ABSTRACT
OBJECTIVE To establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determinations of concentrations of levonorgestrel (LNG) and ethinylestradiol (EE) in New Zealand rabbit plasma, and to study their pharmacokinetics in New Zealand rabbits after multiple dosing. METHODS Six female New Zealand rabbits were given LNG/EE patches ten times (5 cm × 4 cm, each patch contained LNG 5.35 mg and EE 0.11 mg), 1 patch every 3 d, for 30 consecutie days. Blood samples were collected at different time points before and after drug administration. The plasma samples were derived with dansyl chloride and then analyzed by HPLC-MS/MS method. The main pharmacokinetic parameters were calculated using DAS3.0 software. RESULTS The linear concentra-tion range of LNG was 0.10-20.00μg · L-1. The lower limit of quantitation was 0.10μg · L-1. The extrac-tion recovery was more than 78.30％. The intra-day and inter-day precisions were both less than 12.89％. The first-dosing pharmacokinetic parameters for LNG were as follows:Cmax (8.10±2.38)μg·L-1, Tmax (2.38±1.45) h, and AUC(0-768) (142.35±36.99) h·μg·L-1. The last administration pharmacokinetic parameters for LNG were as follows:Cmax (7.05±1.07)μg·L-1, Tmax (2.71±1.83) h, and AUC(0-768) (141.95±22.31) h·μg·L-1. The linear concentration range of EE was 0.02-5.00μg·L-1. The lower limit of quantitation was 0.02μg·L-1. The extraction recovery was above 79.99％. The intra-day and inter-day precisions were both less than 12.76％. The first-dosing pharmacokinetic parameters for EE were as follows: Cmax (0.18 ± 0.04)μg · L-1, Tmax (2.50±1.30) h, and AUC(0-768) (2.65±0.56) h·μg·L-1. The last administration pharmacokinetic parame-ters for EE were as follows:Cmax (0.17 ± 0.07)μg · L-1, Tmax (2.17 ± 0.26)h, and AUC(0-768) (2.02 ± 0.82) h ·μg · L-1. CONCLUSION The HPLC-MS/MS determination method is accurate and sensitive, which can be used to simultaneously determine the concentration of LNG and EE. There are no significant differences in main pharmacokinetic parameters between the first dose and the last dose. After repeated administration of this contraceptive patch, there is no accumulation of blood concentration in the rabbit body.
ABSTRACT
<p><b>OBJECTIVE</b>To explore the molecular mechanism of dutasteride inhibiting fertility by studying its effects on the expressions of the epididymal epithelial junction proteins Claudin1 and β-catenin in rats.</p><p><b>METHODS</b>Sixteen 3-month-old SD male rats were equally divided into an experimental and a negative control group to be treated intragastrically with dutasteride at 40 mg/kg per day and the same dose of solvent, respectively, for 14 consecutive days. Then, the sperm motility and morphology of the rats were detected by computer-assisted sperm analysis, the serum levels of testosterone (T) and dihydrotestosterone (DHT) measured by ELISA, changes in the tight junction of epididymal cells observed under the transmission electron microscope, the protein and gene expressions of Claudin1 and β-catenin determined by RT-PCR and immunohistochemistry, and the conception rate of the mated female rats calculated.</p><p><b>RESULTS</b>Dutasteride significantly suppressed the serum DHT level, sperm motility, and fertility of the rats (P <0.05). Interspaces between epididymal epithelial cell tight junctions were observed, the volume of epididymal fluid obviously increased, and the expressions of Claudin1 and β-catenin gene and protein remarkably downregulated in the experimental rats (P <0.05).</p><p><b>CONCLUSION</b>Dutasteride can significantly inhibit the fertility of male rats by reducing the serum DHT level, suppressing Claudin1 and β-catenin expressions, and damaging epididymal epithelial cell junctions.</p>