All animal experiments were performed with the approval of Institutional Animal Care and Use Committee of Pharmaron Lab Animal Research in an AAALACi-accredited facility (Certification No.: 001760, Date: 4 November 2019). Male beagle dogs (approximate 10 kg each) were kept under standard conditions (temperature: 23 ± 2 °C, moisture: 55 ± 10%, and a controlled 12 h light/dark cycle) with free access to food and water. Dogs were fasted overnight prior to the experiments. Thirteen dogs were randomly assigned to three treatment groups. Three dogs were administrated intravenously (IV) icaritin solution (2 mg/mL) which consisted of 10% (v/v) DMSO, 20% (v/v) Solutol HS15 and 70% (v/v) saline solution at a dose of 2 mg/kg. Four dogs were given IPMs intragastrically (aqueous solution, 20 mg/mL) at a dose of 20 mg/kg. Six dogs were administered orally the oily suspension of icaritin (soft capsule, 100 mg/capsule) used in the clinic at a dose of 20 mg/kg. Blood samples (1 mL) from the IV group were collected from venipuncture of peripheral veins at 0, 0.083, 0.167, 0.5, 1, 2, 4, 6, 8, 12, and 24 h following the single administration in heparinized polypropylene tubes. Blood samples (1 mL) from the oral group were collected from venipuncture of peripheral veins at 0, 0.167, 0.5, 1, 2, 4, 6, 8, 12, and 24 h post-dose in heparinized polypropylene tubes. All the collected blood samples were immediately centrifuged at 2000× g for 10 min at 28 °C, and the plasma samples were then stored at −80 °C until analysis.

The plasma (55 μL) was incubated with acetic acid (0.1 mol/L, 10 μL) and phosphate buffered saline (pH 5.0, 25 μL) containing β-Glucuronidase (25 μL, 10,000 U/mL) for 1 h at 37 °C. The internal standard solution (200 μL, containing 500 ng/mL of dexamethasone) was then added and vortexed for 2 min. The resultant mixture was centrifuged at 4000× g for 15 min, and the 5 μL aliquot of supernatant was subjected to LC/MS/MS analysis using an UHPLC-MS/MS system consisting of an ultra HPLC system (LC-30D, Shimadzu, Japan) coupled with a LC/MS/MS mass spectrometer (AB API 5500, Agilent, Palo Alto, CA, USA) equipped with electrospray-ionization (ESI) source. The chromatographic separation was carried out on a Gemimi C18 (3 μm, 100 A, 50 × 2.1 mm, Phenovmenex, Torrance, CA, USA) column at the column temperature of 40 °C using a mobile phase consisting of 5% acetonitrile in 0.1% formic acid (A) and 95% acetonitrile in 0.1% formic acid (B) pumped at a flow rate of 0.6 mL/min. The program of gradient eluent was set as follows: 0–0.2 min with 20% B, 0.2–1.9 min with 20–100% B, 1.9–2.1 min with 100% B, and 2.1–2.5 min with 20% B. The eluent was directly introduced into an ESI interface. The ESI parameters were set as follows: the temperature of the drying gas (nitrogen), 400 °C; Curtain gas, 45 psi; nebulizer pressure, 50 psi; ion spray voltage, 4500 V for negative ion mode. Multiple-reaction monitoring detection was employed using nitrogen as the collision energy. The icaritin and dexamethasone (internal standard) were monitored at m/z 367.12–297.00 and m/z 391.14–361.20, respectively. The linearity of the calibration curve was excellent (R = 0.9957) within the concentration range from 1 to 1000 ng/mL. The coefficient of variation of the intra- and inter-day precisions (relative standard deviation, RSD) of the quality control samples were lower than 15% and the accuracy was in the range of (87.10 ± 0.46)–(107.0 ± 0.05)%. The limit of quantification (LOQ) was 1 ng/mL.

For the IV dosing group, pharmacokinetic parameters were calculated using two compartment analysis with the WinNonlin (Phoenix, version 6.1) program. The main pharmacokinetic parameters were calculated. The area under the blood drug concentration-time curve from 0 to t (AUC0–t) was calculated with the trapezium method. Total clearance (CL), and mean residence time from 0 to t (MRT0–t) were obtained.

For the oral dosing groups, the main pharmacokinetic parameters were obtained from blood drug concentration–time data. The AUC0–t was estimated by using the noncompartmental model in the WinNonlin (Phoenix, version 6.1) program.

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