2.6. Pharmacokinetic Studies

Twelve male Sprague–Dawley rats (Nara Biotech., Seoul, Korea), 7–8 weeks old and weighing 270 ± 20 g, were used for the in vivo pharmacokinetic studies. All of the animals were caged in controlled conditions of 23–25 °C/50–55% relative humidity (RH), and were freely provided with standard laboratory food and water. The protocols for the animal studies were also approved by the Institutional Animal Care and Use Committee at Hanyang University (permit number: 2014-0190A).

The rats were randomly divided into two groups of 6 rats per cage and left to acclimatize to the controlled conditions for 3 days. Prior to the pharmacokinetic experiment, the animals were fasted for 12h, but were allowed free access to water. Each rat was anaesthetized with diethyl ether and fixed on a surgical board. For cannulation, a polyethylene tube containing 50 IU/mL heparin in saline was placed into the right femoral artery of the rat. Then, each of the rats was orally administered with 1 mL aqueous suspension of MTX powder or MTX-loaded solid SASD at a dose of 20 mg/kg using an oral gavage. At predetermined time intervals, ~0.3 mL blood was collected via the cannulated tube and transferred into microtubes containing heparin, which were then centrifuged at 3000× g for 10 min to isolate the plasma.

To the collected plasma samples (90 μL), 100 μL methanol and 10 μL internal standard solution i.e., theophylline (1 mg/mL in methanol) was added. The mixture was vortexed for a few seconds and then centrifuged at 13,000× g for 10 min, and the supernatant was immediately transferred to vials for quantification by HPLC, as described in Section 2.2. A six-points calibration plot was constructed by the analysis of the working solution over the range 25–2000 ng/mL, and the response was linear throughout the range with R² = 0.997. Moreover, the intra-day and inter-day variations were within the applicable limits (R² = 0.998). The pharmacokinetic parameters of the area under the blood concentration–time curve (AUC), maximum plasma concentration (C_max), time to reach the C_max (T_max), half-life (t_1/2), and elimination rate constant (K_el) were derived using the WinNonlin software (Pharsight Corp., Mountain View, CA, USA). The values are reported as mean ± SD (n = 6), and the data were considered statistically significant at a p-value lower than 0.05 (p < 0.05) between the two formulations, as determined with Student’s t-test.