| Proliferative enteropathy caused by Lawsonia intracellularis?LI?can spread all over the world,which seriously affects the development of animal industry.However,because LI is an obligate intracellular parasitic bacteria,it is difficult for conventional antibacterial drugs to enter cells,which is an important problem for the treatment of LI infection.Tilmicosin?TMS?is widely used in veterinary clinic because of its low inhibitory concentration,wide distribution volume and rapid accumulation in deep tissues.However,its poor intracellular accumulation ability and bitter taste limit its clinical use.In the early stage of our laboratory,tilmicosin oral suspension was prepared with carnauba wax as solid lipid matrix.The prepared suspension can effectively mask the bitter taste of TMS,improve its ability to penetrate the body barrier,and improve the ability of drug sustained release,thus enhancing the antibacterial effect against LI.In this study,the safety range of tilmicosin oral suspension in clinical application was judged by the safety test and the drug residue elimination rule in pigs.And an effective drug withdrawal period was established to provide clinical trial data for new veterinary drug declaration.1 Clinical safety of tilmicosin oral suspension in pigs32 crossbred piglets weighing about 40 kg were divided into 4 groups:1-time recommended dose group?15 mg/kg b.w.?,3-time highest recommended dose group?45mg/kg b.w.?,5-time highest recommended dose group?75 mg/kg b.w.?and blank group,with 8 piglets in each group,which were continuously administered by gavage for 9 days.During the experiment,the feed intake and drinking water of the tested pigs were normal,and there was no obvious abnormality.There was no significant difference in growth rate and feed conversion rate between experimental group and blank group.Although there were significant changes in glucose,urea,lymphocytes and eosinophils in pigs before and after administration,all the detected values fluctuated within the normal range,which indicated that oral administration of tilmicosin suspension would not cause significant changes in hematological parameters and serum biochemical indicators of pigs.There was no significant difference in organ coefficient between each experimental group and the blank control group.The tissues and cells of the experimental group were intact in morphology and normal in structure,without obvious pathological changes.The results showed that when the oral suspension of tilmicosin was applied to pigs at 5 times of the highest recommended dose?75 mg/kg b.w.?,it did not cause obvious toxic and side effects in pigs,and it was safe for clinical application.2 Establishment of high performance liquid chromatography?HPLC?for determination of tilmicosin0.1 mol/L ammonium formate solution-acetonitrile-methanol was used as the mobile phase,and the ratio was 61:29:10;Setting the detection wavelength as 290 nm;The amount of injection was 100?L.The linear regression equation of tilmicosin concentration and peak area was y=190953x-5177.7,and the correlation coefficient was R2=0.9999.The tissue samples were extracted twice with acetonitrile and potassium dihydrogen phosphate,and purified by Bond Elut C18?500 mg/6 m L?solid phase extraction column.Using 0.1 mol/L ammonium acetate methanol-acetonitrile solution?20:80?as eluent,the detection limit and quantitative limit of tilmicosin in pig liver and kidney tissues were 50?g/kg and 10?g/kg,respectively.The limits of detection and quantification in muscle and adipose tissue were 20?g/kg and 50?g/kg,respectively.The average recovery rate in each tissue is more than 78%,and the coefficient of variation is less than 10%.3 Residues Elimination and formulation of the withdrawal period of Tilmicosin Oral Suspension in PigsTilmicosin oral suspension?15 mg/kg b.w.?was given to pigs for 3 days.At the same time of 0.5 d,3 d,7 d,14 d and 21 d,4 pigs were randomly killed,liver,kidney,muscle and fat tissues were homogenized and extracted,and then processed by HPLC.Tilmicosin has the highest initial concentration?1.80±0.12?g/g?,short half-life?2.5 d?and fast elimination rate in liver tissue.In muscle and fat,the concentration was 0.38±0.01?g/g and 0.23±0.03?g/g,respectively,and the half-life was long,which was 4.3 d and 5.2 d,respectively,and the elimination curve decreased slowly.After stopping the drug for 3days,the concentration of tilmicosin in the liver of the tested pigs was lower than the maximum residue limit prescribed by the state The concentration of tilmicosin in other tissues of tested pigs was lower than the national maximum residue limit at 7 days after drug withdrawal.After stopping the drug for 14 days,tilmicosin could not be detected in muscle and adipose tissue.Tilmicosin could not be detected in liver and kidney tissue 21days after drug withdrawal.WT 1.4 fitting results showed that the drug withdrawal period of tilmicosin was 6.72d in pig liver,7.05 d in kidney,10.69 d in muscle and 11.21 d in fat.According to the 95%confidence interval of one side and two sides,it is determined that the recommended withdrawal period in pig tissues of tilmicosin oral suspension was 12 d when pigs be administered continuously for 3 d with recommended dosage?15 mg/kg b.w.?. |
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