*2.4. Pharmacokinetic Study*

The experiment was performed in a parallel design. A total of 20 rats were randomly divided into two groups. The control group, Group N (n = 10, non-pretreated rats), was orally administered for 7 days only with the vehicle (normal saline/polyethylene glycol 400/dimethyl sulfoxide = 40:20:20). Group T (n = 10, ticagrelor-pretreated rats), an experimental group, received 10 mg/kg of ticagrelor in the vehicle once a day for 7 days orally. Thereafter, on the seventh day, tadalafil (2 mg/kg) was orally administered 30 min after the final administration of the vehicle or ticagrelor. Doses of all samples were calculated according to the weight of the rats and administered using gavage. The dose of ticagrelor and tadalafil given to rats were determined by converting the human doses (90 mg for ticagrelor and 20 mg for tadalafil) to animal doses with body surface area, according to the United States Food and Drug Administration guidelines [31]. Blood (0.3 mL) was collected from the retro-orbital plexus or the jugular vein at predetermined time-points (0, 0.33, 0.67, 1, 1.5, 2, 4, 6, 8, 12, and 24 h) after the oral administration. Samples were centrifuged at 15,000× *g* for 5 min at 4 ◦C. The plasma was collected and stored at −20 ◦C until the concentration of tadalafil was analyzed by LC-MS/MS.

#### *2.5. Sample Preparation for LC-MS*/*MS Analysis*

Protein precipitation was applied to extract tadalafil from plasma. Briefly, 200 μL of acetonitrile (0.2 *v*/*v*% formic acid) containing 500 ng/mL of IS was added to 20 μL of the plasma sample. After shaking for 5 min, the mixture was centrifuged at 15,000× *g* for 5 min. The supernatant (150 μL) was transferred and 5 μL of the sample was injected into the LC-MS/MS.

#### *2.6. Pharmacokinetic Data Analysis*

Pharmacokinetic data were analyzed based on the non-compartment analysis of WinNonlin software 8.1 (Pharsight Corp., Sunnyvale, CA, USA). The maximum plasma concentration of tadalafil (maximum concentration (Cmax)) and the time to reach the maximal plasma concentration (Tmax) of tadalafil were determined from the plasma concentration-time profiles. The area under the plasma concentration vs time curve from 0 to 24 h (AUC0–24) was calculated by the linear trapezoidal rule and the area under the plasma concentration vs time curve from 0 h to infinite time (AUC0– ∞) was estimated by extrapolating time to infinity. The elimination half-life (T1/2) and apparent total clearance (CL/F) were determined from the ln 2/elimination rate constant and dose/AUC0– ∞, respectively [32].
