Tissue Pharmacokinetics of SH-BC-893 in Mice

Tissue PK studies were performed at Pharmaron (Beijing, China) on a fee-for-service basis and were approved by their Institutional Animal Care and Use Committee. Six- to eight-week old male or female CD1 mice were treated daily for 5 days with 120 mg/kg SH-BC-893 dissolved in H₂O (P.O., stock = 12 mg/ml) and then sacrificed 0.5, 1, 2, 4, 8 or 24 h after the last dose. Tissues were perfused with 10 mL saline and prior to collection and snap frozen in liquid nitrogen. Frozen tissue was homogenized in PBS (W/V 1:4). 10 μl of tissue or plasma homogenate was mixed with 10 μl blank solution and added to 200 μl acetonitrile containing FTY720 as an internal standard. Samples were vortexed and then spun for 30 min at 4700 rpm at 4°C to precipitate protein. The resultant supernatant was diluted 2-fold with water and 10 μl of diluted supernatant was injected into the LC–MS/MS for quantitative analysis. SH-BC-893 was quantified by LC–MS/MS using a HALO 90A AQ-C18, 2.7 μm 2.1 × 50 mm column and an AB Sciex Triple Quad 5500 LC–MS/MS instrument (serial no. BB214861610) and corrected for extraction efficiency using the FTY720 internal standard.