In vivo pharmacokinetic study

All animal experiments in this study were approved by the China Pharmaceutical University Ethics Committee and carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Male Sprague–Dawley rats of 200–250 g were raised at a temperature of 25°C±2°C and the relative humidity was 45%–50%. All rats were randomly divided into two groups (n=7), and fasted for 12 hours (free access to water) before the experiment. NMP suspension was prepared by dissolving NMP powder in a 0.5% CMC-Na solution.28 Then, NMP-NLC and NMP suspensions were administered orally to rats at a dose of 40 mg/kg, respectively. Blood samples (0.5 mL) were collected via the suborbital vein at 0, 0.25, 0.5, 1, 1.5, 2, 2.5 3, 3.5, 4, 8, 12, and 24 hours after administration. All the blood samples were instantly centrifuged at 5,000 rpm for 5 minutes to acquire the transparent plasma, followed by storing at −20°C before HPLC analysis.29

Diazepam solution (50 µL) was used as the internal standard (50 µg/mL) in this study and mixed a 100 µL plasma sample. After being vortexed for 1 minute, 500 µL of tertbutyl methyl ether were added into the mixture and vortexed for 5 minutes at room temperature to extract the drugs. Then, the mixture was centrifuged at 3,000 rpm for 10 minutes. The organic layer was transferred to a new vessel, followed by evaporation in the vacuum desiccator. The residue was dissolved with 200 µL of the mobile phase. The final mixture was centrifuged at 3,000 rpm for 5 minutes to obtain the supernatant for HPLC analysis.30

The main pharmacokinetic parameters were calculated by a pharmacokinetic program PK solver 2.0.31 The maximum concentration (C_max) and time of C_max (T_max) were obtained by the experimental data. The area under the concentration–time curve (AUC) was calculated by the linear trapezoidal method. The relative bioavailability of NMP-NLC was calculated by using the following formula:

where F_r represents the relative bioavailability, AUC is the area under the blood concentration curve, D is the dosage of administration, T is the determinant formulation (NMP-NLC), and R is the reference formulation (NMP suspension).