Preparation, Bioavailability And Residue Depletion Of Danofloxacin Mesylate Injectable Suspension In Swine

Danofloxacin（DANO） is a third generation synthetic antibiotic of the fluoroquinolone group developed specifically for use in veterinary medicine following Enrofloxacin（ENO）.Danofloxacin is one of the effective antimicrobial activities of the fluoroquinolones,it was widely used in veterinary field,but usually the danofloxacin mesylate were used.The main dosage of DFM is DFM injection,DFM soluble powder and DFM solution,which often need multiple dosing in usual,but DFM injectable suspension could reduce dosing times which brought significant convenience.DFM injectable suspensions were made by dissolving danofloxacin after adding the suspending agent in this study.After the optimization of the best formula and the best technology, several danofloxacin injectable suspensions had been developed successfully.A quality criterion was made with determination results of several productions after studying qualities and stability of these preparations.The pharmacokinetics and residue depletion of DFM injectable suspension in swine were studied to elucidate the bioavailability and residue depletion characteristic,based on which we make clinical dosage program and food safety standard to afford foundation on reasonable use in clinical and residue monitoring of DFM injectable suspension.Preparation and stability of DFM injectable suspension was studied.Screened formula by L₉3³ orthogonal design with suspending agent aluminium monostearate, dispersing agent lecithinum and emulsifying agent Span-80.There was no interaction with expecipent and DFM.Research on technology find that if the disperse rate was below 6000 r/min,DFM couldn’t be dispersed sufficiently,if the rate was above 10000 r/min,there were much sedimentation of injectable suspension,and more air bubble were generated.When the disperse time less than 10min,DFM couldn’t be dispersed sufficiently,while more than 20min,the injectable suspension was a little dark.It had no difference among 3min,5min and 10min,when injectable suspension was redispersed in colloid mill.The best disperse condition was 8000 r/min,15 min in dispersing machine,3 min in colloid mill.Prepared injectable suspension with defined technology,then considerated the appearance characteristic,syringeability,volume ratio of sedimentation and redispersibility of DFM injectable suspension to define formula.The stability test was needed.HPLC-UV method was developed for determining DFM content in preparation samples.The content of DFM was between 90%～110%of labeled amount,related substance＜1.5%,the ratio of sedimentation＞0.9,particle diameter＜3μm.Influencing factor test suggested the color of injectable suspension had a little change at high temperature and illumination,but content,the ratio of sedimentation and redispersibility had no changes.So DFM injectable suspension should better be preserved in amber glass away from light at room temperature.The experimental results indicated that the preparation had good stability.We suggest that the period of validity about the injectable suspension could be temporarily determined at 2 years at room temperature.HPLC method for the determination of DFM in plasma and edible tissues of swine was established.Plasma samples were extracted with ACN which conclude 0.2% glacial acetic acid,before detection by HPLC.Recovery of DFM was＞93%,coefficient of variance＜6%,limit of determination were 0.02μg/mL and limit of quantitation were 0.04μg/mL,which satisfied with detecting bioavailability.Edible tissues of swine were extracted by PBS,then cleaned up through HLB columns,eluted with ACN,and evaporated to dry,before detected by HPLC.The working curve in muscle was y=103.28x+0.84,the recovery＞93%,coefficient of variance＜4.5%;in liver,the working curve was y=98.55x+1.18,the recovery＞83%,coefficient of variance＜2.8%;in kidney the working curve was y=111.74x+1.25,the recovery＞94%,coefficient of variance＜2.0%.The LOD and LOQ in edible tissues were 15 and 20μg/kg respectively.Bioavailability of DFM injectable suspension in healthy pigs was studied.The study was randomized,two-period,two-group crossover trial with a washout time of 7d. 8 mixed breed swine（Yorkshire-Landrace-Doluk cross 15±2kg） were administered DFM injection and DFM injectable suspension in the neck at the dose of 2.5mg/kg body weight.About 3mL of blood samples were collected from front cavity vein before 0h and at 0.167h,0.33h,0.5h,0.75h,1h,1.5h,2h,4h,6h,12h,24h,36h and at 0.167h,0.33h,0.5h,1.5h,2h,4h,6h,12h,24h,36h,48h,72h separately. The concentrations of DFM in plasma were determined by high performance liquid chromatography（HPLC）.Pharmacokinetics parameters were calculated by soft ware WinNonlin5.2 for pharmacokinetics,the result indicate DFM injection and DFM injectable suspension were best described by two-compartment open model with the first-order rate absorption.The main pharmacokinetics parameters of DFM injection were t_1/2 K_a=0.09±0.02h,t_1/2β=8.41±0.91h,T_max=0.57±0.11h,C_max=0.69±0.06mg/L,Vd=9.36 L/kg.The main pharmacokinetics parameters of DFM injectable suspension is t_1/2K_a=0.21±0.05 h,t_1/2β=30.25±2.79h,T_max=1.16±0.11h,C_max=0.38±0.44μg/mL, Vd=10.17L/kg,relative bioavailability were114.2%±2.3%.Compared with the DFM injection,t_1/2 K_a,t_1/2βof DFM injectable suspension were both extended,its T_max was delayed,C_max was reduced,and the relative bioavailability was improved,its effective time could be maintained more than 60h.The experiment results showed that the DFM injectable suspension had significant slow-released efficiency.The dosage regimen was 5 mg/kg B W though intramuscle once.Residue depletion of DFM injectable suspension in swine was studied.20 mixed breed swines（Yorkshire-Landrace-Doluk cross 30±2kg） were administered DFM injectable suspension in the neck at the dose of 5mg/kg body weight once with a washout time of 7d.After 0d（12h）,3d,7d,21d and 42d after injection,each group of 4 pigs were slaughtered.Longissimus muscle of back,liver,kidney and the injection location were collected and then were homogenated at 10000 r/min for 3min.Concentration of the DFM was determined by HPLC.The concentration of DFM residue in tissues from high to low were liver,kidney,muscle,the dephine rate was kidney faster than muscle,liver. After 42d post-withdraw time,concentration in kidney and muscle couldn’t be detected; concentration of liver was similar to LOQ.The half life of liver,kidney,muscle were 9.0 d、6.9d、7.6 d.We used WT1.4 soft to caculated the WDT in tissues,the WDT of them were 32.3d,14d,32d.Because of the target tissue of DFM in swine were liver and muscle according to FAO.So we suggested making WDT of liver 33d as the final WDT of DFM injectable suspension in swine.In conclusion,in this study,twice dispersion was used.During the first time dispersed DFM powder,adjuvant and dissolvant sufficiently with dispersion machine to get crude product.During the second time dispersed the crude product with collid mill. The DFM injectable suspensions were well-distributed,and had the similar particle size. The preparation of DFM injectable suspension could be used with convenience in veterinary clinic for its characteristic of delay-release,which resulted to the decrease of administration times.DFM injectable suspension had high bioavailability in swine,which could also improve therapeutic effect.