| Chicken coccidiosis is a common parasitic disease caused by coccidia,which is more common in farms and seriously damages the economic efficiency of farms.Toltrazuril is a new type of triazine anticoccidial drug in the market now.Its mechanism of action is to kill coccidia by interfering with the normal division process of coccidia nuclei and inhibiting the respiratory metabolism of cell mitochondria,mainly in the cleavage and gamete reproduction stages of coccidia.It has the advantages of wide safety range,high bioavailability,long drug half-life and low drug resistance.The final prescription was determined by screening the types and ratios of raw and auxiliary materials,and the production process of 2.5%toltrazuril solution was initially optimized.Meanwhile,the prescription was scaled up for small test samples under laboratory conditions,and the quality and stability studies were conducted.Finally,the pharmacokinetics of Bacitracin solution and 2.5%toltrazuril homemade solution were carried out in adult yellow-finned broiler chickens and adult King’s Red laying hens,respectively.The above experiments provided data for the development of 2.5%toltrazuril solution and also provided reference for the clinical application of this drug.1.Prescription and process study of toltrazuril solution formulation1.1 Prescription screeningAccording to the original prescription,polyethylene glycol 200 and triethanolamine were selected as the main excipients,and the prescription composition was screened by single-factor test;and the antioxidants were screened by antioxidant properties.The results determined that the addition amount of triethanolamine was 40g per 100mL of toltrazuril solution and the addition amount of toltrazuril API was 2.5g,and the volume was fixed to 100mL with polyethylene glycol 200,and the quality was not affected by not applying antioxidants,and no antioxidant was added in the prescription.1.2 Process studyThe key factors such as dissolution temperature and time,filtration method were investigated.The dissolution temperature was determined to be 60℃ by single-factor test.Also compare the effective content before and after filtration and determine the process adding 0.45μm filter membrane filtration.The preparation process of the solution is as follows:(in 100mL)weigh 60g polyethylene glycol 200 and heat to 60℃,add 2.5g toltrazuril,stir for 10min,add 40g triethanolamine,continue to stir for 150min to dissolve the raw material completely,add the remaining amount of polyethylene glycol 200 and fix the volume to 100mL.pass 0.45μm filter membrane,sterilize and fill,press the cap,pack,that is.2.Quality evaluation and stability study of toltrazuril solutionThe solution was prepared as a colorless and clear solution with the specification of 2.5%(2.5g of toltrazuril in 100mL of preparation),pH 8.0-10.0,content between 92%105%,total unknown impurities not more than 2.0%,hydrolysis products not more than 1.0%,moisture all below 2.0%,relative density in 1.120-1.140.The test of influencing factors shows that the solution can be stored and transported stably under the condition of dryness and avoiding light at room temperature,and the appearance of the solution is still clarified colorless or light yellow solution under the experimental conditions of accelerated test and long-term test,and the change of the content of toltrazuril solution is less than 5.0%,and the range of pH change is less than 0.5,and the content of relevant substances is in accordance with the regulations,which indicates that the storage of toltrazuril solution under the conditions of accelerated test and long-term test is The stability is good.3.Pharmacokinetic study of toltrazuril solution in chickensIn this study,the in vivo pharmacokinetics of toltrazuril solution and homemade toltrazuril solution were investigated in adult Peking red laying hens and adult yellowfinned broiler chickens,respectively.The sample pretreatment method was as follows:500 μL of plasma sample was added with 2000 μL of acetonitrile to precipitate protein,vortexed and mixed for 5 min,centrifuged at 10,000 rpm for 10 min,and the supernatant was extracted and blown dry using a water bath with nitrogen blowing concentrator at 45℃ and then re-dissolved with 500 μL of mobile phase.Vortex and mix for 5 min,centrifuge at 10,000 rpm for 10 min.0.22 μm filter membrane filtration.Chromatographic conditions:mobile phase:potassium dihydrogen phosphate solution(pH=3.0):acetonitrile(43:57),flow rate:1.0 mL/min,detection wavelength:240 nm,injection volume:20 μL,column temperature:30℃.The results of methodological validation were:the detection limit of toltrazuril in chicken plasma was 0.01 μg/mL,and the quantification limit was 0.05 μg/mL;the average recoveries were in the range of 100.79%-114.15%,and the relative standard deviations of intra-batch precision and inter-batch precision RSD were less than 10%for medium and high concentrations,and less than 20%for low concentrations.A total of 48 healthy yellow-feathered broilers and adult laying hens,weighing about 2±0.5 kg each,were selected,while each chicken was randomly numbered into two groups of twelve birds each,totaling four groups.The groups were A(adult yellowfeathered broilers with paclobutrazol),B(adult laying hens with paclobutrazol),C(adult yellow-feathered broilers with homemade toltrazuril solution),and D(adult laying hens with homemade toltrazuril solution),respectively.The administered dose was the clinically recommended dose of 25 mg/kg,and blood was collected at time points of 0.25,0.5,1,2,4,8,12,18,24,48,72 and 120 h.Blood concentrations were measured and pharmacokinetic parameters were fitted using Winnonlin software.The results showed that the area under the drug-time curves of Bacitracin solution and toltrazuril autologous solution in adult King Red laying hens were 329.55 h-μg/mL and 221.01 h-μg/mL,respectively,and the peak drug concentrations of both were 16.23 μg/mL and 10.43μg/mL,with peak times of 4.09 h and 3.25 h,respectively,and the mean drug retention times of The mean retention times were 22.5 1h and 24.24h,elimination phase half-lives were 24.84h and 33.37h,clearance rates were 0.08L/h/kg and 0.11L/h/kg,and apparent volume of distribution were 3.13L/kg and 5.71L/kg,respectively.the area under the drug-time curves of paclobutrazol and toltrazuril autologous solutions in adult yellowfinned broiler chickens were 637.81h-The peak drug concentrations were 23.48 μg/mL and 25.11 μg/mL,peak times were 8.00 h and 3.17 h,mean drug retention times were 24.47 h and 25.27 h,elimination phase half-lives were 19.01 h and 25.83 h,and clearance rates were The differences between the pharmacokinetic parameters of paclobutrazol and toltrazuril were not significant in adult King’s Red laying hens,and the pharmacokinetic parameters of both solutions were not significant in adult yellow-finned broilers.The differences in the pharmacokinetic parameters of AUC and Cmax were significant in adult Peking red poults and adult yellow-finned broiler chickens,and the differences in the pharmacokinetic parameters of AUC and Cmax were significant in toltrazuril homemade solution in both.Adult yellow-finned broiler chickens were more bioavailable and could reach higher peak hour concentrations compared to adult King’s Red poults. |
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