Pharmacokinetics Of Tiopronin Preparation

1. The establishment of analytical method of tiopronin biological matrixTo develop and validate two HPLC-ultraviolet method for determination of tiopronin concentration in rat plasma and mice tissues.A Diamonsil C18(200×4.6 mm,5μm)column with a mobile phase composed of methanol-water-phosphoric acid (60:40:0.1,v/v/v/v)was used in separation.After derivatization by p-BPB,the biological samples were pretreated by Liquid-liquid extraction with acetoacetate.The concentration of tiopronin was calculated by calibration curve accompanied. The calibration curve of tiopronin in rat plasma have a good linear between 0.5-32.0μg·mL-1. The intra-day precision were 3.23%,6.00% and 4.92%, respectively. The inter-day precision were 9.04%,4.60% and 10.7%, and the accuracy were 103.3%,101.7% and 100.1%, respectively. The recovery of extraction was more than 70%. The calibration curve of tiopronin in mice tissues was linear between 5.0-500.0μg·g-1. The intra-day precision of QC samples were 4.98%,7.49% and 8.86%, respectively. The inter-day precision were 9.79%, 4.48% and 8.61%, and the accurate were 96.8%,101.6% and 99.5%, respectively. The recovery of extraction was more than 70%.2. The pharmacokinetics and tissue distribution study of tiopronin in animalTo study the pharmacokinetics of the injection of tiopronin only or combined with salviamiltiorrhiza in rat plasma,and the distribution of the injection of tiopronin only or combined with salviamiltiorrhiza in mice tissues.The results of pharmacokinetics of tiopronin only showed that the ratio between Cmax,AUC0-t and dose had a linear correlation in rat plasma,which showed insignificant difference after analysis of variance (P>0.05).There was significant difference in the pharmacokinetics parameters between the single middle and combination administration group, in which AUC0-t went down by 45.0%(P<0.05), AUC0-∞went down by 53.2%(P<0.05) and Cl went up by 53.4%.Tiopronin distributed rapidly in tissues after administrated i.v to mice. The highest concentration of tiopronin was in lung; the next was in liver which was the target tissue of tiopronin;the least were in heart and spleen.The result also showed that tiopronin was easier to absorb in intestine than in stomach.3. The pharmacokinetics and bioavailability of tiopronin in humanTo establish a LC-MS/MS method for determination of tiopronin in human plasma and to study pharmacokinetics and relative bioavailability of tiopronin enteric capsule in healthy male volunteers.Chromatographic separation was performed at 25℃on a Zorbax Eclipse C18-column (150×4.6 mm,5μm, Agilent Technologies). The mobile phase consisted of a mixture of acetonitrile-water containing 1% formic acid (95:5,v/v) at a flow rate of 0.5 mL-min-1. Liquid-liquid extraction was used to pretreat plasma sample. The calibration curve in plasma was linear between 25.0-5000.0 ng-mL-1. Accuracy and precision of method were as follows: The intra-day precision of QC samples were 7.66%,6.83% and 7.84%, respectively;the inter-day precision were 2.51%,14.1% and 11.8%, respectively; the accurate were 98.9%,96.3% and 100.1%, respectively. The recovery of extraction of tiopronin and internal standard were more than 80%.A single oral dose of 200 mg tiopronin test and reference preparations were administrated to 20 healthy volunteers in a randomized cross-over design to study pharmacokinetics and relative bioavailability of tiopronin enteric capsule in healthy male volunteers. Main pharmacokinetic parameters for single dose were as follows:Tmax were (4.20±1.01) and (3.85±0.99) h; Cmax were (4456±2447) and (4765±2015) ng-mL-1;t1/2 were (5.96±4.37) and (5.61±4.42) h; AUC0-t were (20903±8085) and (20566±9902) ng·h·mL"1; AUCo-∞were (22088±8914)and(21696±11054) ng-h·mL"1.The results showed the relative bioavailability of the test was (96.6±15.0)%. The present study indicated that new T was bioequivalent to R.