Clinical drug-drug interactions（DDIs） induced by CYP3A may reduce the exposure and pharmacological activity of CYP3A substrate.Up-regulation of CYP3A mRNA is often used to evaluate inductive effect of test compounds on CYP3A. A quantitative real time PCR assay was developed and validated for the absolute quantification of CYP3A1 and CYP3A2 mRNA.Specific primers of CYP3A1,CYP3A2 and GAPDH（glyceraldehyde-3-phosphate dehydrogenase,as a house-keeping gene） were well designed.The relationship between threshold cycle（Ct） and logarithm of the concentrations of CYP3A1, CYP3A2 and GAPDH was linear ranged from 1 attomol/μL to 1×10~6 attomol/uL with great inter- and intra-assay reproducibility. This method was successfully applied to investigate the time courses of CYP3A1 and CYP3A2 mRNA induction in rat liver after 100 mg/kg dexamethasone（DEX） administration by intraperitoneal（i.p.） injection.The baseline levels of CYP3A1 and CYP3A2 mRNAs were 37.78 attomol/ug（total RNA） and 252.31 attomol/ug（total RNA）,respectively.CYP3A1 and CYP3A2 mRNA values increased gradually to their peak levels（19- and 8- fold vs.baseline） within 24 h and 42 h,respectively,and then returned to their baseline 60 h after DEX administration.
An efficient and sensitive ion-pair HPLC-UV method using atenolol as internal standard （IS） was developed and validated for the determination of metformin in the plasma of diabetic rats. Plasma samples were deproteinated with 10% （v/v） perchloric acid. Separation was achieved on a UltimateTM AQ-C18 column （250 mm×4.6 mm, 5 μm） with a mobile phase （pH 5.05） composed of acetonitrile-water （31：69, v/v, containing 0.002 M sodium dodecyl sulfate, 0.0125 M potassium dihydrogen phosphate, 0.015 M triethylamine） at a flow rate of 1.0 mL/min. The calibration curve was linear （r〉0.994） between 7.5 and 4000 ng/mL. The lower limit of quantification （LLOQ） was 7.5 ng/mL. The precision was validated and the relative standard deviation was in the range of 1.87% to 15.70%; the accuracy was between 93.98%-106.89%. The mean recoveries were 95.40% and 95.31% for metformin and IS, respectively. The relative error （RE） of stability at different storage conditions was within ±9.00%. This method was used to determine the concentration-time profile of metformin in diabetic rat plasma following an oral administration of metformin at the dose of 10 mg/kg. Our results indicated that ion-pair HPLC-UV method using UltimateTM AQ-C18 column was effective for the pharmacokinetic studies of high polarity compounds like metformin.
Sulfotransferases（SULTs） are one of the most important phase II drug-metabolizing enzymes. Among them, aryl sulfotransferases（SULT1A1） and estrogen sulfotransferase（EST, SULT1E1） belong to the rSULT1 family that catalyzes sulfation of phenolic compounds. Dopamine（DA） acts as a vital neurotransmitter in central nervous system（CNS） and regulates a variety of activities in periphery. In our study, we aim to detect the effects of exogenous DA and levodopa（L-DOPA） on rSULTs（rSULT1A1 and rSULT1E1） in rat liver. In order to achieve this target, varying doses of DA（0, 2, 10 and 100 mg/kg/d） and L-DOPA（0, 5, 25 and 125 mg/kg/d） were provided to male and female rats, respectively. Real-time PCR assay and western blot were used in the determination of the mRNA and protein expression of rSULTs. PNPS assay and radioactivity assay were applied to the detection of enzyme activity of rSULT1A1 and rSULT1E1, respectively. Our results showed that DA induced the expression and activity of rSULT1A1 in the liver of male and female rats and DA had little effect on rSULT1E1. However, L-DOPA caused no evident change of rSULT1A1 in both sex and had no significant effect on rSULT1E1 in female rat liver, but increased rSULT1E1 expression and activity only in male rat liver when administered at high dose. Our results suggest that DA plays different roles in the regulation of rSULT1A1 and rSULT1E1 when it is in periphery or in the CNS.
