*2.2. Animals and Ethical Approval*

Male Institute of Cancer Research (ICR) mice (7 or 8-weeks-old, weighing 34–37 g) and male Sprague-Dawley (SD) rats (7 or 8-weeks-old, 230–270 g) were purchased from Samtako Co. (Osan, Korea). The animals were acclimatized for 1 week at an animal facility at Kyungpook National University. Food and water were available ad libitum. All animal procedures were approved by the Animal Care and Use Committee of Kyungpook National University (Approval No. KNU 2018-192, 19 December 2018) and carried out in accordance with the National Institutes of Health guidance for the care and use of laboratory animals. An overview of the study design and methods is provided in Table 1.



IV, intravenous injection; PO, per oral administration, PPD, 20(S)-protopanaxadiol; PK parameters, pharmacokinetic parameters; AUC, area under plasma concentration.

#### *2.3. Pharmacokinetic Study*

ICR mice and SD rats received compound K intravenously at a single dose of 2 mg/kg via the tail vein and were returned to the metabolic cage with water and chow ad libitum. Before administration, the compound K was dissolved in a vehicle containing DMSO: saline (2:8, *v*/*v*) (vehicle volume, 1 mL/kg for mice and 0.4 mL/kg for rats). Blood samples (approximately 100 µL) were collected at 0, 0.17, 0.5, 1, 2, 4, 8, 24, and 48 h following intravenous injection of compound K with no sign of hemoglobinemia and hemoglobinuria. Blood sampling was performed using a sparse sampling method (time schedule is given in Table 2). The blood samples were centrifuged at 16,000× *g* for 10 min to separate the plasma. An aliquot (50 µL) of each plasma sample was stored at −80 ◦C until the analysis. Urine and feces samples were collected from 0–24 h and 24–48 h following the compound K administration. Aliquots (50 µL) of urine and 10% feces homogenates were stored at −80 ◦C until the analysis.

SD rats were randomly divided into two groups: the non-bile cannulated control group and the bile cannulated group. For the bile cannulated rats, the femoral artery, femoral vein, and bile duct were cannulated with polyethylene tubes (PE-50 and PE-10; Jungdo, Seoul, Korea) under light anesthesia with isoflurane. For the control group, the femoral artery and femoral vein were cannulated with PE-50. Compound K was injected intravenously via the femoral vein at 2 mg/kg. Blood samples (approximately 200 µL) were collected from the femoral artery at 0, 0.25, 0.5, 1, 2, 4, and 8 h after the compound K injection. After each blood sampling, normal saline was injected into the femoral vein to compensate for blood loss. Bile samples were collected every 2 h for a total of 12 h. The blood samples were centrifuged at 16,000× *g* for 10 min, and 50 µL aliquots of plasma and 50 µL aliquots of bile were stored at −80 ◦C until the compound K analysis. In the non-bile cannulated rats, the complete contents of the entire gastrointestinal tract were collected using a 10 mL syringe filled with 30 mL pre-warmed saline. The contents were homogenized with tissue homogenizer, and 50 µL aliquots of intestinal fecal homogenates were stored at −80 ◦C until the analysis.

The 50 µL samples of plasma, urine, 10% fecal homogenates, bile, and intestinal fecal homogenates were each mixed with 60 µL of IS (20 ng/mL 13C-caffeine in water) and 600 µL of methyl *tert*-butyl ether (MTBE). The mixtures were vortexed vigorously for 10 min and centrifuged at 16,000× *g* for 5 min. After centrifugation, the samples were frozen at −80 ◦C for 2 h. For each sample, the upper layer was transferred to a clean tube and evaporated to dryness under a nitrogen stream. The residue was reconfigured with 200 µL of 80% methanol consisting of 0.1% formic acid, and a 10 µL aliquot was injected into the liquid chromatography–tandem mass spectrometry (LC–MS/MS) system.


**Table 2.** Blood sampling schedule for the pharmacokinetic study of compound K in mice and rats following intravenous injection of compound K.

<sup>1</sup> RO-right: retro-orbital blood sampling—right eye under anesthesia with isoflurane; <sup>2</sup> RO-left: retro-orbital blood sampling—left eye under anesthesia with isoflurane; <sup>3</sup> AA: abdominal artery blood sampling.

#### *2.4. Tissue Distribution Study*

ICR mice and SD rats received compound K intravenously at a single dose of 2 mg/kg via tail vein and were returned to the metabolic cage with water and chow ad libitum. The animals were euthanized and blood samples (approximately 200 µL) were collected from the abdominal artery at 0.17, 0.5, 2, 4, 8, and 24 h after intravenous injection. The liver, kidney, brain, heart, lung, spleen, and testis were immediately excised, gently washed with ice-cold saline, and weighed. The tissue samples were homogenized with 4 volumes of saline. Aliquots (50 µL) of plasma and 20% tissue homogenates were stored at –80 ◦C until the analysis of compound K.

To investigate the effect of rifampin (a representative inhibitor of the organic anion-transporting polypeptide (Oatp) transporters [11]) on the hepatic distribution of compound K, ICR mice and SD rats were randomly divided into either control or rifampin groups. The rifampin group was orally administered with rifampin solution (20 mg/kg, dissolved in DMSO: saline = 2:8, *v*/*v*) and the control group received only the vehicle via oral gavage. One hour after rifampin treatment, mice and rats received compound K intravenously at a single dose of 2 mg/kg via tail vein. The animals were euthanized and blood samples (approximately 200 µL) were collected from the abdominal artery at 0.5 and 2 h after intravenous injection. The liver tissues were immediately excised, gently washed with ice-cold saline, and homogenized with 4 volumes of saline. Aliquots (50 µL) of plasma and 20% liver homogenates were stored at −80 ◦C until the analysis of compound K.

The plasma samples and 20% tissue homogenates samples were each mixed with 60 µL of IS (20 ng/mL 13C-caffeine in water) and 600 µL of MTBE. The mixtures were vortexed vigorously for 10 min and centrifuged at 16,000× *g* for 5 min. After centrifugation, the samples were frozen at −80 ◦C for 2 h. The upper layer was transferred to a clean tube and evaporated to dryness under a nitrogen stream. The residue was reconfigured with 200 µL of 80% methanol consisting of 0.1% formic acid, and a 10 µL aliquot was injected into the LC-MS/MS system.
