Pharmacokinetic Characteristics Of Artemisinin In Chickens

In our previous study,ART was found to have a certain therapeutic effect on E.tenella.It is proved that ART has potential therapeutic effect for E.tenella.On this basis,this study focused on the safety and pharmacokinetics of ART and its potential mechanism in chickens,to provide a reference for clinical rational application.Firstly,30-day-old Green-shell laying hens were divided into blank group,solvent group,low?medium and high(15?40?100 mg/kg)dose groups(n=6).oral administration 11 times at 12 hours intervals.The toxicity of ART to chickens was determined by liver index,weight gain rate,serum biochemical index,tissue appearance and microscopic structure.The results showed that chickens grew well and were in normal spirits;there were no abnormalities in heart,liver,spleen,kidney,brain and other organs observed by dissection;there was no significant difference in liver index and weight gain rate between each administration group and the blank group(P>0.05),and the serum biochemical indexes were normal.Microstructural observation revealed no significant differences in liver,kidney,and brain structures from the blank group.These indicates that conventional doses of ART are safer for chickens.Secondly,in order to research the metabolism of ART in chickens,the method of LC-MS/MS was establish for detecting ART in chicken plasma.The electrospray ionization(ESI)source was used as the ion source,artemether(ARM)as the internal standard(IS).The[M+Na]+quasi ion peaks of ART and ARM were scanned by secondary mass spectrometry.Two pairs of ions,m/z 305.2?151.3(ART)and m/z 321.0?163.1(ARM),were selected for quantitative analysis by multi-monitoring reaction(MRM).It was concluded that there was a good linear relationship of ART in the range of 10 ng/mL-1000 ng/mL(R2=0.997).In this method,the minimum quantitative limit and the recovery rate were 10 ng/mL and between 90%and 105%.Meanwhile,the intra and inter day precision(RSD)were less than 15%.This method is simple,sensitive and reproducible,so it can be used to determine ART levels in chicken plasma.The tested chickens were divided into 12 groups(n=6)according to the three doses of 15,40,100 mg/kg and the number of oral administrations(1,3,7,11).Each group was intragastrically administered,and each group was administered separately.At the end of the last administration,300 ?L of blood was collected into the anticoagulation tube at 10 min,30 min,1 h,1.5 h,2 h,2.5 h,3 h,4 h,6 h,and 8 h.After test,it was found that the peak time of ART in chicken plasma was 1 h,1.5 h,1.5 h after oral administration of the above three doses;after 3-7 times,the peak time was maintained at about 1 h.After 11 times oral administrations,the peak time of the 15 mg/kg dose group was advanced to 0.5 h,and the peak of 40-100 mg/kg appeared at 0.167 h.The low-dose(15 mg/kg)ART did not change significantly in the pharmacological parameters between different oral doses;while the medium-high dose(40,100 mg/kg)was administered constantly 11 times compared with the single dose,the oral clearance rate was significantly increased.High,the area under the curve and the plasma concentration were significantly lower.This suggests that ART has significant self-induced metabolism in chickens in a dose-dependent manner.Subsequently,a blank control group,a solvent control group,and ART group were established.Drug is taken orally once every 12 hours for 11 consecutive times.Two research methods,Real-time PCR and in vitro incubation of liver microsomes,were used to explore the mechanism of ART inducing metabolism in chickens.After extraction of liver microsomes,protein concentration and activity were determined.The results showed that the content of liver microsomes and the activities of CYP1A and CYP3A in the ART group were higher than those in the blank group,but there was no significant difference(P>0.05).The qPCR results of CYP-related genes in liver tissue showed that the expression levels of CYP1A4 and CYP3A37 genes in ART group were up-regulated by 6-7 times compared with the blank group(P<0.05).The expression levels of CYP2C23a was up-regulated by about 10 times(P<0.05),and that of CYP2C45 was up-regulated by 70 times(P<0.01).However,there was no significant change in the expression level of CXR,a transcriptional regulator of CYP2C23a and CYP2C45.In addition,the expression levels of MDR1(multidrug resistance 1-gene)and MPR2(multidrug resistance-associated protein 2)did not change significantly either.This indicates that the self-induced metabolism of ART in chickens is mainly achieved by inducing the CYP450 enzyme family.Among them,the main metabolic effect may be CYP2C45 of the CYP2C subfamily,while the CYP1A and CYP3A subfamilies play a supporting role.Finally,we explored the effect of ART on the timeliness of CYP2C23a and CYP2C45 gene expression levels.The chickens for test were divided into 9 groups(n=9).The drugs were fed at different doses(15,40,100 mg/kg)for 1 day,3 days,and 5 days.Each group was sacrificed at 2 h,4 h,8 h after the last dose of the animals.The expression of CYP2C23a and CYP2C45 was detected by qPCR.The results showed that the expression levels of CYP2C23a and CYP2C45 in the liver from different groups usually reached a peak within 4 h after administration,and CYP2C45 in the middle and high dose groups showed high expression after early administration.As the number of administrations increases,the expression level of CYP2C45 in the low dose group also increases.After repeated administrations,different doses can induce high expression of CYP2C45.As the dose and time increased,the expression of CYP2C23a also increased,but the fold was limited.This indicates that CYP2C45 is dominant in ART metabolism in chickens.