Safety Of Levamisole Via Transdermal And Oral Administration

Autoimmune disease is a kind of disease which caused by the immune response of the immune system to autoantigens.For the long-term use of therapeutic drugs such as immunosuppressants was likely to cause side effects,there is no effective drug therapy for the diseases.The clinical research shows that levamisole,which has strong immune regulation,was a potential new therapeutic drug for autoimmune diseases.Considering the serious adverse effects caused by the oral administration of levamisole,the safety of levamisole under the transdermal administration was studied,mainly from the pharmacokinetics of rats,tissue distribution characteristics and subacute toxicity to mice.ChapterⅠ:Pharmacokinetic study of levamisoleIn order to investigate the pharmacokinetics of levamisole close to the oral toxic dose in rats,a LC-MS/MS method with high specificity,simplicity and rapidity for the determination of levamisole in rat plasma was successfully developed.The plasma samples were pretreated by protein precipitation with methanol（plasma:methanol=1:9）.The matrix effect,recovery and stability meet the needs of biological sample analysis.The established method was used to investigate the pharmacokinetics of levamisole in rats after intravenous,transdermal and oral administration at doses of 10,60 and 60mg/kg,respectively.After transdermal administration,the plasma concentration of levamisole peaked at 0.74±0.14 h,the mean retention time was 25.03±17.01 h,the half-life was 5.69±0.69 h,the apparent volume of distribution was 88.70±14.07 L/kg,and the absolute bioavailability was 13.98%.After oral administration,levamisole peaked at 1.58±0.66 h,the mean retention time was 6.21±0.66 h,the half-life was4.89±0.61 h,the apparent volume of distribution was 59.28±9.65 L/kg,and the absolute bioavailability was 68.68%.Compare with oral administration,the time to peak of levamisole was shortened,the mean retention time and half-life were extended,the apparent volume of distribution was increased,and the maximum plasma concentration and the area under the curve were decreased after transdermal administration.Chapter Ⅱ:Tissue distribution study of levamisoleThe established LC-MS/MS method was also applied to quantify the concentration of levamisole in tissues.The compounds were detected in all tissues after the mice were given 130 mg/kg levamisole by transdermally and orally.The tissue distribution results suggest that levamisole reaches a high tissue concentration at 0.5-2 h and the concentration were in the following order:liver>brain>spleen>kidney>heart>lung.The pharmacokinetic parameters of C_max,AUC_0-t,CL_z MRT_0-t,and the ratio of tissue to plasma concentration of AUC_0-t（K_p）were compared.The results showed that K_p in tissues were more than 1.0,showing a good tissue affinity of levamisole.Compared with oral administration,the C_max,AUC_0-t,MRT_0-t and K_p of levamisole in brain,liver and kidney,and the CL_z in liver decreased significantly after transdermal administration.What’s more,the CL_z in kidney and brain increased.After transdermal administration,the distribution of levamisole to the various tissues was changed.Chapter Ⅲ:Subacute toxicity study of levamisoleThe study was conducted to evaluate the safety of levamisole by measuring the body weight,organ coefficient,biochemical indexes and pathological examinations of major organs of mice.The results showed that the organ coefficient and biochemical indexes of the mice in the middle-and high-dose oral levamisole groups were statistically different from those in the control group,and there were obvious pathological changes in the tissue sections.Meanwhile,there was no significant difference between the transdermal and control groups.It was indicated that the toxicity of levamisole by transdermal administration was less than that to the oral administration,and this compound may cause some damages to the liver and kidney of mice after oral administration.Conclusion:A rapid and reliable LC-MS/MS method was developed and validated for the determination of levamisole in plasma and tissues.The pharmacokinetics and tissue distribution characteristics of levamisole were reported,and the preliminary safety of the transdermal and oral administration routes were evaluated.In the study,it was proved that the transdermal route of levamisole was a safe way to drug delivery,which can reduce its toxicity and make it more suitable for clinical use,and also provide a basis for further development of transdermal preparations of levamisole.