Pharmacokinetic experiments

Twelve healthy rabbits (70 days of age, half males and half females) were allowed to acclimate to the facility for 1 week. The rabbits were then weighed and randomly divided into two groups. One group received 10 mg/kg body weight (bw) Tol, whereas the other group received 10 mg/kg bw Tol-HP-β-CD (containing the equivalent doses of Tol). The preparation of Tol/Tol-HP-β-CD is described as follows: the weighted Tol was added to suitable amount of distilled water, and the solution was shaked strongly to form mixed suspension. The preparation of Tol-HP-β-CD solution was similar to that of Tol mixed suspension. Both groups were orally administered a single dose. Blood sample (1 mL) was collected from the ear vein in heparinized vacutainer tubes prior to treatment immediately before drug administration (baseline) and at 0.25, 0.5, 1, 2, 3, 4, 8, 12, 24, 36, 48, 72, 120, 168, and 240 h after drug administration. Blood samples were centrifuged at 1,788.8× g for 10 min within 1 h after sampling, and plasma was collected and stored at −70°C until analysis.

Tol was extracted from rabbit plasma using extraction with acetonitrile following Kim et al4 with slight modifications. In brief, the samples were prepared by adding 200 μL of plasma to 300 μL of acetonitrile after unfreezing. The mixture was vortexed for 2 min and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm polytetrafluoroethylene syringe filter. A 20 μL aliquot was used in a high-performance liquid chromatography (HPLC) system for analysis.

All samples were analyzed on a Shimadzu LC-2010CHT system (Shimadzu Corporation) with a UV detector. HPLC analysis was performed using a 5 μm C18-silica-based stainless-steel Kromasil column (4.6×250 mm; Akzo Nobel, Bohus, Sweden) as a stationary phase. The mobile phase consisted of 0.1% acetic acid aqueous solution and acetonitrile (45:55, v/v), which was ultrasonicated for 10 min and degassed for another 5 min. The flow rate was 1 mL/min at 25°C with the detection wavelength of 240 nm.

A series of stock solutions of Tol standard were prepared, and each standard (20 μL) was mixed with drug-free plasma (180 μL) to prepare calibration curves ranging from 0.2 to 25.6 μg/mL. The slope of the lines between peak areas and drug concentration was determined using least-squares linear regression. Linearity was established to determine the relationship between Tol concentration and detector response. The detection limit of Tol was established with HPLC analysis of blank plasma fortified with the standard and eventually measured to be 0.05 μg/mL. The regression lines between peak areas and drug concentrations show that the correlation coefficient was 0.9996. The mean extraction recovery from plasma was 90.23%±0.73%. The average relative SDs of intraday and interday precision were 1.51% and 2.12%, respectively.

DAS2.0 (Mathematical Pharmacology Professional Committee of China, Shanghai, China) was used to fit the plasma concentration–time curves obtained after single oral administration in each rabbit, and the pharmacokinetic parameters were calculated using the double-compartment method. The peak concentrations (C_max) and time to peak occurrence (T_max) were determined from the plotted concentration–time curve in each rabbit. The area under the concentration–time curve (AUC0-∞) was calculated using the trapezoidal rule.25 Arithmetic mean values and SDs were calculated using Microsoft^® Excel 2007. All statistical evaluations were performed with SPSS for Windows version 19.0 (IBM Corporation, Armonk, NY, USA). Some pharmacokinetic parameters were log-transformed before statistical analysis. Statistical significance was set at p<0.05. Pharmacokinetic variables are reported as mean ± SD.