Toxicokinetic Study Of PBA In SD Rats

Object:1.To observe the plasma concentration change of PBA in rats treated with different doses of PBA orally and intravenously,and study the absorption character of PBA compound via oral and intravenous exposure in rats.Explore and complete the toxicokinetic study of PBA.2.To study the distribution profiles of PBA in rat tissues like heart,liver,spleen,kidney,gastrointestinal tract,thyroid,thyroid,genital,fat and skin after orally giving PBA to SD rats.3.To study the excretionprofiles of PBAin urine,feces and bile after oral PBA exposure in rats.Method:1.Establishedan LC-MS method for the determination of PBA in SD rats plasma.SD rats were divided into 4 groups as low dose single ig(10mg/kg)group,medium dose single ig(30 mg/kg)group,and high dose signle ig(100mg/kg)group,and single iv(5mg/kg)group.Each group contains eight rats(number:males= females).All the rats were banned with food but free access to water before experiment.After oral drug exposure,0.3 mL blood was collected from orbital vein at time intervals of 0min(pre-dose),10min,30min,1h,1.5h,2h,3h,4h,6h,8h,12h and 24h post-dose.Blood samples are all treated with heparin,while in intravenous exposure group time intervals were 0 min(pre-dose),5 min,15 min,30 min,45 min,1h,2 h,4 h,6 h,8 h,12 h and 24 h post-dose.Samples added with internal standard solution(400 ng/ml 4-Methoxybenzoic Acid)were vortexed for 1 min.Then add 0.3 mL acetonitrile,vortexing for 2 min and centrifuged at 13,000rpm for 10 min,and the organic phase was separatedinto another tube.DAS software was used to obtain kinetic parameters such as AUC,Cmax,Tmax,MRT,T1/2and so on.2.Observe the PBA distribution in heart,liver,spleen,kidney,gastrointestinal tract,thyroid,thyroid,genital,fat,skin after ig administrated 100 mg/kg PBA to SD rats.Time intervals to collect samples were 0.5 h,2 h,8 h and 168 h post-dose.3.LC-MS method was set up to determine the PBA concentration in rats' urine,feces and bile after oral drug expose at the dosage of 100 mg/kg PBA for excretion study.Results:1.the plasma concentration change of PBA in SD rats was treated with different doses of PBA orally and intravenously:(1)Endogenous substrate in the plasma didn't interfere the PBA analysis.The extraction recovery rate was at the range of 88.8%-94.4%,the intra-day precision was at the range of 6.4%-8.9%,and the inter-day precision was at the range of 3.85%-5.87%.Accuracy was at the range of 97.8%-105.5%.Linear calibration curves for PBA was observed at 20-2500 ng/ml,with a correlation coefficient r>0.9914.The lowest limit of quantification(LLOQ)for PBA was 20 ng/ml(RSD=9.67%).(1)In female rats,the AUC(0-?),Cmax,Tmax,MRT(0-?),and t1/2values in low dose single ig groupwere 3602.2±672?g/L*h,532±77?g/L,2.625±0.75h,10.19±2.36 h and 7.69±3.44 h,respectively,with an absolute bioavailability of 10.2%.TheAUC(0-?),Cmax,Tmax,MRT(0-?),and t1/2values in middle dose single ig group were 7533.3±3131.7 ?g/L*h,776±179 ?g/L,3±0.816 h,10.54±3.71 h,and 7.039±2.951 h,respectively,with an absolute bioavailability of 7.1%.TheAUC(0-?),Cmax,Tmax,MRT(0-?),and t1/2values in high dose single ig group were 10020.1 ±3225.7 pg/L*h,1120±79 ?g/L,3±0.816 h,and 10.85±3.87 h,6.918±3.32,respectively,with an absolute bioavailability of 2.9%.Moreover,the AUC(0-?),Cmax,Tmax,MRT(0-?)and t1/2values in 5mg/kg single iv group were 17575.2±5151.3 ?g/L*h,3465±1073 ?g/L,0.083±0.00 h,23.81±16.74 h,and 18.29 ± 12.82 h,respectively.(2)In male rats,the AUC(0-?.),Cmax,Tmax,MRT(0?O),and t1/2valuesin low dose single ig group were 2062.4±438.6 ?g/L*h,339±131 ?g/L,2.6±2.3 h,8.00±2.53 h,and 3.97±2.21 h,respectively,with an absolute bioavailability at 8.9%.theAUC(0-?),Cmax,Tmax,MRT(0-?),and t11/2valuesin middle dose single ig group were 6421.5±1654.6 ?g/L*h,775±444 ?g/L,2.625±1.109 h,16.02±8.48 h,and 11.86±8,38 h,respectively,with an absolute bioavailability at 9.2%.The AUC(0-?),Cmax,Tmax,MRT(0-?),and t1/2valuesin high dose single ig group were 9119.9±3432.2 ?g/L*h,1426±866 ?g/L,3±0.816 h,12.43±3.66 h,and 9.22±4.48 h,respectively,with an absolute bioavailability at 3.9%.Moreover,the AUC(0-?)?Cmax,Tmax,MRT(0-?),t1/2values in the 5mg/kg single iv group were 11606.5±3650.5 ?g/L*h,3830±466 ?g/L,0.083±0.00 h,17.37±20.55 h,and 13.92±18.14 h,respectively.2.Establish an LC-MS method for the PBA analysis in rat liver tissue,and the endogenous substrate did not interferethe PBA analysis.The accuracy of PBA in tissue homogenate was ranged from 100.9%to 104.7%,and the intra-day precision was at the range of 8.8%-10.3%,and the inter-day precision was at the range of 5.9%-8.4%.Linear calibration curves for PBA was observed at 20-1600 ng/ml,withacorrelation coefficient r>0.9932.The lowest limit of quantification(LLOQ)for PBA was 20 ng/ml(RSD=1.9%).Therefore,the assay method was accurate and precise for replicate analysis of PBA.Results showed PBA was widely distributed in rat tissue.In four sampling points,PBA was determined in tissues like liver,gastrointestinal tract,kidney,heart,skin and spleen while in thyroid,genital PBA could only be determined 8h after drug exposure.Especially in skin,PBA could be determined at all the sampling points except 0.5h.It is remarkable that 168h post-dose there is still high concentration of PBA in most tissues except thyroid and genital,which indicates PBA has a long residual time.3.Endogenous substrate did not interfere in determination of PBA in the urine,feces and bile.The intra-day precision for PBA analysis in urine was ranged from 6.0%-10.5%while inter-day precision was from 7.3%to 12.0%.The linear calibration curves for PBA was observed at 20-2000 ng/ml.The lowest limit of quantification(LLOQ)for PBA was 20 ng/ml(RSD=9.0%)and the matrix effect was at the range of 92.4%to 111.5%.Researches on stability suggested that urine samples could be stored at-200? for 30 days(freeze-thaw for three cycles)while stored only for 24h at room temperature.Preprocessed samples could be stored for 24h at 4?.After high dose single ig in rats,the cumulate excretion rate in urine,feces and bile acid was very low.The total average excretion rate in urine and feces sample after 168h drug exposure was 0.214±0.128%o?0.295±0.062‰,and the total average excretion rate of PBA in bile 48h post-dose was 0.0607± 0.0212‰.Conclusion:1.Results indicate that there is certain amount of absorption in rats after PBA exposure but its absolute bioavailability in male and female rats is less than 10.2%and 9.2%,which also seemingly declines as the exposure dosages grow.The concentration of PBA in rats has nonlinear dynamics and its systemic exposure doesn't raise as the administered dosage does.2.The distribution results show that PBA has a high distribution in liver,gastrointestinal tract,kidney and hearts which has a long residual time as well.3.The study on excretion suggests that after orally administrated with PBA in rats,the cumulative excretion in urine,feces and bile appear in a quite low stage,ordered as follow:feces>urine>bile.