| Our research was mainly to make an evaluation of pre-clinical pharmacokinetics forGP205, calculate the pre-clinical pharmacokinetics, investigate the relationship betweendosage and parameters of pharmacokinetics, calculate the absolute bioavailability afteroral administration, and study the accumulation effect after oral administration ofmultiple dosage.1. Method validation for GP205in biological samplesIn this work, we developed and validated a sensitive, specific LC-MS/MS methodfor the quantitative determination of the HCV protease GP205and studied thepharmacokinetics of GP205in biological samples. Paclitaxel was selected to be theinternal standard, the analyte and internal standard were isolated from plasma samples bya simple liquid-liduid extraction(LLE) using methyl tertiary-butyl ether. Reverse-phaseHPLC separation was accomplished on a Zorbax SB-C18column(100mm×3.0mm,3.5μm) with a mobile phase composed of methanol-water-formic acid(95:5:0.1,v/v/v) ata flow rate of0.3mL/min. A Varian1200L electrospray tandem mass spectrometerequipped with an electrospray ionization sourse was operated in selected reaction monitor（SRM）with the precursor-to-product ion transitions m/z887.0'787.0(seraprevir) andm/z876.0'307.0(IS) used for quantitation. The collision induced dissociation energyfor two channels was35V and21.5V, respectively.In our study, The linear range for beagle dog plasma samples was1.996~4990.00ng/ml, and1.990~4975.00ng/ml for SD rat plasma samples. The LLOQwas2ng/ml; the RSDs of inter-and intra-precision were all less than15%; the extractionrecovery was more than80%. The result of matrix effect was90%~100%, that is, theendogenous substances don’t interfer the determination of targeted compounds. Thestandard solution of GP205and Paclitaxel kept stable within40days in the condition of-20℃as well as the plasma sample.2.The pharmacokinetics study of GP205after single-dose administration in BeagledogsIn the experiment,24healthy adult Beagle dogs with half male and female weredivided into4groups. They were orall administrated of low-dose0.75mg/kg, medium-dose1.5mg/kg, high-dose3mg/kg and intravenous administration0.5mg/kg, respectively.They were blooded from the vein before administration and at different time points after administration. The drug concentration of Beagle dog plasma was measured by plasmasample analysis methods established in this study.The plasma concentration-time curvewere based on the measured density data, and the pharmacokinetic parameters and theabsolute bioavailability were calculated with the non-compartmental model.The test results showed that pharmacokinetic parameters in Beagle dogs such asCmax±S.D.were35.19±10.6ng/ml,35.19±10.6ng/ml and329.06±52.61ng/ml; tmax±S.D.were3.67±0.52h,3.17±0.75h and3.17±0.75h; t1/2±S.D. were6.48±0.48h,7.22±1.48h and7.56±1.77h; MRT±S.D.were11.45±0.86h,9.84±1.51h and9.48±1.33h;AUC0±S.D.were380.37±131.22ng h/ml,1061.5±331.06ng h/ml and1879.67±381.71ng h/ml; AUC0~∞±S.D. were422.09±144.13ng h/ml,1172.98±359.42ng h/ml and2060.24±393.14ng h/ml after single oral dose of GP205at three grades；the C0、t1/2、MRT、Cl、Vd、AUC0、AUC0~∞were2689.81±487.54ng/ml,12.09±3.13h,8.36±2.82h,0.4±0.05L/h/kg,3.25±0.87L/kg,1120.33±121.04ng h/ml and1275.22±156.2ng h/mlrespectively after intravenous administration. From the data above we can conclude thatit is basic linear dynamics characteristics of the absorption of the drug in Beagle dogs.The absolute bioavailability of GP205is31.53%in Beagle dog.3.The pharmacokinetics study of GP205after multi-dose administration in BeagledogsIn multi-dose test,6healthy adult Beagle dogs were administered middle dose(1.5mg/kg) of GP205orally for seven days continuously. They were took blood beforethe administration in the5th,6th,7thdays. In the7thday, they were took blood inaccordance with the time point to investigate the steady-state, the fluctuation coefficientDF, the steady-state pharmacokinetic characteristics and the accumulation effect aftermultiple dose administration.The results showed that in the fifth day, the plasma concentration of GP205inBeagle dogs reached steady state essentially and the plasma concentration no longerincreased with the frequency of administration.Taking GP205continuously will notproduce a sifnificant accumulation effect.4. The pharmacokinetics study of GP205in SD rats24SD rats with half male and female were divided into4groups. They were orallyadministrated with low-dose2.5mg/kg, medium-dose5mg/kg, high-dose10mg/kg andintravenous administrated1mg/kg, respectively. The drug concentration of SD ratsplasma was measured by plasma sample analysis methods established in this study.The plasma concentration-time curve were based on the measured density data, and thepharmacokinetic parameters and the absolute bioavailability were calculated with thenon-compartmental model.The pharmacokinetic parameters were as following. Cmax±S.D. were4260.34±400.35ng/ml,6782.15±749.52ng/ml and9406.12±582.99ng/ml; tmax±S.D. were6.30.8h,6.31.5h and6.30.8h; t1/2±S.D. were12.38±1.51h,13.91±1.48h and17.13±2.88h; MRT±S.D. were19.35±3.22h,22.17±2.18h and25.56±3.47h; AUC0~∞±S.D.were70136.79±16623.73ng h/ml,135312.81±13439.93ng h/ml and206888.49±19318.30ng h/ml after single oral dose of GP205at three grades; the C0、t1/2、MRT、Cl、Vd、AUC0~∞were9511.56±442.31ng/ml,16.26±2.99h,20.76±3.41h,0.015±0.006L/h/kg,0.31±0.12L/kg and75210.88±23751.74ng h/ml after intravenousadministration. From the data above, we can conclude that it is basic linear dynamicscharacteristics of the absorption of the drug in Beagle dogs. The absolute bioavailabilityof GP205is36.68%in SD rats.The study suggests that innovative drugs GP205present the basic linear dynamicsabsorption after oral administration in animals; accumulation effect in the body will notproduce after long-term administration of the drug; the absolute bioavailability is greaterthan30%in experimental animals after oral administration of the drug. |
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