Study On The Pharmacokinetics And Pharmacodynamics Of A New Anticoccidial Drug Ethanamizuril In Chicken

Chicken coccidiosis is a kind of intracellular parasitic protozoa caused by Eimeria coccidia parasitizes in different parts of the intestinal tract of chickens.After the chickens are infected,they show clinical symptoms such as weight loss,loss of appetite,anemia,bloody stools,reduced weight gain,and decreased feed conversion rate,and even cause the chickens to die.According to relevant statistics,the cost of China’s livestock and poultry industry for the prevention and control of chicken coccidiosis can reach hundreds of millions of yuan each year,accounting for nearly one-third of the total cost of chicken disease prevention and control.To deal with the harm of chicken coccidiosis,some chicken industries use a lot of anti-coccidiosis drugs or even abuse them,which has caused serious drug resistance problems of anti-coccidiosis drugs.At present,to deal with the problem of coccidia resistance,on the one hand,it is necessary to continuously develop new anti-coccidial drugs,and on the other hand,it is necessary to propose a reasonable anti-coccidial drug administration plan.Ethanamizuril（EZL）is a new type of triazine anticoccidial drug independently developed by the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.It has the characteristics of high anticoccidial activity,broad-spectrum,high efficiency,and low toxicity.This thesis is to rationally use ethanamizuril,provide a reasonable dosage of ethanamizuril,delay the development of drug resistance,and carry out pharmacokinetics and pharmacodynamic research of ethanamizuril.According to the experimental results of pharmacokinetics and pharmacodynamics,the optimal dosage is given,to provide data support for the rational use of ethanamizuril in veterinary clinics and delay the emergence of ethanamizuril resistance.The results are as follows:1.One method for the detection of ethanamizuril in chicken plasma and cecal contents was established,respectively,and the method was evaluated separately.The plasma samples were extracted with ethyl acetate,blow-dried,and reconstituted for testing.The cecal content sample was extracted with dichloromethane,and the extract was purified with Waters Oasis HLB solid-phase extraction cartridges.The experimental results showed that the recovery rate of Ethanamizuril in plasma ranged from 90.19%to 116.72%,and the inter-assay and intra-assay coefficient of variation between batches were 1.62%to 8.57%and 2.64%to 7.41%,respectively.Within the linear range of 0.04 to 10 mg/L,the standard curve is y=0.8532x-0.0337,R2=0.9998.The detection limit and quantification limit of ethanamizuril in plasma were 0.02 mg/L and 0.04mg/L,respectively.The recovery rate of ethanamizuril in the cecum content ranged from 98.07%to 110.30%,and the inter-assay and intra-assay coefficient of variation between batches were 5.46%to 8.73%and 5.52%to 8.16%,respectively.Within the linear range of 0.1 to 10 mg/L,the standard curve was y=0.9882 x-0.028,R2=0.9993.And the detection limit and quantification limit of ethanamizuril in the cecal contents were 0.04 mg/kg and 0.1 mg/kg,respectively.The method can be used to determine the concentration of ethanamizuril in chicken plasma and cecum.2.Two hundred and ten healthy chickens were selected and the infected models were established,and they were divided into 3 groups.Ethanamizuril was administered by gavage at the dosage of 2 mg/kg.bw,4 mg/kg.bw and 8 mg/kg.bw,respectively.Cardiac blood was collected at0,0.5,1,2,3,4,6,8,12,24,36,48,60,72 hours before and after administration,and the cecum was collected by dissection.Five chickens were taken at each time point.Plasma samples and cecal contents were subjected to sample pre-processing and then detected using UPLC method to obtain the concentration of ethanamizuril in chickenplasma and cecal contents at different time points.The concentration data were analyzed with Win Nonlin 5.2 software with non-compartment model.The experimental results showed that when the chickens were administered at the dosage of 2 mg/kg.bw,4 mg/kg and 8 mg/kg,the area under the curve（AUC）of ethanamizuril in plasma was 68.59 mg/Lh and 186.06 mg/Lh and 377.25 mg/Lh,respectively.The half-lives(t_1/2z)of ethanamizuril were 9.18h,20.05h,and 18.05h,respectively.The peak time(T_max)of the drug was3h,3h,and 8h respectively,and the corresponding maximum plasma concentration(C_max)was3.97mg/L,6.52 mg/L,and 10.13 mg/L,respectively.At the three dosages,the AUC of ethanamizuril in cecal contents was 36.93 mg/kg h,96.90 mg/kg h,and 262.76 mg/kg h,respectively.The t_1/2zof the drug was 10.82 h,10.53h,10.60h respectively.The T_max of ethanamizuril was 4h,5.2h,and 22.8h,respectively and the corresponding C_max was 2.43 mg/kg,4.28 mg/kg,and 8.57 mg/kg,respectively.3.Different dosage regimens were used to evaluate the therapeutic effect of ethanamizuril on Eimeria tenella challenged chickens.First,chicken challened models were established.Each healthy chicken was infected with 80,000 Eimeria tenella sporulated oocysts.The first dosing regimen was that the challenged chickens were administered ethanamizuril at a single dosage by gavage on the 3rd day after infection with the dosages of were 0.5,1,2,4,8,10,12,16mg/kg.bw,respectively.The second dosing regimen was that the chickens were administered by gavage on the 3rd and 4th day after infection with the dosages of 0.5,1,2,4,and 8 mg/kg.bw,respectively.The results showed that the weight of chickens in the challenged control group was significantly lower than that of the healthy group and the challenged administration group.When the single doses were 1mg/kg.bw and 2mg/kg.bw,the oocyst value and lesion value had no significant difference with the challenged control group,and the anticoccidial index（ACI）was126 and 143,respectively,indicating ineffective or inefficient.The oocysts value and lesions value of 4mg/kg.bw grouphad significant differences with the challenged group with the ACI of175,which showing a moderate anti-coccidial activity.When the twice-administered dose was1mg/kg.bw,the oocyst value and lesion value were not significantly different from the challenged control group,and the anti-coccidial index（ACI）was 128.when the two-administered dose was of 2mg/kg.bw,the oocyst value and lesion value were significantly different from the challenged control group with the ACI of 162,showing moderate anticoccidial activity.When the two doses were 4mg/kg.bw and 8mg/kg.bw showed highly effective anticoccidial activity,and the ACI were184 and 191,respectively.4.In this experiment,bovine kidney cells（MDBK）were selected to culture the sporozoites of Eimeria tenella and then different concentrations of ethanamizuril were added to evaluate the inhibitory effect of ethanamizuril on sporozoites in vitro.The experimental results showed that there was no significant difference in the invasion rate of sporozoites with different concentrations of ethanamizuril and the infection control group.The invasion rate of the infection control group and the experimental group were in the range of 10%to 17%.This study showed that ethanamizuril had no obvious inhibitory effect on the sporozoite stage of Eimeria tenella,which is consistent with the results of previous studies that ethanamizuril mainly acts on the second merozoites of Eimeria tenella.