CN103285013B - Medical composition for preventing and treating pneumonia disease of cattle and hogs and preparation method thereof - Google Patents

Abstract

The invention discloses a medical composition for preventing and treating pneumonia disease of cattle and hogs and a preparation method thereof. The medical composition based on 1000ml of liquid medicine comprises medicines and auxiliary materials: 250-350g of florfenicol, 2-8g of meloxicam, 40-80ml of 1, 2-propylene glycol, 80-250ml of 2-pyrrolidone, 650-900ml of polyethylene glycol-400 and 10-15ml of benzyl alcohol. The preparation method comprises the following steps: preparing a mixed solvent; primarily preparing a liquid medicine; preparing an injection. Massive clinical application verifies that the medicine has anti-microbial, anti-inflammatory and anti-mycoplasma functions and has the characteristics of high and long effects to treat and prevent infectious diseases of the upper respiratory tract of cattle and hogs, especially various pneumonia diseases including mycoplasma pneumonia syndrome. The medical composition is good in treatment effect, and has a positive effect to drive development of cattle and hog raising industry in China.

Description

A kind of pharmaceutical composition of preventing and treating cattle hyopneumoniae disease and preparation method thereof

Technical field

The invention belongs to a kind of florfenicol of preventing and treating cattle hyopneumoniae disease and meloxicam pharmaceutical composition and preparation method thereof.

Background technology

Cattle hyopneumoniae disease is one of common disease in cattle and pig aquaculture, and calf and piglet especially easily send out, and mortality rate is higher.The cause of disease of such disease is generally antibacterial or antibacterial and mycoplasma mixed infection etc., conventionally adopts antibacterials and anti-inflammatory drug therapeutic alliance to reach the object for the treatment of both the principal and secondary aspects of a disease on veterinary clinic, has also obtained gratifying effect.But, on veterinary drug market, there is not yet and be specifically designed to the antibacterials of cattle hyopneumoniae disease treatment and the compound preparation of anti-inflammatory drug at present, while causing antibacterials and anti-inflammatory drug use in conjunction, need multiple dosing, bring many unfavorable and inconvenient impacts to animal and veterinarian, producer.On the other hand, the mixed infection pneumonia disease such as emerging Mycoplasma bovis pneumonia syndrome, mycoplasma pneumoniae of swine, also without active drug, is seriously restricting the sound development of cattle-raising and pig industry in recent years.Therefore, formulate treatment cattle hyopneumoniae disease antimicrobial antiphlogistic compound preparation novel, efficient, long-acting, low toxicity very urgent.

Florfenicol is animal specific medicine; its mechanism of action be can be in animal body the intracellular 70S ribosome of pathogenic microorganism and 50S subunit be combined and play inhibitory action; peptidyl transferase is suppressed; thereby suppress the extension of aminoacid peptide chain; protein synthesis is interfered, thereby performance suppress the effect of microbial reproduction growth.The has a broad antifungal spectrum of florfenicol, all has stronger bactericidal action to the clinical multiple Gram-negative of separating and gram positive bacteria, mycoplasma is also had to killing action, and most of bacterial strain of resistance to chloromycetin and thiamphenicol is still had to higher antibacterial activity.Therefore, florfenicol has higher killing action to most pathogen of cattle hyopneumoniae disease, can be used as the first-selected antibacterials for the treatment of cattle hyopneumoniae disease.Meloxicam is a kind of non-steroid antiinflammatory (NSAIDs) of enol amide-type, COX-2 is had to high selectivity, there is good antiinflammatory, analgesia, refrigeration function, smaller to gastrointestinal harmful effect, for various inflammation diseases, effect is splendid clinically.

Summary of the invention

Deficiency based on existing cattle hyopneumoniae disease medicament and the both at home and abroad demand to the product development of control cattle hyopneumoniae disease medicament, the present invention, taking modern medicines compatibility theory as guidance, has developed a kind of pharmaceutical composition of preventing and treating cattle hyopneumoniae disease.

Another object of the present invention is to provide the preparation method of this pharmaceutical composition.Pharmaceutical composition has both adjuvant taking florfenicol and meloxicam as main, is made into injection, is used for the treatment of and prevents cattle hyopneumoniae disease, especially mycoplasma pneumonia syndrome, has the features such as efficient, long-acting, low toxicity, convenience.

The object of the invention is to be achieved through the following technical solutions:

Prevent and treat a pharmaceutical composition for cattle hyopneumoniae disease, calculate with 1000ml medicinal liquid, containing each medicine and adjuvant amount proportioning be:

Florfenicol 250～350g, meloxicam 2～8g, 1,2-PD 40～80ml, 2-Pyrrolidone 80～250ml, PEG-4000 650～900ml, benzyl alcohol 10～15ml.

The preparation method of aforementioned pharmaceutical compositions, the steps include:

1. mixed solvent preparation, according to formula composition, measures respectively 1,2-PD 40～80ml, 2-Pyrrolidone 80～250ml, and PEG-4000 650～900ml, benzyl alcohol 10～15ml, is uniformly mixed;

2. the first registration of medicinal liquid really takes florfenicol 250～350g, and meloxicam 2～8g, adds the 800ml mixed solvent 1. obtaining, and at 45-55 DEG C, constant temperature is stirred to into clear solution.

3. 2. medicinal liquid preparation will obtain 1. mixed solvent of solution and be settled to 1000ml, be filtered to clear and bright with No. 3 sintered glass funnels or other filters.Embedding, 121 DEG C of sterilizings 15 minutes and get final product.

The dosage form of product:

Injection

Physical and chemical parameter:

PH value: 7.0 ± 0.2;

Proportion: 1.05 ± 0.05;

Character: weak yellow liquid, mildly bitter flavor.

Product assay: according to high effective liquid chromatography for measuring.

Chromatographic condition and system suitability test are taking octadecylsilane chemically bonded silica as filler; Taking methanol-water (pH phosphoric acid is adjusted to as 3.0) (40: 60) as mobile phase; Detect wavelength: florfenicol is 222nm, and meloxicam is 355nm.Theoretical cam curve is calculated and all should be not less than 6000 by florfenicol and meloxicam peak.

Florfenicol is got respectively in the preparation of reference substance solution and meloxicam reference substance is appropriate, accurately weighed, respectively puts in brown measuring bottle, adds methanol and makes the solution of every 1ml containing 100 μ g, to obtain final product.

The preparation of need testing solution is taking liquid 1ml respectively, respectively with 3000 times of methanol and 40 times of dilutions, obtains the need testing solution of florfenicol and meloxicam.

Algoscopy is accurate reference substance solution 10 μ l and the need testing solution 10 μ l of drawing respectively, and injection liquid chromatography, measures, and to obtain final product.

The every 1ml of this product, containing florfenicol and meloxicam, is respectively 300.0 ± 30.0mg and 4.0 ± 0.4mg.

The beneficial effect of advantage of the present invention and generation is:

It is raw material that the present invention selects the antibacterials florfenicol of definite effect and anti-inflammatory drug meloxicam, has both adjuvant, belongs to anti-infection drug.By a large amount of clinical uses, prove that this medicine has very good therapeutic effect to cattle pig especially calf and the various pneumonia diseases of piglet, pharmaceutical dosage form is injection, easy to use, simple to operate, its application is for control cattle pig antibacterial or antibacterial and mycoplasma mixed infection pneumonia disease, and the development that promotes China's cowboying pig industry will produce positive role;

This product primary treatment and the various pneumonia diseases of prevention cattle pig, can replace existing antimicrobial antiphlogistic pharmaceutical preparation, and clinical use has advantages of efficient, long-acting, safe, easy to use.

Brief description of the drawings

Fig. 1 chromatogram, wherein: figure lA: be blank plasma; Figure 1B adds chloromycetin in white blood plasma; In Fig. 1 C blank plasma, add florfenicol; Fig. 1 D adds chloromycetin in blood sample after administration; Florfenicol and chloromycetin retention time are respectively 3.87lmin, 5.342min

Fig. 2 chromatogram, wherein: Fig. 2 A is blank plasma; Fig. 2 B adds piroxicam in blank plasma; Fig. 2 C adds meloxicam in blank plasma; Fig. 2 D adds piroxicam in blood sample after administration; Meloxicam and piroxicam retention time are respectively 14.299min, 10.100min

Fig. 3 healthy rabbits single dose intramuscular injection 30mgkg ^-1the blood plasma drug-time curve of fluorine Ben Nikao

Fig. 4 healthy rabbits single dose intramuscular injection 0.4mgkg ^-1the blood plasma drug-time curve of meloxicam

Detailed description of the invention

Below, in conjunction with pharmacology test, safety testing, clinical trial, the present invention is further described again:

Embodiment 1

1. mixed solvent preparation, according to formula composition, measures respectively 1,2-PD 50ml, 2-Pyrrolidone 240ml, and PEG-4000 700ml, benzyl alcohol 10ml, is uniformly mixed;

2. the first registration of medicinal liquid really takes florfenicol 280g, and meloxicam 5g, adds the 800ml mixed solvent 1. obtaining, and at 45-55 DEG C, constant temperature is stirred to into clear solution;

3. 2. medicinal liquid preparation will obtain 1. mixed solvent of solution and be settled to 1000ml, be filtered to clear and bright with No. 3 sintered glass funnels or other filters.Embedding, 121 DEG C of sterilizings 15 minutes and get final product.

Embodiment 2

1. mixed solvent preparation, according to formula composition, measures respectively 1,2-PD 60ml, 2-Pyrrolidone 100ml, and PEG-4000 830ml, benzyl alcohol 10ml, is uniformly mixed;

2. the first registration of medicinal liquid really takes florfenicol 300g, and meloxicam 4g, adds the 800ml mixed solvent 1. obtaining, and at 45-55 DEG C, constant temperature is stirred to into clear solution.

3. 2. medicinal liquid preparation will obtain 1. mixed solvent of solution and be settled to 1000ml, be filtered to clear and bright with No. 3 sintered glass funnels or other filters.Embedding, 121 DEG C of sterilizings 15 minutes and get final product.

Above-mentioned two schemes all meets product quality requirement, but from solvent zest, price and medicine carrying capacity and two kinds of drug ratios of preparation, embodiment 2 schemes are more rational.

Test example 1

The pharmacokinetic of single dose intramuscular injection pharmaceutical composition in rabbit body

1 materials and methods

1.1 animal

5 of healthy new zealand white rabbits, body weight 4～5kg, buys in Lanzhou Veterinary Inst., Chinese Academy of Agricultural Science, raises routinely, and concentrated feed is the complete feed that does not contain antibacterials, freely drinks water, clinical observation 2 weeks before test.

1.2 main medicine and reagent

Florfenicol standard substance (content 99.8%), chloromycetin reference substance (content 99.4%), meloxicam standard substance (content 99.3%), piroxicam reference substance (content 99.4%) is purchased from Nat'l Pharmaceutical & Biological Products Control Institute; Pharmaceutical composition (being prepared by Lanzhou Livestock and Animal Drug Inst., Chinese Academy of Agricultural Science by the embodiment of the present invention 2); Chromatographic grade acetonitrile, methanol are purchased from fisher chemical; Ethyl acetate, DMF, purchased from Tianjin chemical reagent factory; Sodium ethylene diamine tetracetate (EDTA) is purchased from Shanghai uncle biological Be Able LLC difficult to understand.

1.3 main experimental instrument and equipments

Waters2695 high performance liquid chromatogram (UV 2489, containing Waters chromatograph station working software); Sartorius BS110S electronic balance (Beijing Sai Duolisi balance company limited); Biofuge Fresco high speed centrifuge (13000rmin ^-1, Kendro Lab Products LP of the U.S.); WR-2001 solvent filter (Shanghai Jia Peng Science and Technology Ltd.); XW-80A eddy mixer (Qingpu Shanghai Hu Xi instrument plant); Nitrogen dries up instrument: BF2000 type, and manufacture in Beijing from all directions Centrix Technology Ltd.; Vortex oscillation device: Mslminishaker, IKA company manufactures; Ultrasonic cleaner: KQ3200E type, Kunshan Ultrasonic Instruments Co., Ltd.; Ultralow temperature-80 DEG C refrigerator: MDF-192 type, Japan manufactures; The triple pure water distillators of SZ-BIII type (Shanghai Jia Peng Science and Technology Ltd.).

1.4 chromatographic condition

1.4.1 florfenicol chromatographic condition

Chromatographic column: Hypersil ODS ₂(250mm × 4.6mm, 5 μ are m); Mobile phase: methanol: water (50: 50, v/v); Flow velocity 1.0mlmin ^-1; Column temperature: 30 DEG C; Detect wavelength 223nm; Sample size: 50 μ l.

1.4.2 meloxicam chromatographic condition

Chromatographic column: C Hypersil ODS ₁, (250mm × 4.6mm, 5 μ m), mobile phase: acetonitrile: acetic acid water (65: 35) flow velocity: 1.0mlmin ^-1; Detect wavelength: 355nm; Sample size: 50 μ l.

1.5 medicinal liquid preparations

1.5.1 florfenicol medicinal liquid preparation

Florfenicol titer, dissolves florfenicol standard substance and makes 1mgml with chromatographic grade acetonitrile ^-1standard stock solution, be diluted to before use standard solution (the 2.5-500 μ gml of series mass concentration with distilled water ^-1) preparation standard curve.After the sterilizing of florfenicol compound injection as trial target.Chloromycetin inner mark solution, dissolves chloromycetin reference substance with appropriate chromatographic grade acetonitrile, be made into 500 μ gml with distilled water ^-1solution as interior mark liquid, 4 DEG C of preservations.

1.5.2 meloxicam medicinal liquid preparation

Meloxicam titer, dissolves meloxicam standard substance with DMF, then by chromatographic grade dilution in acetonitrile and make 1mgml ^-1standard stock solution, be diluted to before use standard solution (the 1-300 μ gml of series mass concentration with distilled water ^-1) preparation standard curve.Piroxicam inner mark solution, dissolves piroxicam reference substance with appropriate DMF, then is made into 5 μ gml by chromatographic grade dilution in acetonitrile ^-1solution as interior mark liquid, 4 DEG C of preservations.

1.6 administrations and blood specimen collection

Before administration, white rabbit is weighed, numbers, and single dose intramuscular injection pharmaceutical composition is (containing florfenicol 30mgkg ^-1with meloxicam 4mgkg ^-1) after, respectively at 0,0.133,0.167,0.250,0.5,0.75,1,1.5,2,4,6,8,12,24,48,72 and 96h blood sampling.All adopt white rabbit ear edge vein exploitating blood, each about 1.5ml, EDTA anticoagulant, 3000rmin ^-1centrifugal 5min, separated plasma, to be measured in-20 DEG C of Refrigerator stores.

1.7 determination of plasma concentration

1.7.1 florfenicol determination of plasma concentration

Accurately pipette plasma sample 0.5ml, add 500 μ gml ^-1chloromycetin in mark liquid 10 μ l, the PBS liquid (pH7.0) of 0.5ml, vortex 30s mixes.Then add 2.5ml ethyl acetate, vibration 1min, ultrasonic 5min, 4000rmin ^-1centrifugal 10min, draws upper strata ethyl acetate in 5ml centrifuge tube, and same method repeats to extract once, combined ethyl acetate extracting solution, 45 DEG C of water-bath N ₂dry up, and 200 μ l (acetonitrile: water=20: 80) dissolve, vibration 30s, ultrasonic 5min, 0.22 μ m membrane filtration is inserted in sample injection bottle, 50 μ l sample introductions.

1.7.2 meloxicam determination of plasma concentration

Accurately pipette plasma sample 0.25ml, add mark liquid 5 μ gml in piroxicam ^-1the second acid liquid (pH4.0) of volume 10 μ l and 0.15ml, vortex 30s mixes.Then add 2.5ml ethyl acetate, the ultrasonic 5min of concussion 1min, 4000rmin ^-1centrifugal 10min, draws upper strata ethyl acetate 45 DEG C of water-bath N in 5ml centrifuge tube ₂dry up 200ul (acetonitrile: methanol=1: 1) dissolve vibration 30s, ultrasonic 5min, 50 μ l sample introductions.

The preparation of 1.8 standard curves and method research

1.8.1 the preparation of florfenicol standard curve and minimal detectable concentration are determined and determination of recovery rates

Accurately pipette blank plasma 0.5ml, add mark liquid 10 μ l in florfenicol series standard solution 10 μ l and chloromycetin, accurately test according to 1.7.1 aforesaid operations, taking the ratio (S) of florfenicol and interior mark chloromycetin peak area as vertical coordinate, corresponding florfenicol concentration (C) is made equation of linear regression for abscissa, obtains equation of linear regression and correlation coefficient.Determination of recovery rates adds florfenicol titer in plasma sample, is made into 0.1,1 and 10 μ gml ^-1the sample of three kinds of drug level.Then every kind of concentration is measured respectively three times, gets the pharmaceutical analysis method response rate of this concentration sample of mean value computation.The coefficient of variation, determines lowest detectable limit.

1.8.2 the preparation of meloxicam standard curve and method research

Pipette blank plasma 0.5ml, add mark liquid in 10 μ l meloxicam series standard solution and 10 μ l piroxicams, accurately test according to 1.7.2 aforesaid operations, ratio (S) with meloxicam and piroxicam peak area is made linear regression to corresponding meloxicam concentration (C), asks regression equation and correlation coefficient.Determination of recovery rates adds florfenicol titer in plasma sample, is made into 0.1,1 and 5 μ gml ^-1the sample of three kinds of drug level.Then every kind of concentration is measured respectively three times, gets the pharmaceutical analysis method response rate of this concentration sample of mean value computation.The coefficient of variation, determines lowest detectable limit.

1.9 date processing

The data of plasma drug level utilize the processing of WinNonlin5.2 pharmacokinetics program software to obtain pharmacokinetic parameter, judge optimal pharmacokinetic model according to WSS and AIC value.

2 results

2.1, standard curve, the response rate and the coefficient of variation

2.1.1 florfenicol standard curve and the response rate

Florfenicol is 0.05-10 μ gml in concentration ^-1scope internal linear relation good, regression equation is S=0.178C-0.003, coefficient R ²=0.999.With 0.10 μ gml ^-1, 1.0 μ gml ^-1with 10 μ gml ^-1in 3 interpolation concentration determination plasma samples, the florfenicol response rate is in table 1, and lowest detection is limited to 0.05 μ gml ^-1.

The response rate of table 1 florfenicol in rabbit blood plasma

2.1.1.1 the impurity interference test of florfenicol

In test, florfenicol and chloromycetin retention time are respectively 3.871min, 5.342min, and other components in florfenicol, chloromycetin and blood plasma separate well, and without obvious Interference Peaks, chromatographic peak is sharp-pointed and symmetrical (seeing Fig. 1).

2.1.2 meloxicam standard curve and the response rate

Meloxicam is 0.02-6 μ gml in concentration ^-1scope internal linear relation good, regression equation is S=3.821C+0.171, coefficient R ²=0.994.With 0.1 μ gml ^-1, 1.0 μ gml ^-1with 5 μ gml ^-13 are added the concentration determination plasma sample Meloxicam response rate in table 2.Lowest detection is limited to 0.02 μ gml ^-1.

The response rate of table 2 meloxicam in rabbit blood plasma

2.1.2.1 the impurity interference test of meloxicam

This test meloxicam and piroxicam retention time are respectively 14.299min, 10.100min, and other components in meloxicam, piroxicam and blood plasma separate well, and without obvious Interference Peaks, the sharp-pointed and symmetry of chromatographic peak is shown in Fig. 2.

The characteristics of pharmacokinetics of 2.2 injections in rabbit body

2.2.1 the characteristics of pharmacokinetics of florfenicol in rabbit body

After rabbit intramuscular injection pharmaceutical composition, record different time florfenicol blood drug level, corresponding blood drug level-time graph is shown in Fig. 3.The average pharmacokinetic parameter of blood drug level-time data gained is in table 3.

Table 3 healthy rabbits single dose intramuscular injection 30mgkg ^-1the pharmacokinetic parameter of florfenicol

2.2.2 the characteristics of pharmacokinetics of meloxicam in rabbit body

After rabbit intramuscular injection pharmaceutical composition, record different time meloxicam blood drug level, corresponding blood drug level-time graph is shown in Fig. 4.The average pharmacokinetic parameter of blood drug level-time data gained is in table 4.

The pharmacokinetic parameter of table 4 healthy rabbits single dose intramuscular injection 0.4mg/kg meloxicam

3 discuss

In this invention, the rabbit of all clinical trials is all healthy, and after intramuscular injection pharmaceutical composition, does not occur any obvious side effect.

Rabbit absorbs two compartment model by the florfenicol medicine-time data fit one-level after every kg body weight single dose intramuscular injection 30mg pharmaceutical composition, and there were significant differences to absorb one-compartment model with the one-level of the research report horse, chicken, pig and sheep.In this research after rabbit intramuscular injection pharmaceutical composition, T _{i/2 α}1.983 ± 0.278h, T _{1/2 β}35.54 ± 2.804h, AUC64.48 ± 4.308 μ gml ^-1h, T _max1.577 ± 0.065h, C _max5.943 ± 0.211 μ gml ^-1, wherein absorption halftime (T _{1/2 α}) and Chen Hongwei (Chen Hongwei, Li Yinglun. single dose florfenicol takes orally and intramuscular injection pharmacokinetic in rabbit body. Chinese veterinary's journal, 2008, 166～169) and (the F Koc such as F.Koc 28 (2):, M Ozturk, Y Kadioglu.Pharmacokinetics of florfenicol after intravenous and intramuscular administration in New Zealand White rabbits.Research Veterinary science, 2009, (8): 102～105) 0.802 ± 0.098h that partly declines of the absorption in for oral administration and pharmacokinetic of intramuscular injection in rabbit body of the single dose florfenicol of report compares and extended to 1.983 ± 0.278h with 0.83 ± 0.24h, and elimination half-life (T _{1/2 β}) compare with 1.49 ± 0.23h and extended to 35.54 ± 2.804h with 2.317 ± 0.136h of the reports such as Chen Hongwei, eliminate the half-life much larger than absorption halftime, peak time 1.577 ± 0.065h is similar with 1.56 ± 0.13h with peak time 1.805 ± 0.103h that F.Koc etc. reports to Chen Hongwei, and it is little to reach peak concentration difference, illustrate pharmaceutical composition in rabbit body, reach peak fast, eliminate slowly, discharge completely.Show that medicine has reached object long-acting, slow release, realize long-acting, reduce the advantage of frequent drug administration number of times, obtain more stable blood level, reduce side effect, reduce drug dose and more effective.

Rabbit absorbs two compartment model, T by every kg body weight intramuscular injection containing medicine-time data fit one-level of the meloxicam after the pharmaceutical composition of 0.4mg meloxicam _{1/2 α}3.529 ± 0.083h, T _{1/2 β}138.4 ± 18.32h, AUC25.44 ± 9.059 μ gml ^-1h, T _max5.155 ± 0.077h, C _max1.016 ± 0.058 μ gml ^-1, wherein eliminate the half-life (T _{1/2 β}) with Xia Yunyue etc. (Xia Yunyue, Jiang Kaiyu thank to plum forests, etc. the pharmacokinetic of domestic meloxicam in rat body. Chinese Pharmacological Society's communication, 2000, the ten seven volumes, three phases .) 1mgkg of report ^-143.25 ± 5.5h in the pharmacokinetic of administration meloxicam in rat body has extended to 138.4 ± 18.32h.With (Gu Shifen such as Patricia V Turner, Jin You, Yang Xiaoyan, Deng. meloxicam tablet, capsules in healthy volunteers pharmacokinetics and relative bioavailability. Chinese Journal of New Drugs, 2004,13 (1): the 60) pharmacokinetic of the oral meloxicam of report in rabbit body, its dosage is 0.3mgkg ^-1, its main medicine is T for parameter _max6.4 ± 0.82h, AUC2.57 ± 0.21 μ gml ^-1h, C _max0.14 ± 0.02 μ gml ^-1, T _{1/2 β}8.16 ± 2.19h, CL/V 0.12 ± 0.01mlmg ^-1h ^-1compare, peak time is more approaching, and maximum peak concentration C _maxbe about 6 times of left and right of oral administration, intramuscular injection meloxicam is eliminated half-life (T _{1/2 β}) than large 15 times of oral meloxicam, illustrate that the half-life of intramuscular injection meloxicam in rabbit body is much larger than oral administration.

By each pharmacokinetic parameter that the pharmacokinetic model in laboratory animal rabbit body draws to this pharmaceutical composition, illustrate that injection of the present invention has efficient, long-acting effect, has important theoretical and practical significance.

Test example 2

Irritation test

1 rabbit quadriceps femoris method

1.1 material

1.1.1 experimental animal

Rabbit (new zealand white rabbit), 2-3kg, purchased from Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences.

1.1.2 test drug

Pharmaceutical composition, by being prepared by Lanzhou Livestock and Animal Drug Inst., Chinese Academy of Agricultural Science by the embodiment of the present invention 2.

1.1.3 reagent and instrument

Syringe, syringe needle, 75% ethanol.

1.1.4 method

Get 4 of body weight 2～3kg healthy rabbits, inject for reagent liquid 1.0～2.0ml with sterile working's method respectively at a side quadriceps femoris position, without death, longitudinally cut injection site muscle 48～72h planted agent, observe injection site irritant reaction.According to the form below is converted into corresponding reaction score value.

The irritant reaction standards of grading of table 5 injection

Result: average response score value person below 2 of 2 rabbit, can think and conform with the regulations; As average response score value person between 2-3, should separately get 2 rabbit retrials (4 rabbit can COMPREHENSIVE CALCULATING), result judgement is the same, as exceedes 2 and think against regulation, can not be for subcutaneous and intramuscular injection.

1.1.5 result

After 48 hours, analyse as seen, medicine-feeding part is without obvious reaction.Nonirritant effect.

2 rabbit eye drip methods

2.1 material

Animal: rabbit (new zealand white rabbit).Medicine: pharmaceutical composition, by being prepared by Lanzhou Livestock and Animal Drug Inst., Chinese Academy of Agricultural Science by the embodiment of the present invention 2.

2.2 method

Select two of the healthy rabbit not coming to harm, binding box is fixed, and carefully checks two of every rabbit, and normal person can be used to test.Slowly pull open palpebra inferior, allow it leave after eyeball, by 1～2 of the normal saline of florfenicol compound injection and 0.9% sterilizing (0.05～0.1ml), drip successively in the images of left and right eyes conjunctival sac of White Rabbit, should ensure to splash into two medicament for the eyes liquid measures equates, the slowly about 1-2 of drawing eyelid second, prevents the loss of florfenicol compound injection, affects experimental result.

2.3 evaluation criterion

After administration 0.5,1,1.5 and examine conjunctiva when 2h, checks whether conjunctiva has hyperemia, edema, sheds tears and the phenomenon of the irritant reaction such as photophobia.If do not have above several phenomenon to occur, can assert that zest conforms with the regulations, or standards of grading corneal, iris and the conjunctiva of according to the form below mark respectively, calculate meansigma methods, and compare with the eye that contrasts of same animal.

Evaluation result:

Table 6 eye irritation evaluation criterion

2.4 result

Select 4 rabbits, before experiment, half an hour observation eyes are all normal, can be used for test.Eyes splash into after medicine and normal saline, and the eyes that splash into medicine close, and after 2-3 minute, open, and matched group is opened immediately.

Left eye: medicine 0.1mL right eye: normal saline 0.1mL

Stimulate as can be seen from the table according to eye, with medicine left eye in the scoring of cornea, iris, conjunctiva, comprehensive mean scores 0 < 3 < 3.9 of 4 rabbits, prove this injection nonirritant according to eye irritation evaluation criterion.

Test example 3

The clinical test of pesticide effectiveness

1, the result of the test of medicine composition injection treatment hyopneumoniae disease of the present invention

Select the sick pig of hyopneumoniae natural occurrence.The case of selecting should, through clinical and laboratory diagnosis, be diagnosed as hyopneumoniae disease.By every day neopathy, qualified sick pig bring at random in 4 groups and treat, 15～20 of every group of cumulative cases.If (1) embodiment of the present invention 2 pharmaceutical composition dosage groups, (2) florfenicol dosage group, (3) meloxicam dosage group, (4) Positive of penicillin skin-test medicine matched group.Route of administration and dosage: 1,2,3 groups adopt intramuscular injection, 1 time/3 days, successive administration 1-2 day.4 groups adopt intramuscular injection, 2 times/day, successive administration 3 days.Refer to table 7.

Table 7 grouping arranges table with dosage

Result:

The curative effect of table 8 medicine composition injection to hyopneumoniae disease

Remarks: curative effect determinate standard recovery from illness: treat after 10 days, transference cure, it is normal that indices recovers; Effective: treat after 10 days, indices is improved or obviously improves; Invalid: treat after 10 days, indices is showed no and improves or death.

Above result shows: pharmaceutical composition of the present invention is better than existing two kinds of medicines and contrast positive drug penicillin to the curative effect of hyopneumoniae disease.

2, the result of the test of the scorching disease of medicine composition injection treatment pulmonis Bovis seu Bubali

Select the sick cattle of the scorching natural occurrence of pulmonis Bovis seu Bubali.The case of selecting should be through clinical and laboratory diagnosis, is diagnosed as the scorching disease of pulmonis Bovis seu Bubali.By every day neopathy, qualified sick cattle bring at random in 4 groups and treat, 8～12 of every group of cumulative cases.If be (1) that the embodiment of the present invention 2 pharmaceutical composition dosage groups, (2) are that florfenicol dosage group, (3) are that meloxicam dosage group, (4) are Positive of penicillin skin-test medicine matched group.Route of administration and dosage: 1,2,3 groups adopt intramuscular injection, 1 time/3 days, successive administration 1-2 day.4 groups adopt intramuscular injection, 2 times/day, successive administration 3 days.Refer to table 7.

Table 9 grouping arranges table with dosage

Result:

The curative effect of table 10 medicine composition injection to the scorching disease of pulmonis Bovis seu Bubali

Remarks: curative effect determinate standard recovery from illness: treat after 10 days, transference cure, it is normal that indices recovers; Effective: treat after 10 days, indices is improved or obviously improves; Invalid: treat after 10 days, indices is showed no and improves or death.

Above result shows: pharmaceutical composition of the present invention is better than existing two kinds of medicines and contrast positive drug penicillin to the curative effect of the scorching disease of pulmonis Bovis seu Bubali.

Using method explanation:

Function: antibacterial, antiinflammatory.

Cure mainly: treatment and prevention cattle, pig upper respiratory tract infection disease, the especially various pneumonia diseases including mycoplasma pneumonia syndrome.

With metering method: cattle: 0.1ml/kg, cervical region subcutaneous injection; Pig: 0.1ml/kg, intramuscular injection, 1 time on the every 3rd, is used 1-2 time continuously.Preventive dose reduces by half.

Specification: 50ml/ bottle.

Storage: airtight, lucifuge, puts shady and cool place.

Effect duration: 2 years.

Claims (2)

1. a pharmaceutical composition of preventing and treating cattle hyopneumoniae disease, is characterized in that:

Calculate with 1000ml medicinal liquid, containing each medicine and adjuvant amount proportioning be:

Florfenicol 250～350g, meloxicam 2～8g, 1,2-PD 40～80ml, 2-Pyrrolidone 80～250ml, PEG-4000 650～900ml, benzyl alcohol 10～15ml.

2. the preparation method of a kind of pharmaceutical composition of preventing and treating cattle hyopneumoniae disease as claimed in claim 1 is:

1. mixed solvent preparation, according to formula composition, measures respectively 1,2-PD 40～80ml, 2-Pyrrolidone 80～250ml, and PEG-4000 650～900ml, benzyl alcohol 10～15ml, is uniformly mixed;

2. the first registration of medicinal liquid really takes florfenicol 250～350g, and meloxicam 2～8g, adds the 800ml mixed solvent 1. obtaining, and at 45-55 DEG C, constant temperature is stirred to into clear solution;

3. 2. medicinal liquid preparation will obtain 1. mixed solvent of solution and be settled to 1000ml, be filtered to No. 3 sintered glass funnels or other filters clear and bright, embedding, 121 DEG C of sterilizings 15 minutes and get final product.