofloxacin 25g in step ⑵ solution, stir;
⑷ add lactic acid 15g in step ⑶ solution, be stirred to the solution clarification;
⑸ add residue water for injection to 100g in step ⑷ solution, filtration, packing, sterilization promptly get ENNUOSHAXING ZHUSHEYE.
Embodiment 6
ENNUOSHAXING ZHUSHEYE of the present invention is to the clinical efficacy test of colibacillosis of pigs.
(1) material and method
1, experiment material
The ENNUOSHAXING ZHUSHEYE of trial drug: embodiment 5, intramuscular injection, every day 1 time, logotype three days;
Control drug: ENNUOSHAXING ZHUSHEYE (baytril): lot number KP06H8U, content 5%, Bayer (Sichuan) animal health company limited;
2, experimental animal: pig farm, Jining City, Shandong Province, 30 have been diagnosed as the pig that colibacillosis infects, morbidity age in days 80 days.
3, test method: with 30 pigs of natural occurrence, be divided into three groups of I, II, III, every group of 10 pigs carry out administered intramuscular to the experimental group pig respectively by table 1.Measure rectal temperature before each medication, observe and the record clinical symptoms.Observation period is 15d, and to every pig before and after making an experiment weighing and body condition is observed.
Table 1 test pig is divided into groups and the dosage regimen table
(2) therapeutic evaluation index
1, mortality rate
At duration of test; The classical symptom of colibacillosis only appears in pig, and symptoms such as fervescence, appetite decline, dysentery are also dead, and necropsy mesentery etc. locates to occur suppurating the appearance material; And be dispersed in the petechia, can isolate the stronger escherichia coli of a large amount of hemolytics from this simultaneously.Calculate mortality rate according to the death toll in every treated animal.
Mortality rate (%)=every group of death toll (head)/each group sum (head) * 100%.
2, cure rate
At duration of test, the pig of a medication clinical symptom disappearance, anus is dry, does not have redness, and feces and mental restoration are normal, and the inoculation bacterium separates negative, all belongs to healing.Count the only a few cure rate according to the healing in every treated animal.
Cure rate (%)=each group is cured number (head)/each group sum (head) * 100%.
3, effective percentage
Body temperature reduces or recovers normal after the pig of curing and the administration, and appetite increases, and dysentery alleviates or the pig of symptom such as disappearance, all is judged as effectively, calculates effective percentage in view of the above.
Effective percentage (%)=each group survival number (head)/each group sum (head) * 100%.
4, average weight gain
The body weight of every pig during according to on-test and during off-test is calculated the relative weight gain amount of every pig, calculates average gain in weight.
5, data analysis and processing
With PASS 16.0for Windows result of the test is carried out statistics biology.
(3) experimental result
After the medication of the sick pig difference of 30 hairs, observe clinical effectiveness day by day, its result sees table 2.
Table 2 suspension of the present invention is to the clinical trial result of colibacillosis
From table 2, can find out:
⑴ cure rate and effective percentage: embodiment does not have animal dead for 1 group, and its effective percentage and cure rate are significantly higher than medicine matched group (P < 0.05);
⑵ average weight gain amount: the average weight gain of 1 group of 15d of embodiment is higher than the medicine matched group.
Above presentation of results ENNUOSHAXING ZHUSHEYE of the present invention has excellent curative for the diarrhoea that the pig ehec infection causes, its effective percentage and cure rate are significantly higher than the medicine matched group, can effectively alleviate the swine diseases weightening finish slowly, and significantly reduce mortality rate.
Embodiment 7
ENNUOSHAXING ZHUSHEYE of the present invention is to the clinical trial of Pullorum Disease.
(1) material and method
1, experiment material
The ENNUOSHAXING ZHUSHEYE of test drug: embodiment 1 preparation, every day 1 time, logotype three days;
Control drug: ENNUOSHAXING ZHUSHEYE (baytril): lot number KP06H8U, content 5%, Bayer (Sichuan) animal health company limited;
Strain: Pullorum Disease Salmonella virulent strain C79-21, available from China Veterinary Drugs Supervisory Inst..
1, experimental animal and grouping
Select 216 of healthy 1 age in days AA carnivorous (public female half and half).Raise 3d before the test, free choice feeding and drinking-water, the mental status and the diet situation of observing chicken, the discovery spiritedness is depressed, appetite descends or feces is abnormal all eliminates
2, test method
⑴ the preparation of bacterium liquid
At first, the bacterial classification inoculation of preserving is dull and stereotyped in Mai Kangkai, behind the cultivation 24h, pick out single colonies typical with the disinfection inoculation ring, be inoculated in the 3mlMH meat soup, under 37 ℃ of environment, cultivate 18h and take out.Then, inoculate chicken, after rejuvenation, from dead chicken liver, isolate Salmonella, and be inoculated in the dull and stereotyped cultivation of Mai Kangkai with broth culture.At last, select colonies typical and be inoculated in the nutrient broth medium, cultivate 24h down for 37 ℃, as merit toadstool liquid.
⑵ the mensuration of minimal infecting dose (MID) (MID)
16 chickens are divided into 4 groups, 4 every group, find chicken morbidity and death condition through the test organisms liquid of chest muscle injection 6 * 107,6 * 108,6 * 109,6 * 1010CFU/ml respectively.The inoculum concentration of antibacterial is 6 * 109CFU/ml when testing surely.。
⑶ test method
200 chickens are divided into 4 groups (seeing table 3) 50 every group at random, and except that the normal healthy controls group, each organizes chicken through chest muscle test injection bacterium liquid 0.5ml.The treatment group is behind inoculation bacterium liquid, and per os is irritated the clothes administration, and concrete dosage regimen is seen table 3.Behind the counteracting toxic substances, examine and note down incidence, clinical symptoms and the death condition of respectively organizing chicken.Dead chicken is cutd open inspection, get liver, the heart, spleen and carry out bacteriology's separation and Culture and identify that whether to confirm chicken death because the inoculation bacterium infects, the clinical observation time is 15d.When inoculated bacteria front and back and off-test, weigh respectively and respectively organize the body weight of chicken, and calculate the relative weight gain rate.
Table 3 test chicken divides into groups and dosage regimen
(2) therapeutic evaluation index
1, mortality rate: all at disease symptoms such as duration of test appearance row loose stools, cuing open inspection after the death has the typical cytopathic characteristic again, and isolates corresponding pathogen person and think that infection is dead, calculates mortality rate according to death toll.
Computing formula: mortality rate (%)=is respectively organized death toll (only)/each group sum (only) * 100%
2, cure rate: all at duration of test, the back spirit of taking medicine, appetite recover normal, clinical symptoms such as row's loose stool no longer occurs and belong to and curing, and figure out cure rate according to curing.
Computing formula: cure rate (%)=each group is cured number (only)/each group sum (only) * 100%
3, effective percentage: all at duration of test, after medication, cure fully, and clinical symptoms is arranged but the dead not, think that administration is effective to it, calculate effective percentage in view of the above.
Computing formula: effective percentage (%)=each group survival number (only)/each group sum (only) * 100%
4, the relative weight gain rate: when reaching off-test before the administration each group chicken is weighed one by one, calculate the gain in weight of every chicken.The relative weight gain rate is the ratio of the weightening finish of each medication treatment group, and wherein the normal healthy controls group is made as 100%.
Computing formula: the relative weight gain rate (%)=is respectively organized average weight gain/matched group average weight gain * 100%
(3) result of the test
The ENNUOSHAXING ZHUSHEYE intramuscular administration is seen table 4 to the therapeutic effect of Pullorum Disease.
Table 4 ENNUOSHAXING ZHUSHEYE of the present invention is to the comparative test result of Pullorum Disease
Can know that by table 4 enrofloxacin can effectively reduce the mortality rate that chicken infects Salmonella.All more than 70%, the relative weight gain rate is more than 90% to the effective percentage of Pullorum Disease, cure rate for 1 group of embodiment, control drug group, and 1 group of embodiment is significantly higher than control drug group (P < 0.05).Explain that ENNUOSHAXING ZHUSHEYE of the present invention can effectively treat Pullorum Disease, and compare, significantly reduced mortality rate with ENNUOSHAXING ZHUSHEYE.
Embodiment 8
ENNUOSHAXING ZHUSHEYE of the present invention and 5% ENNUOSHAXING ZHUSHEYE are in the intravital relatively pharmacokinetic studies of pig.
The relevant requirements that new veterinary drug is declared by following experimental basis country; Research and inquirement ENNUOSHAXING ZHUSHEYE of the present invention and 5% ENNUOSHAXING ZHUSHEYE in intravital relatively pharmacokinetic studies of health pig and relative bioavailability investigation; Be intended to for science, reasonably estimate ENNUOSHAXING ZHUSHEYE of the present invention; For new veterinary drug examining provides necessary material, also scientific basis is provided on veterinary clinic for this injection uses, have the important clinical meaning.
One, materials and methods
1, medicine and reagent
ENNUOSHAXING ZHUSHEYE: lot number 20110829, content 25%, Tiaozhan provides, and pH is 5.3;
ENNUOSHAXING ZHUSHEYE (baytril): lot number KP06H8U, content 5%, Bayer (Sichuan) animal health company limited;
Enrofloxacin standard substance: lot number H010102, content 99.9%, China Veterinery Drug Inspection Office;
Triethylamine: lot number 20100721, content>99%, Fu Yu Fine Chemical Co., Ltd in Tianjin provides;
Phosphoric acid: lot number 20101102, content>85.0%, Tianjin good fortune chemical reagent in morning factory.
2, key instrument
Highly effective liquid phase chromatographic system: the Agilent1200 type is equipped with quaternary gradient pump, automatic sampler, post constant temperature system, FLD detector, chromatographic work station, U.S. Agilent company;
Electronic analytical balance: BP121S type, sensibility reciprocal 0.0001g, German Sartorius company;
Adjustable micropipettor: EppendorfResearch type, 10 ~ 100 μ L, 20 ~ 200 μ L, 100 ~ 1000 μ L, 500 ~ 5000 μ L, German Eppendorf company;
Refrigerated centrifuger: TDL-5000B type, Anting Scientific Instrument Factory, Shanghai;
Whirlpool vortex mixer: VXH-3 type, the Shanghai medical apparatus and instruments factory that jumps;
The syringe-type nuclepore membrane filter: the aperture is 0.45 μ m, the Tianjin filter element factory of rising.
3, the configuration of medicinal liquid
The enrofloxacin storing solution: accurately take by weighing enrofloxacin reference substance 50.05mg, place the brown volumetric flask of 50mL, to scale, promptly process the enrofloxacin storing solution of 1.0mg/mL with methanol constant volume, 4 ℃ of preservations are subsequent use.Face with preceding and be made into series standard liquid with mobile phase.
0.05mol/L phosphoric acid solution: get 85% phosphoric acid 3.4mL, be diluted with water to 1000mL, stir down and drip triethylamine, transfer pH to 2.4.
4, animal and grouping
Healthy long white * 16 of DABAI binary pigs; 60 ages in days; Body weight 22.53 ± 0.40
kilogram, plant the pig farm and provide in Guangdong Province.Be divided into 2 groups at random, 8 every group.Raise by routine before the test, freely drink water and search for food, feedstuff is adequate diet (not containing antibacterials), observes for two weeks, clinical observation health.
5, EXPERIMENTAL DESIGN
Two groups of pig medications are respectively that musculi colli is injected 5% ENNUOSHAXING ZHUSHEYE, musculi colli is injected 25% ENNUOSHAXING ZHUSHEYE (acidity).Before experiment,, carry out clinical administration according to actual drug content to supplying the reagent thing to carry out assay.Before the test 16h rise and administration after fasting during the 4h, only freely drink water.
6, administration and blood specimen collection
Administration: press 2.5mg/kg body weight single-dose.
Sampling time: on the basis of prerun, confirm the sampling time, adopt blank plasma before the administration, after the administration 0.1,0.25,0.5,0.75,1,1.25,1.5,2,3,4,6,8,12,24,36,48h gathers blood sample respectively.
The blood sampling approach: pig Baoding of only lying on the back, the vena cava anterior blood sampling is taken a blood sample about 5mL at every turn.
Sample preservation: gather venous blood and place the centrifuge tube that contains heparin, mixing, the centrifugal 10min of 4000r/min, separated plasma is put-20 ℃ of refrigerators and preserved, and is to be measured.
7, the assay method of blood drug level
⑴ plasma sample pre-treating method
After treating that blood plasma thaws, shake up, accurately draw 0.5mL in the 2mL centrifuge tube, add the methanol of 1mL, the whirlpool mixing, the centrifugal 10min of high speed centrifuge 12000r/min draws supernatant and does the HPLC analysis.
⑵ liquid phase chromatogram condition
Highly effective liquid phase chromatographic system: Agilent 1200 highly effective liquid phase chromatographic systems.
Chromatographic column: phenomenex luna-C18 (2) (4.6mm * 250mm, 5 μ m).
Mobile phase: 0.05mol/L phosphoric acid solution: second eyeball=82:18 (V:V).
Flow velocity: 0.8mL/min.
Detect wavelength: excitation wavelength 280nm, emission wavelength 450nm.
Sample size: 20 μ L.
⑶ confirming of detectability and quantitative limit
Get blank porcine blood plasma; Add the sample (n=20) that the enrofloxacin titer is prepared into 0.005,0.01,0.02,0.05 μ g/mL, by " " 7 ⑴ plasma sample pre-treating methods in " are handled sample, again by " carrying out HPLC under " 7 ⑵ liquid phase chromatogram conditions in " measures; Record the baseline level of noise; Asking its meansigma methods, is detectability (LOD) by signal to noise ratio S/N=3, and S/N=10 is quantitative limit (LOQ).
⑷ the standard curve and the range of linearity
In 9 plastic centrifuge tubes, respectively add the 0.48mL blank plasma; Add 20 μ L enrofloxacin titers series (0.5 ~ 100 μ g/mL) more successively, the vortex mixing, making the drug level in the plasma sample is 0.02 ~ 4 μ g/mL; The 1st pipe does not add medicine and compares, and specifically sees table 1.Handle by " 7 ⑴ plasma sample pre-treating methods in ", last machine is measured.Enrofloxacin chromatographic peak area (S) that obtains and concentration (C) are returned, try to achieve standard curve regression equation and correlation coefficient, the result sees table 5.
The standard curve series concentration of enrofloxacin in table 5 slurry
⑸ the response rate and the coefficient of variation are measured
Quantitative enrofloxacin titer is added in the pig blank plasma; Make low (0.02 μ g/mL), in (0.2 μ g/mL), high (2 μ g/mL) three sample concentrations; Handle sample by " 7 ⑴ plasma sample pre-treating methods in ", measure by carrying out HPLC under " 7 ⑵ liquid phase chromatogram conditions in " again; After getting concentration simultaneously respectively and be 0.02 μ g/mL, 0.2 μ g/mL, 2 μ g/mL enrofloxacin titers and handling, carry out HPLC and analyze, calculate with peak area ratio and ask its response rate by " in one 7 (1) " method.5 repetitions of every concentration (batch in), 3 totally batches (between batch), in calculating batch and batch between the coefficient of variation.
⑹ the mensuration of plasma drug level after the administration
The blood plasma that different time points is gathered after the administration; Handle sample by " 7 ⑴ plasma sample pre-treating methods in ", analyze by carrying out HPLC under " 7 ⑵ liquid phase chromatogram conditions in " again, try to achieve the peak area of enrofloxacin; Bring the standard curve regression equation into, converse the concentration of enrofloxacin in the blood plasma; Surpass 4 μ g/mL blood drug level samples simultaneously, be diluted to corresponding multiple and make it in 0.02～4 μ g/mL scope.
⑺ data analysis is handled
Adopt the non-compartment model of the pharmacokinetics software Winnonlin5.2.1 of U.S. Pharsight company to handle the moving data of medicine; Calculate the pharmacokinetic parameters of every test pig; Calculating mean value
and standard deviation (S.D.) then, simultaneously with the blood drug level meansigma methods to the time catch cropping drug-time curve figure.
Two, result and analysis
1, the methodology that enrofloxacin detects in the blood plasma
⑴ chromatographic behavior
Carry out HPLC according to " 7 ⑵ liquid phase chromatogram conditions in " and measure, enrofloxacin separates well with other components in the blood plasma, and the chromatographic peak retention time of enrofloxacin is respectively about 9.8min.
Actual measurement plasma sample chromatogram was seen Fig. 1-4 respectively after enrofloxacin reference substance, pig blank plasma, blank plasma added medicine, administration.
⑵ detectability and quantitative limit
Measuring according to " 7 ⑶ in ", is detectability (LOD) by signal to noise ratio S/N=3, and S/N=10 is quantitative limit (LOQ); Try to achieve that the detection of enrofloxacin is limited to 0.01 μ g/mL in the porcine blood plasma; Quantitatively be limited to 0.02 μ g/mL, show the highly sensitive of method, can satisfy the pharmacokinetic studies requirement.
⑶ the standard curve and the range of linearity
Measure according to " 7 ⑷ in ", obtain standard curve and the range of linearity of enrofloxacin in blood plasma and see table 6.The enrofloxacin standard curve is in 0.02 ~ 4 μ g/mL concentration range, and the phase relation number average is greater than 0.9990, and linear relationship is good.
Table 6 enrofloxacin standard curve regression equation and correlation coefficient
⑷ the response rate and the coefficient of variation
Measure according to " 7 ⑸ in ", in batch and batch between the response rate and coefficient of variation result see table 7.This method is in porcine blood plasma; Enrofloxacin basic, normal, high three response rate of adding levels respectively 73.50～113.23%, 78.54 ~ 100.27% and 83.77 ~ 99.38%; 3.02 ~ 10.34%, 2.44 ~ 10.54% and 1.98 ~ 7.19%, interassay coefficient of variation is 5.36 ~ 12.90% respectively for the variation within batch coefficient.The method response rate, accuracy, precision all satisfy the requirement of residue detection.
The response rate of enrofloxacin and the coefficient of variation in table 7 porcine blood plasma
2, the concentration of enrofloxacin in the blood plasma
Press 2.5mg/kg b.w. to behind two kinds of ENNUOSHAXING ZHUSHEYE of pig single dose intramuscular injection; Method according to " 7 ⑹ in "; Enrofloxacin concentration in the blood plasma is detected; Enrofloxacin concentration determination result sees table 8 and table 9 in each time point blood plasma, and two kinds of injection are seen Fig. 5 at intravital blood drug level of pig and time history.Test simultaneously finds that behind the pig intramuscular injection ENNUOSHAXING ZHUSHEYE, knock-on (flip-flop) phenomenon appears in blood drug level, and possibly there is the liver sausage circulation in the prompting enrofloxacin in the pig body.
The blood drug level (μ g/mL) of table 8 health pig single dose (2.5mg/kg b.w) intramuscular injection 5% ENNUOSHAXING ZHUSHEYE
ND: expression does not record (Not Detectable), is lower than quantitative limit 0.02 μ g/mL.
The blood drug level (μ g/mL) of table 9 health pig single dose (2.5mg/kg b.w) intramuscular injection speed 25% ENNUOSHAXING ZHUSHEYE (acidity)
ND: expression does not record (Not Detectable), is lower than quantitative limit 0.02 μ g/mL.
3, enrofloxacin is in the intravital relatively characteristics of pharmacokinetics of pig
Method according to " 7 ⑺ in " />; Data when adopting the non-compartment model of Winnonlin5.2.1 to handle in the blood plasma enrofloxacin medicine calculate the pharmacokinetic parameters of every test pig, and the result sees table 11 and table 12; Main pharmacokinetic parameters utilizes Excel to carry out medical statistics t check analysis; The result sees table 10, and parameter MRT, Cmax statistical difference heteropole be (P < 0.01) significantly, V parameter d, Kel, t12 statistics variant (P < 0.05); Parameter A UC statistics there was no significant difference (P>0.05); The relative bioavailability of 25% ENNUOSHAXING ZHUSHEYE (acidity) is 98.83%, and bioavailability is suitable, shows that there is potential bioequivalence in two kinds of ENNUOSHAXING ZHUSHEYE.
The main pharmacokinetic parameters of two kinds of ENNUOSHAXING ZHUSHEYE of table 10 is (
n=8) relatively
Annotate: * *: P 0.01, explain that difference has the height statistical significance; *: p 0.05, explain that difference has statistical significance.
The pharmacokinetic parameter of table 11 health pig single dose (2.5mg/kg b.w) intramuscular injection 5% ENNUOSHAXING ZHUSHEYE
The pharmacokinetic parameter of table 12 health pig single dose (2.5mg/kg b.w) intramuscular injection speed 25% ENNUOSHAXING ZHUSHEYE (acidity)
Three, conclusion
1, two kinds of ENNUOSHAXING ZHUSHEYE at the intravital pharmacokinetics of pig relatively
Enrofloxacin all has report both at home and abroad at the pharmacokinetic study of pig, and the dosage form that relates to has injection (Ceng Zhenling etc., 2001; Zhang Xiaohui etc., 2010; Zhu Wanju, 2003; Xiao Tianan etc., 2002; Zhou Yunbo etc., 2005), microcapsule (Luo Ya strength etc., 2010), suspension (once five and etc., 2002) etc., but Shang Weijian has the pharmacokinetics report of acid pH injection.
This test is pressed 2.5mg/kg b.w. to behind pig single dose intramuscular injection 5% ENNUOSHAXING ZHUSHEYE, and the non-compartment model of Winnonlin5.2.1 is handled the moving data of medicine, and main pharmacokinetic parameters is: AUC is 8.23 ± 1.63 μ gh/mL; Vd is 4.78 ± 0.92L/kg, and MRT is 14.24 ± 3.63 (mL/kgh), and Cmax is 0.65 ± 0.17 μ g/mL; Tmax is 1.45 ± 1.25h; Kel is 0.06 ± 0.021/h, and t1/2 is 11.56 ± 3.56h, and Cl is 0.30 ± 0.07 (L/ (kgh)); Press 2.5mg/kg b.w. to behind pig single dose intramuscular injection 25% ENNUOSHAXING ZHUSHEYE (acidity), the non-compartment model of Winnonlin5.2.1 is handled the moving data of medicine, and main pharmacokinetic parameters is: AUC is 8.14 ± 1.91 μ gh/mL; Vd is 3.04 ± 1.13L/kg, and MRT is 7.67 ± 2.31 (mL/kgh), and Cmax is 1.11 ± 0.23 μ g/mL; Tmax is 0.95 ± 0.44h; Kel is 0.11 ± 0.041/h, and t1/2 is 7.07 ± 2.60h, and Cl is 0.31 ± 0.07 (L/ (kgh)).Parameter MRT, Cmax statistical difference heteropole be (P < 0.01) significantly; Show that 25% ENNUOSHAXING ZHUSHEYE (acidity) reaches peak concentration and significantly raises in the pig body, average residence time significantly reduces, V parameter d, Kel, t1/>2 statistics variant (P < 0.05); Parameter A UC statistics there was no significant difference (P>0.05); The relative bioavailability of 25% ENNUOSHAXING ZHUSHEYE (acidity) is 98.83%, and bioavailability is suitable, shows that there is potential bioequivalence in two kinds of ENNUOSHAXING ZHUSHEYE.
2, the suggestion of ENNUOSHAXING ZHUSHEYE clinical administration scheme of the present invention
Along with the chemical sproof generation of FQNS, its clinical application effect weakens.FQNS belongs to concentration dependent form medicine; The research of (1993) such as George L Drusano shows; Cmax/MIC has the sign effect to the drug effect of FQNS; Become positive correlation with drug effect, having identical Cmax/MIC value, to show drug effect approaching, and Cmax/MIC has clinical therapeutic efficacy preferably greater than 8.The chemical sproof generation of sensitive organism mainly is because Cmax/MIC is less than normal.The Cmax of 25% ENNUOSHAXING ZHUSHEYE (acidity) is 1.11 ± 0.23 μ g/mL; Be significantly higher than the Cmax (0.65 ± 0.17 μ g/mL) of 5% ENNUOSHAXING ZHUSHEYE; Near 8 or greater than 8, prompting can effectively prevent and treat clinical various bacterial disease to the Cmax/MIC of various antibacterials.
At present, on the domestic market in the veterinary drug preparation similar injection more, it is bigger that drug effect differs, most injection do not carry out the animal experiment of system, have gap with the state external preparation.Blood drug level and the delivery times of the MIC of antibacterial being confirmed this medicine of adopting during domestic formulation dosage regimen more; Or with reference to state's external preparation; Can provide the time-dependent medicine than scientific basis, however the formulation of concentration dependent form drug administration scheme is lost objective.Cmax/MIC and AUC/MIC have the better prediction effect to the FQNS curative effect, when formulating dosage regimen, should give to take into full account, and strengthen the research (Zhang Xiaohui etc., 2010) of related fields.Existing research proof, FQNS can produce long antimicrobial drug after effect (PAE), is 2 ~ 6h, when designing the therapeutic scheme of antibacterials in the past; Emphasical blood drug level is higher than MIC, the existence prompting of PAE, and when blood drug level was lower than MIC, bacterial growth still continued to be suppressed; So according to PAE and pharmacokinetic parameters, when clinical design dosage regimen, but proper extension administration interval; Reduce administration number of times and dosage, still can keep antibacterial effect (Fang Yi etc., 1997; Gicquel M et al, 1997; Once five and etc., 2002).Various factorss such as preparation, species variation, age, different physiological situation, route of administration all can exert an influence to pharmacokinetics, formulate rational dosage regimen according to clinical practice.
Enrofloxacin is low to the minimal inhibitory concentration (MIC) of sensitive organism; Like enrofloxacin the MIC of swine escherichia coli, bacillus rhusiopathiae suis is respectively 0.05 and 0.1 μ g/mL; To the MIC90≤0.06 μ g/mL of actinobacillus pleuropneumoniae, the MIC90 of clinical isolating actinobacillus suis, escherichia coli, Krefeld Salmonella, Klebsiella pneumonia, pasteurella multocida all is lower than 0.128 μ g/mL (Gutierrez et al.1993; Ceng Zhenling, 1996).After pig is pressed 2.5mg/kg b.w single dose intramuscular injection 25% ENNUOSHAXING ZHUSHEYE (acidity); Blood drug level rises rapidly; Administration after 3 minutes blood drug level be 0.33 ± 0.22 μ g/mL, reach the above blood drug level of MIC of most of sensitive organisms, promptly can suppress to kill pathogen effectively; 1h reaches 0.98 ± 0.21 μ g/mL; Blood drug level begins to descend subsequently, drops to 0.15 ± 0.06 μ g/mL to 14h blood drug level, with the blood drug level of 5% ENNUOSHAXING ZHUSHEYE 14h quite (0.17 ± 0.04 μ g/mL).The blood drug level of 25% ENNUOSHAXING ZHUSHEYE (acidity) before sampled point 9h all is higher than the blood drug level of corresponding time 5% ENNUOSHAXING ZHUSHEYE.Enrofloxacin belongs to concentration dependent form medicine, points out 25% ENNUOSHAXING ZHUSHEYE (acidity) more can effectively prevent and treat clinical various bacterial disease than 5% ENNUOSHAXING ZHUSHEYE.The clinical sensitive organism of pig is infected, advise according to 2.5mg/kg b.w. administered intramuscular 1d administration 1～2 time, continuous use 2～3d.
To sum up:
Set up the HPLC detection method of enrofloxacin in the porcine blood plasma.The detection of method is limited to 0.01 μ g/mL, quantitatively is limited to 0.02 μ g/mL.Standard curve is in 0.02 ~ 4 μ g/mL concentration range; The phase relation number average is greater than 0.9990, and linear relationship is good, enrofloxacin basic, normal, high three response rate of adding levels 73.50 ~ 113.23%; Variation within batch coefficient＜10.54%, interassay coefficient of variation＜12.90%.
Studied the comparison pharmacokinetics of two kinds of ENNUOSHAXING ZHUSHEYE of health pig intramuscular injection (2.5mg/kg b.w).The result shows that 25% ENNUOSHAXING ZHUSHEYE (acidity) is compared with 5% ENNUOSHAXING ZHUSHEYE, reaches peak concentration and significantly raises, and average residence time significantly reduces, and bioavailability is suitable.
The clinical sensitive organism of pig is infected, advise according to 2.5mg/kg b.w. intramuscular injection 25% ENNUOSHAXING ZHUSHEYE (acidity) 1d administration 1～2 time, continuous use 2～3d.
Claims (6)
1. ENNUOSHAXING ZHUSHEYE, it is characterized in that: its composition and parts by weight are following:
21～25 parts of enrofloxacins;
5～15 parts of lactic acid;
0.1～0.5 part of sodium sulfite;
59.5～73.9 parts of waters for injection.
2. ENNUOSHAXING ZHUSHEYE according to claim 1 is characterized in that: its composition and parts by weight are following:
21 parts of enrofloxacins;
9 parts of lactic acid;
0.2 part of sodium sulfite;
69.8 parts of waters for injection.
3. ENNUOSHAXING ZHUSHEYE according to claim 1 is characterized in that: its composition and parts by weight are following:
25 parts of enrofloxacins;
15 parts of lactic acid;
0.5 part of sodium sulfite;
59.5 parts of waters for injection.
4. according to each described ENNUOSHAXING ZHUSHEYE of claim 1 to 3, it is characterized in that: the pH of said ENNUOSHAXING ZHUSHEYE is 5.0～6.9.
5. ENNUOSHAXING ZHUSHEYE according to claim 1 is characterized in that: the usage of said ENNUOSHAXING ZHUSHEYE and consumption are: according to 2.5mg/kg b.w. intramuscular injection, and 1d administration 1～2 time, continuous use 2～3d.
6. method for preparing like each described ENNUOSHAXING ZHUSHEYE of claim 1 to 5, it is characterized in that: step is following:
⑴ take by weighing and be used for dissolved water for injection;
⑵ add sodium sulfite by weight in step ⑴ solution, be stirred to dissolving fully;
⑶ add enrofloxacin by weight in step ⑵ solution, stir;
⑷ add lactic acid by weight in step ⑶ solution, be stirred to the solution clarification;
⑸ add remaining water for injection to full dose in step ⑷ solution, filter, sterilize, and promptly gets ENNUOSHAXING ZHUSHEYE.
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