| Ivermectin(IVM)is widely used to prevent and control parasites of livestock and companion animals.Traditional oral and injection formulations of IVM required to administrate frequently,and blood concentration was fluctuated greatly,which affected the prevention and treatment of parasitic diseases.At present,although some long-acting formulations or sustained-release bolus reported,they all had different defects.In this study,a IVM sustained-release bolus contained soluble silicate glass was prepared to avoid the defects of the existing IVM preparation.The specific contents are reported as follows:1 IVM suatained-release bolus preparationSoluble glass is prepared by high temperature melting using inexpensive sodium carbonate and silica.Fourier transform infrared spectroscopy characterization showed that the carbonate characteristic peak of soluble glass disappeared,and only the characteristic peak of silicate of about 1000 wave number was obtained.Scanning electron microscopy revealed irregular pores on the surface of soluble glass powder.Soluble glass with good biocompatibility was used as one of the excipients of IVM sustained-release bolus.The disintegration time of the boluses in the artificial rumen juice were used as an index,and the components of the IVM sustained-release bolus and the preparation process parameters were screened by singal factor,partial factors and full factors test.The response surface were optimized by Box-Behnken with microcrystalline cellulose,starch and LS-HPC as factors.The optimum composition of the bolus were 2:1 for barite powder and soluble silicate glass powder,8%for microcrystalline cellulose,0.5%for starch and 0.25%low-substituted hydroxypropyl cellulose.The optimum preparation process wet granulation using 12 mesh sieve,drying at 60? for 30 min,12 mesh sieve whole grain,37.5 Ton pressure,and consolidating at 30? for 24 h.Two specifications of IVM sustained-release bolus were prepared.The length,width,and height of the IVM sustained-release boluses were 28.12 mm±0.14 mm,16.1 mm±0.13 mm,and 13.03 mm±0.05 mm,respectively.The IVM sustained-release boluses weighed 11.4842 g±0.1675 g and its density was 1.95 g/cm3.2 Establishment of related detection methods for IVM sustained-release bolusA HPLC-UV method for the detection of IVM content in sustained-release bolus was established.After the bolus was pulverized,it was extracted with methanol,and the sample is diluted and detected by HPLC.The chromatographic conditions were performed on a Waters Symmetyr C18 column(4.6×250 mm,5 ?i)with a mobile phase of methanol-water(85:15,v/v),a detection wavelength of 245 nm,a flow rate of 1.0 mL/min and a column temperature of 30?.The injection volume was 10 ?L.The results showed a good linear relationship in the linear range of 0.5?g/mL-50 ?g/mL(R2=0.9999).The recovery was between 95.47%and 106.72%.The intra-assay coefficient of variation ranged from 1.41%to 6.44%,and the coefficient of variation between batches ranged from 1.21%to 2.28%,both less than 10%.The methods for the detection of IVM in artificial rumen juice and plasma by HPLC-FLD were established.The samples of artificial rumen juice and plasma were extracted with ethyl acetate and acetonitrile,dried at 50? under nitrogen,and derivatizated by N-methylimidazole and trifluoroacetic anhydride.The chromatographic conditions were performed on mobile a phase of methanol-water(97:3,v/v),a flow rate of 1 mL/min,a column temperature of 40?,a fluorescence detector excitation wavelength of 365 nm and emission wavelength of 475 nm.The IVM linear relationship was good in the concentration range of 0 ng-20 ng(R2=0.9984)and 1 ng-100 ng(R2=0.9992)in artificial rumen juice.The IVM linear relationship was good in the concentration range of 0?100 ng(R2=0.9993)in plasma.The recovery rate of IVM was between 96.65%and 99.76%in artificial rumen juice.The mean coefficient of variation within the batch was between 1.52%and 5.46%,and the coefficient of variation between batches was 3.73%±2.01%.The limit of detection was 0.3 ng/mL,and limit of quantitation was 0.5 ng/mL in artificial rumen juice.The recovery rate of IVM was between 93.61%and 99.79%in plasma.The mean intra-assay coefficient of variation was between 2.73%and 3.74%.The coefficient of variation between batches was 3.34%±0.53%.The limit of detection was 0.1 ng/mL and the limit of quantification was 0.3 ng/mL in plasma.The IVM concentration of high,medium,low and LOQ in the samples maintained good stability under different conditions.The established methods had specificity,precision,recovery and stability that meet the criterion of methodology.3 Study on release kinetics of IVM sustained-release bolusThe IVM sustained-release bolus were placed in a dissolution cup containing 900 mL of artificial rumen juice containing 0,5%SDS,and in vitro release test was carried out with the condition of at temprerature of 39.5? and totation speed of 100 rpm.1 mL of sample was collected at different times,and dissolution medium was replaced every day until the test completely.The IVM boluses added 0.50 g IVM had a burst release at 1st day.The release of IVM reached 48.37 mg±3.04 mg,accounted for 10.56%of the total IVM in the bolus.From 7 d to 53 d,the sustained release phase of the bolus was between 2 mg to 9 mg per day.At 54 d to 57 d,the release of IVM increased to 10 mg to 13 mg per day.At 58 d to 62 d,IVM release decreased to the end of release.The IVM cumulative release of the bolus in artificial rumen juice reached 423.72 mg±5.49 mg,and the cumulative percent release was 92.52%.The IVM boluses added 0.25 g IVM had a burst release too at 1st day.The release of IVM reached 27.74 mg±1.62 mg,accounted for 12.27%of the total IVM in the bolus.From 7 d to 59 d,the sustained-release phase of the bolus was between 1 mg?6 mg per day.The IVM cumulative release of the bolus reached 209.10 mg±4.56 mg,and the cumulative percent release was 92.49%.The best-fit release kinetic equations were all the Korsmeyer Peppas equations,and the n-values of parameter of the release mode were 0.5180 and 0.4581 repectively,between 0.45 and 0.89.It indicated that IVM sustained-release bolus released IVM in vitro by diffusion and erosion.Six sheep(Small Tailed Han Sheep)weighted 21.20 to 24.20 kg,one IVM sustained-release bolus administrated orally per sheep.A 2.5 mL blood sample was taken at different times,and centrifuged at 3000 rpm for 10 min to separate and store at-20?.The bolus stayed in the rumen nearly 1 month with an average peak concentration of 6.69 ng/mL.The IVM concentration was relatively stable form 1 d to 14 d,and maintained at 1 ng/mL?6 ng/mL in plasma.At 28 d,the concentration of IVM in plasma was 0.63 ng/mL±0.12 ng/mL.The pharmacokinetic parameters of the compartmental and non-compartmental models were calculated using WinNolin 5.2 software,respectively.According to the Akaike's information Criterion(AIC)and the Schwarz_Bayesian Criterion(SBC),the release of IVM sustained-release bolus in vivo was consistent with the 1st order and 1 compartment model(no Lag Time).The Ka,Ke,Tmax,Cmax,AUC and CL of compartment model were 1.69 d,0.06 d,2.33 d,4.42 ng/mL,86.86 day·ng/mL and 122.26 L/day/kg respectively.The Ke,T1/2?,Tmax,Cmax,AUC,Vd,CL and MRT of non-compartment model were 8.33 d,6.37 d,1.5 d,7.53 ng/mL,76.63 day·ng/mL,2501.93 L/kg,270.31 L/day/kg and 12.71 d respectively.Comparing the pharmacokinetics parameters of two model,it was found that the parameters of non-compartmental model is more coincident with the pharmacokinetics of the IVM bolus with oral administration at a once dose.4 Preliminary quality evaluation of IVM sustained-release bolusThe IVM sustained-release boluses content is between 98.87%and 102.06%of the labeled amount,which meets the requirement of between 90%and 110%.The weight difference within the IVM sustained-release boluses meet the relevant regulations.Under the identification item,the time of peak in the IVM bolus was consistent with that of the control.The IVM sustained-release boluses showed good stability against high temperature,high humidity and light.It was indicated that the blouses were not moisture absorption property because the weight of IVM sustained-release boluses were increased less than 3%in high-humidity test.The accelerated test of the boluses,packaged in aluminum foil bag,were stable with the condition at 40? and a relative humidity of 75%.The IVM sustained-release bolus for sheep prepared in this study has a simple prparation method,is safe,high-efficiency and ahs no foreign matter residue in the animal body.IVM sustained-release boluses are continuously released in sheep nearly one month,which is superior to conventional IVM preparations.It has important clinical application value for the prevention and control of parasitic diseases in sheep breeding in China,especially in northern pastoral area. |
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