*2.3. Animal Plasma Samples*

Rat and dog plasma samples were obtained from Biotoxtech Co. Ltd. (Cheongju, Korea). Plasma samples for oral pharmacokinetic study were obtained from the 4-week repeated oral toxicity study. The information on the plasma samples were provided in the Supplementary data. Briefly, for the pharmacokinetic analysis in rats, HSG4112 was orally administered HSG4112 at a dose of 100 mg/kg/day for 4 weeks and the plasma samples were collected on the 28th day. The blood drain time point was 0, 0.5, 1, 2, 4, 6, 10, and 24 h (*n* = 3; Table S1). In addition, rats were intravenously administrated HSG4112 at a dose of 10 mg/kg and blood was collected at 0, 0.083, 0.25, 0.5, 0.75, 1, 2, 3, 6, 12, and 24 h (*n* = 3). For the pharmacokinetic analysis in dogs, HSG4112 was orally administered at a dose of 100 mg/kg/day for 4 weeks (*n* = 3), and the plasma samples were collected on the 28th day. Blood samples were taken at 0, 1, 2, 4, 6, 10, 12, and 24 h. In addition, dogs were administrated HSG4112 intravenously at a dose of 2 mg/kg (*n* = 2) and blood was collected at 0, 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 12, and 24 h. All animal studies were performed according to the guidelines of the Ethics Committee for Use of Experimental Animals and approved by the Institutional Animal Care and Use Committee of Biotoxtech Co. Ltd. (Approval ID and date: 2016-05-160252, 2016-05-160276).

#### *2.4. Sample Preparation*

The plasma (30 µL) sample was put into a 1.5 mL tube and added with 60 µL IS solution. The tube was vortexed and centrifuged at 13,200 rpm for 5 min at room temperature. The supernatant was transferred to an LC vial for LC-MS/MS analysis.

#### *2.5. In Vitro Metabolic Stability*

Incubation mixture containing liver or intestinal microsomes (1 mg/mL), and HSG4112 (10 µM) in potassium phosphate buffer (0.1 M, pH 7.4) were preincubated at 37 ◦C for 5 min. The reactions were initiated by the addition of an NADPH-generation solution in a final incubation volume of 100 µL (*n* = 3). For glucuronide conjugate formation, incubation mixture containing microsomes (1 mg/mL), magnesium chloride (2.5 mM), UDPGA (2 mM), alamethicin (25 µg/mL), and drug (10 µM) in potassium phosphate buffer (0.1 M, pH 7.4) were preincubated at 37 ◦C for 5 min. The reactions were initiated by the addition of the NADPH-generation solution in a final incubation volume of 200 µL (*n* = 3). After the incubation for 0, 30, 60, and 120 min, the reaction was stopped by the addition of 200 µL ACN with IS. The samples were vortex-mixed and centrifuged at 13,200 rpm for 5 min. The supernatant (5 µL) was injected on to ultra-performance liquid chromatographic (UPLC) column for LC-MS/MS analysis. The experiment was performed in triplicate.
