| Respiratory and digestive diseases, there are currently two important aquaculture diseases, seriously affecting the economic benefits of China’s aquaculture industry. As the blind in the treatment of animal diseases a large number of drugs, casual and high-dose and long-term medication to induce resistance in many bacteria, animals, once the disease, using a single drug for treatment to be ineffective.3rd generation fluoroquinolones danofloxacin mesylate (DFM) andβ-lactams amoxicillin (AMO) in the treatment of respiratory and digestive tract diseases of livestock and poultry, there are very good results. A result of fluoroquinolones and P-lactam drugs in the clinical synergy is usually presented, therefore intends to danofloxacin mesylate and amoxicillin compound preparation is made with a view to improving the treatment of animal respiratory tract diseases and digestive diseases. Due to a shorter half-life of amoxicillin in the treatment of livestock and poultry diseases delivery interval is short, require multiple doses, which in large-scale farms, the workload will be very huge, based on such considerations, this study developmented DFM and AMO into a long-acting injection. At the same time, bioavailability and residue depletion of DFM-AMO suspension in pigs were studied to clarify the pharmacokinetic characteristics, bioavailability and elimination rules on residues of DFM and AMO.The DFM-AMO suspension was developmented. DFM-AMO suspension was made by suspending the AMO & DFM in soybean oil after adding lecithin, Span 80, BHT and BHA. After selecting the best formula, DFM-AMO suspension has been developed successfully. Three batches of pilot products were prepared for further quality and stability study. The preparation of DFM-AMO suspension had been carried on a stability study. The accelerated testing and and long term testing were carried. The AMO and DFM concentrations of preparation samples were determined by HPLC-UV. In the accelerated testing and and long term testing, the content of DFM-AMO suspension decreased less than 5%, and the related substance slightly increased. At the end of the test, all indexs had not significant changes, such as the concentration, the color, the particulate and so on. The experimental results indicated that the preparation was stable to light and heat.Pharmacokinetics of DFM-AMO suspension in pigs was studied. A HPLC detection method was developed to detect DFM and AMO in plasm. DFM extraction method:The sample was precipitated with ACN, the supernatant was evaporated to dryness. The residue was dissolved in mobile phase. AMO extraction method:The sample was precipitated with trichloroacetic acid (20%), the supernatant by adding 7% formaldehyde solution for derivatives, derivatives were extracted by ether and evaporated to dryness. The residue was dissolved in mobile phase. The standard curves for DFM were liner in a range of 0.05～5μg/mL(r=0.9998) and the standard curves for AMO were liner in a range of 0.10～20μg/mL (r=1). The limit of quantitation of DFM and AMO were 0.05μg/mL and 0.10μg/mL respectivly. The recoveries of DFM at 0.05μg/mL、0.5μg/mL and 5μg/mL three concentration levels were more than 89.0% with relative standard deviation(RSD) less than 3.0% and the recoveries of AMO at 0.10μg/mL、2μg/mL and 20μg/mL three concentration levels were more than 88.0% with relative standard deviation(RSD) less than 13.0%.18 male three-way cross pigs (Duroc×Landrace×Large White,15±5kg), randomLy separated the pigs into 3 groups. Each group has 6 animals. According to the cross-over test design, intramuscular DFM aqueous solution and AMO aqueous solution and DFM-AMO suspension respectively. The dosage was DFM 2.5mg/kg, AMO 15mg/kg. Blood samples were collected up to 24h,48h and 96h respectively after administration and plasma concentrations of DFM and AMO were determined by a high performance liquid chromatography (HPLC). Pharmacokinetic analysis for DFM and AMO were carried out by using WinNonlin software (Version 5.2.1) with a non-compartmental mode. The main pharmacokinetic parameters for DFM aqueous solution were as follows:λ, 0.067±0.01h-1; T1/2λ,10.42±1.19h; Tmax,0.88±0.21h; Cmax,0.70±0.13μg/mL; AUC, 8.45±0.90h·μg/mL; V,4.62±0.60L/kg; CL,0.31±0.04L/h/kg; MRT,14.23±1.65h. The main pharmacokinetic parameters for AMO aqueous solution were as follows:λ, 0.40±0.04h-1; T1/2λ,1.74±0.16h; Tmax,1.53±0.40h; Cmax,5.62±0.74μg/mL; AUC, 25.35±2.25h·μg/mL; V,1.64±0.38L/kg; CL,0.65±0.12L/h/kg; MRT,3.20±0.41h. The main pharmacokinetic parameters for DFM-AMO suspension were as follows:DFM:λ, 0.022±0.002h-1; T1/2λ,31.31±2.21h; Tmax,2.03±0.50h; Cmax,0.44±0.04μg/mL; AUC, 13.89±0.95h·μg/mL; V,8.07±0.74L/kg; CL,0.18±0.02L/h/kg; MRT,45.58±2.90h; F, 166.2±20.9. AMO:λ,0.028±0.002h-1; T1/2λ,24.60±2.04h; Tmax,1.20±0.25h; Cmax, 1.50±0.10μg/mL; AUC,37.41±2.34h·μg/mL; V,14.27±1.35L/kg; CL,0.40±0.02L/h/kg; MRT,37.65±1.6h; F,148.6±15.7. The pharmacokinetic characteristics of single intramuscular administration of prepared DFM-AMO suspension in this study indicated slow absorption, wide distribution and slow elimination. It was reasonable that the prepared suspension was injected one time in every two days in thet reatment of sensitive bacterial infectious diseases in pigs.Residue depletion of DFM-AMO suspension in pigs was studied. A HPLC detection method was developed to detect DFM and AMO in muscle, liver, kindey and fat. DFM extraction method:tissues of pigs were extracted with phosphate buffers and directly cleaned up. The SPE eluate was acetonitrile, The eluate was evaporated to dry under a stream of nitrogen, The residue was dissolved in mobile phase. AMO extraction method: The residues of AMO in pigs muscle and fat were extracted with phosphate buffers and directly cleaned up; Those in pigs liver and kidney were first extracted with acetonitrile followed by phosphate buffers, The solution of acetonitrile was evaporated to dry under a stream of nitrogen and redissolved by the second extraction for SPE clean-up. All extraction was cleaned up with Accubond SPE, The SPE eluate was acetonitrile, The eluate was evaporated to dry under a stream of nitrogen, redissolved with standard dilute solution, derivatized with benzoic anhydride and 1,2,4-triazole mercury reagent. In four tissues, the limit of quantitation of DFM and AMO were 20μg/kg and 25μg/kg respectivly. The recoveries of DFM in four tissues were more than 83.0% with relative standard deviation(RSD) less than 9.0% and the recoveries of AMO in four tissues were more than 66.0% with relative standard deviation(RSD) less than 8.0%.Twenty healthy three-way cross pigs (Duroc×Landrace×Large White,30±5kg), were used in this study. Intramuscular DFM-AMO suspension, The dosage was DFM 2.5mg/kg, AMO 15mg/kg. Four animals were randomLy chosen and slaughtered on each occasion at 1,3,7,21 and 42 days after drug withdrawal. The tissue samples (muscle, liver, kidney and fat) were collected and then were homogenated at 10000r/min for 3min. Tissue concentrations of DFM and AMO were determined by HPLC. In descending order, the DFM residues concentrations found in all examined tissues were injection site> liver > kidney> muscle> fat and the AMO residues concentrations found in all examined tissues were injection site> kidney> fat> liver> muscle. The withdrawal period was estimated by WT1.4 software, and determined at the time when the upper one-sided tolerance limit, with a confidence of 95%, was below the MRL established by the European Union for DFM and AMO in pigs. The statistically estimated withdrawal period was calculated to be 34 days.In summary, the DFM-AMO suspension prepared in this study has excellent performance. The stability of the preparation is good, simple process. Suspension pharmacokinetic results showed that, DFM and the AMO are widely distributed in pigs, a high bioavailability, long elimination half-life to be effective in the treatment of livestock and poultry of the respiratory and digestive diseases. In practical applications, as compared with the normal preparation, suspension increased the dose interval, reduced delivery times, thereby reducing labor costs and increase the economic benefits of aquaculture. |
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