Preparation Of TCBZ And LMS Compound Dry Suspension As Well As Study On Its Pharmacokinetics And Deworming Effect In Sheep

China’s stockbreeding has been developing rapidly in recent years,but parasitic diseases have seriously harmed the stockbreeding,especially the cattle and sheep that are susceptible to parasites like trematode and nematode,usually in a mixed manner.The parasites however have developed different degrees of resistance to most deworming drugs on the market,for which to develop an easy-to-use and effective deworming drug that can treat parasitic mixed infection is the future direction of veterinary drug research and development.Triclabendazole(TCBZ),as a clinically high-efficiency anti-fasciola hepatis drug,has obvious killing effect on both adult and larva of fasciola hepatica.For evident efficacy in the treatment of gastrointestinal nematodiasis and capability to improve animal immunity as an immune enhancer,Levamidazole(LMS)was selected to be combined with TCBZ as the main drug for preparation of dry suspension in this study.Not only can the main drug drive and kill fasciola hepatica and gastrointestinal nematodes,it also improves animal immunity.The most suitable formula was selected by single factor test and orthogonal test of suspension aid,emulsifier and flow aid.The contents of TCBZ and LMS in pharmaceutical preparations and plasma were detected by HPLC-MS/MS.Besides,Drug stability experiments,pharmacokinetic studies of TCBZ and LMS in sheep and their repellent effects were conducted in sequence to verify the feasibility of preparation of dry suspension by combining TCBZ and LMS.In this test,a single variable was controlled by a single factor experiment.Xanthan gum,hydroxypropyl methyl cellulose and sodium carboxymethyl cellulose were selected as suspension aids for the single factor test of the dry suspension;while poloxamer 188 and micropowder silica gel were used as emulsifier and flow aid,respectively.Xanthan gum was selected as the optimal suspension aid from xanthan gum,hydroxypropyl methyl cellulose and sodium carboxymethyl cellulose based on the sedimentation volume ratio and redispersability as indicators,and 3 groups of optimal doses were selected from the 9 groups of applicable doses;3 groups of optimal doses were selected from 9 groups of commonly used doses of poloxamer 188 based on the sedimentation volume ratio and floating drug condition as indicators;3 groups of optimal doses were selected from 9 groups of commonly used doses of micropowder silica gel based on the sedimentation volume ratio and angle of repose as indicators;an orthogonal experiment was performed on xanthan gum,poloxamer and micropowder silica gel at the optimal prescription ratio,based on the sedimentation volume ratio and redispersability as indicators.The final results of the study determined that the optimal prescription was recorded by a weight ratio of 4%TCBZ,3%LMS,2.4%xanthan gum,1.0%poloxamer,1.4%micropowdered silica gel and 88.2%sucrose(gross weight:1g).In this study,HPLC-MS/MS was used to test the contents of TCBZ and LMS in pharmaceutical preparations.HPLC-MS/MS was performed on an ACQUITY UPLC BEN C18column(1.7μm,2.1 mm×50 mm)with gradient elution in acetonitrily-0.1%formic acid aqueous solution.Positive ion scanning mode(ESI+)and multiple reaction monitoring(MRM)mode were used for the scanning of parent and daughter ions.The TCBZ-LMS linear relationship was investigated as good in the concentration of 1-20 ng/m L(regression equations are y=72262x+29486,~2=0.9986;y=7034.2x-47.835,~2=0.9987,respectively).The recovery rate and precision of the two main drugs were examined respectively to show that both were less than2%.The detection limit and quantitation limit of TCBZ and LMS in the reagent of this method were tested respectively.The results showed that the detection limit and quantitation limit of TCBZ were 0.2 ng/m L and 0.6 ng/m L respectively,while those of LMS were 0.2 ng/m L and 0.8ng/m L respectively.This simple,feasible and highly reproducible detection method can be used for in vitro drug content determination and drug stability experiment of TCBZ and LMS for follow-up compound dry suspension.Stability tests were carried out on the optimal prescription,including 60℃high temperature test,RH90%high humidity test,4500 Lx strong light test and accelerated stability test.The results prove that:the drug appearance changed obviously,white caking appeared,and the drug content decreased by more than 5%in the high temperature test and high humidity test;there was no significant change in drug appearance,and the drug content decreased by no more than 5%in the strong light test and accelerated stability test;high temperature and humidity should be avoided during storage of TCBZ-LMS compound dry suspension.This test investigates the linear relationship between the concentrations of TCBZ and LMS in plasma,showing a good linear relationship between TCBZ and LMS at a concentration of 1-20ng/m L(regression equations are y=71862x+35163,~2=0.9978;y=6586.9x-498.89,~2=0.9989respectively).The recovery rate and precision of the two main drugs were examined respectively to show that both were less than 10%.The detection limit and quantitation limit of TCBZ and LMS in plasma were examined respectively.The results showed that the detection limit and quantitation limit of TCBZ were 0.5 ng/m L and 1.5 ng/m L respectively,while those of LMS were 0.2 ng/m L and 1.7 ng/m L respectively.The influence of sheep plasma and compound dry suspension excipients on the pharmacokinetic test could be excluded with a highly exclusive and reproducible method that is applicable for the follow-up pharmacokinetic tests of TCBZ and LMS.In this experiment,3 m L of cervical venous blood was collected according to the time point of blood collection to separate the plasma after sheep administration,HPLC-MS/MS was applied to detect drug time concentration with DAS2.0 software introduced.The results showed the main pharmacokinetic parameters of TCBZ and LMS:t1/2z was 35.706 h and 72.850 h,AUC(0-t)was6124.816 ng/m L*h and 31210.379 ng/m L*h,AUC(0-∞)was 6498.652 ng/m L*h and 35143.592ng/m L*h,MRT(0-t)was 16.296 h and 24.680 h,MRT(0-∞)was 22.634 h and 43.939 h,Tmax was8 h and 18 h,Cmax was 509.668 ng/m L and 1098.415 ng/m L,Cl＿F＿obs was 0.041 L/h/kg and0.001 L/h/kg,and the Vz＿F＿obs was 0.158 L/kg and 0.057 L/kg,respectively.The results prove that,TCBZ-LMS combined dry suspension features earlier peak time and lower Cmax and AUC compared with TCBZ suspension,but features later peak time,higher Cmax and AUC,and prolonged t1/2z compared with LMS suspension.In this study,a deworming effect experiment was conducted on 20 sheep with fasciola hepatica and gastrointestinal nematodes in groups,so as to calculate the negative conversion rate and reduction rate of eggs.The results showed that the compound suspension of TCBZ and LMS had good repellent effect on fluke and nematode:the egg conversion rates of fluke and nematode were 80%and 0%,respectively(Day 7);the egg conversion rates of fluke and nematode were100%and 40%,respectively(Day 15);the egg loss rates of fluke and nematode both exceeded90%(Day 7);the egg loss rates of fluke and nematode both exceeded 99%(Day 15).In conclusion,the TCBZ-LMS compound suspension prepared in this study is characterized by various and convenient administration modes,high reliability and excellent deworming effect,which provides the possibility and new idea for preparation of dry suspension formulation with the combination of TCBZ and LMS.