Studies On The Quality Control Methods And Pharmacokinetic Of Tulobuterol Patches

Tulobuterol patch is the only transdermal delivery of drugs through the treatment of asthma so far.Tulobuterol patch was recommend as the first choice in asthma patients with significantly treatment efficacy of drug.The Significance of the study was to establish the Quality control methods of Tulobuterol patch,pharmacokinetic research and bioequivalence study of tulobuterol patch,providing a reference for subsequent research and clinical application.1.Quality control methods of Tulobuterol patchThe chemical,UV spectroscopy,thin layer chromatography and high performance liquid chromatography methods were used to identify it.Detect methods were established of the patch release,content uniformity,related substances and residual solvents examination,respectively.The methods were used to examine 3 batches of 3 specifications Tulobuterol test patch corresponding examination,the results show that:The release of tulobuterol patch at 2,8 and 24 hours were in 25%to 55%,60%to 90%and more than 80%which met the requirements;the results of content uniformity examination satisfied the standard of Japanese Pharmacopoeia as AV values were less than 15;in related substances examination,the largest single impurity contents were no more than 0.23%and the total impurity contents were no more than 0.73%;the content of Ethyl acetate was less than 0.5%and the content of residual solvent passed the inspection.A high performance liquid chromatographic(HPLC)method was developed and validated to determine tulobuterol in patch and the result was 98.2%to 100.5%of 3 batches of 3 specifications tulobuterol patch.2.Tulobuterol pharmacokinetic studies in rats patchA sensitive and rapid ultra-fast liquid chromatography/tandem mass spectrometry(LC-MS/MS)method has been developed and validated for determination of tulobuterol in rat plasma.After addition of the internal standard,clenbuterol,plasma samples were extracted by liquid-liquid extraction method with methyl tert-butyl ether.The linear range was 0.5-100.0 ng/mL for tulobuterol with the lower limit of quantitation of 0.5 ng/mL.The analyte and the internal standard extraction recoveries were more than 70.0%,specificity,matrix effects methods,precision,accuracy and stability were in line with the requirements of the biological sample analysis.The validated method has been successfully applied to a linear pharmacokinetic study of tulobuterol patch in rat plasma.The validated method was applied to a pharmacokinetic study of tulobuterol after transdermal administration to rats.Calibration levels were sufficient to quantitate plasma samples obtained in pharmacokinetic study.The relevant pharmacokinetic parameters of dose 2 mg,1 mg and 0.5 mg:Cmax were 71.02 ± 13.84,38.78 ± 3.00,20.50 ± 2.59 ng/mL;AUC(0-?)were 638.0 ± 69.9,335.9 ± 28.9,168.4 ± 18.7 ng·h/mL;t1/2 were 3.67 ± 1.24,2.96 ± 0.60,3.44 ± 0.46 h.The results showed that the AUC(0-48),AUC(0-?)and Cmax of tulobuterol patches made by pharmaceutical laboratories were proportional to doses.The pharmacokinetic properties of tulobuterol exhibited good linear trend.3.Bioequivalence study Tulobuterol patchThe design of the study was an open label,balanced,randomized two-treatment,two-period,two-sequence,crossover,single-dose bioequivalence study under normal conditions in 6 healthy adult Bama miniature pigs,rabbits and rats.Therefore,our study focused on a preclinical bioequivalence study of tulobuterol patch in pig plasma.Meanwhile,we also use the same method to validate for quantification of tulobuterol in plasma of rabbit and rat,and the method has been successfully applied to the preclinical bioequivalence study of tulobuterol patch in rabbit and rat,respectively.The pharmacokinetic parameters and bioequivalence experimental results were then compared among three kinds of animals.The validated method was applied to a pharmacokinetic study of tulobuterol after transdermal administration to rats.Calibration levels were sufficient to quantitate plasma samples obtained in pharmacokinetic study.Pharmacokinetic parameters of the test patch and the reference patch were as follows:test patch of pigs t1/2:5.61h,Cmax:3.73 ng/mL,Tmax:6.33 h,AUC(0-?):48.05 ng·h/mL;reference patch of pigs t1/2:5.64 h,Cmax:3.73 ng/mL,Tmax:7.50 h,AUC(0-?):53.57 ng-h/mL;test patch of rabbits t1/2:7.56 h,Cmax:4.30 ng/mL,Tmax:3.40 h,AUC(0-?)1.84 ng·h/mL;reference patch of rabbits t1/2:6.14 h,Cmax:4.20 ng/mL,Tmax:2.00 h,AUC(0-?):32.63 ng·h/mL;test patch of rats t1/2:4.08 h,Cmax:38.09 ng/mL,Tmax:2.83 h,AUC(0-?):317.5 ng-h/mL;reference patch of rats t1/2:4.09 h,Cmax:40.38 ng/mL,Tmax:2.75 h,AUC(0-?):351.2 ng·h/mL.By calculating AUC(0-48),the relative bioavailability of tulobuterol was 93.7±11.5%of pigs,97.7± 20.6%of rabbits,and 90.4± 6.9%of rats compared to reference patch.According to the results of statistics analysis,there were no significant differences in main pharmacokinetic parameters between test tulobuterol patch and reference patch and they were biological equivalent in Bama miniature pigs,rabbits and rats.