2.3. Pharmacokinetic Study

For the development of 5 CYP cocktail (Figure 1), rats were randomly divided into the 5 CYP cocktail group (n = 6) and single groups (n = 6 each for individual probe substrate administration). The femoral arteries and femoral veins of rats were cannulated with PE50 polyethylene tubing (Jungdo, Seoul, Korea) under anesthesia with zoletil and lompun (50 and 5 mg/kg, respectively, intramuscular injection) and heparinized saline (10 U/mL) was used to prevent blood clotting. Pharmacokinetic studies were initiated after the recovery from anesthesia. Each rat in the 5 CYP cocktail group received the 5 CYP cocktail solution (2 mL/kg), including caffeine (1 mg/kg), diclofenac (2 mg/kg), dextromethorphan (10 mg/kg), omeprazole (2 mg/kg), and nifedipine (0.5 mg/kg). The probes were dissolved in a saline solution containing 10% DMSO. The rats in the single groups received individual CYP probe substrate solution orally with the same dose and volume as the cocktail solution. Blood samples were collected via the femoral artery at 0, 0.25, 0.5, 1, 2, 4, 8, and 24 h following the oral probe substrate administration, and a normal saline solution was administered via the femoral vein to compensate for blood sampling. After the centrifugation of blood samples at 8000× g for 1 min, 50 μL aliquots of plasma samples were stored at −80 °C until the analysis of probe substrates and their metabolites.

For the 5 transporter cocktail development (Figure 1), the rats were randomly divided into the 5 transporter cocktail (n = 6) and single groups (n = 6 each) for individual probe substrate studies. The femoral arteries and veins of the rats were cannulated with PE50 polyethylene tubing (Jungdo, Seoul, Korea) under anesthesia with Zoletil and lompun (50 and 5 mg/kg, respectively, intramuscular injection), and heparinized saline (10 U/mL) was used to prevent blood clotting. Pharmacokinetic studies were initiated after recovery from the anesthesia. Each rat in the 5 transporter cocktail groups received an intravenous cocktail (1 mL/kg) of 5 solutions, which included digoxin (2 mg/kg), furosemide (0.1 mg/kg), metformin (0.5 mg/kg), methotrexate (0.5 mg/kg), and valsartan (0.2 mg/kg). The drugs were dissolved in saline solution containing 10% DMSO. The rats within the single group studies received individual substrate solutions intravenously with an equivalent dose and volume to the cocktail solution. Blood samples were collected via the femoral artery at 0, 0.25, 0.5, 1, 2, 4, 8, and 24 h following the intravenous injection of probe substrates. Normal saline solutions were administered via the femoral vein to compensate for blood sampling. After the centrifugation of blood samples at 8000× g for 1 min, 50 μL aliquots of plasma samples were stored at −80 °C until the analysis of probe substrates.

For the dual cocktail development (Figure 1), the rats were randomly divided into three groups to receive the 5 CYP cocktail (n = 6), 5 transporter cocktail (n = 6), and dual cocktail set (n = 7). The femoral arteries and veins of rats were cannulated with PE50 polyethylene tubing (Jungdo, Seoul, Korea) under anesthesia with Zoletil and lompun (50 and 5 mg/kg, respectively, intramuscular injection), and heparinized saline (10 U/mL) was used to prevent blood clotting. Pharmacokinetic studies were started after the recovery from the anesthesia. The rats in the 5 CYP cocktail group received the 5 CYP substrate mixture orally. The rats in the 5 transporter cocktail group received the 5 transporter substrate mixture intravenously as previously described. The rats in the dual cocktail group simultaneously received the oral 5 CYP cocktail solution and the intravenous 5 transporter cocktail solution at an equivalent dose and similar method previously described. Blood samples were collected via the femoral artery at 0, 0.25, 0.5, 1, 2, 4, 8, and 24 h following the probe substrate administration.

For the CYP3A inhibition (Figure 1), the rats were randomly divided into the control (n = 3) and single ketoconazole (10 mg/kg) groups (n = 4). The rats in the ketoconazole group received ketoconazole solution (10 mg/mL/kg) via oral gavage, while the control rats received vehicle (1 mL/kg) also via oral gavage. After 1 h of ketoconazole treatment, the dual cocktail mixture solution was administered as previously described. Blood samples were collected via the femoral artery at 0, 0.25, 0.5, 1, 2, 4, 8, and 24 h following the probe substrate administration. For the OATPs inhibition (Figure 1), the rats were randomly divided into the control (n = 3) and single rifampin (20 mg/kg) groups (n = 4). The rats in the rifampin group received rifampin solution (20 mg/mL/kg) via oral gavage, while the control rats received vehicle (1 mL/kg) also via oral gavage. After 1 h of rifampin treatment, the dual cocktail mixture was administered as previously described. Blood samples were collected via the femoral artery at 0, 0.25, 0.5, 1, 2, 4, 8, and 24 h following the probe substrate administration. For the induction study (Figure 1), the rats were randomly divided into the control (n = 4) and multiple rifampin (20 mg/kg/day for 5 days) groups (n = 4). The rats in the rifampin group received rifampin solution (20 mg/mL/kg) via oral gavage, while the control rats received vehicle (1 mL/kg for 5 days) also via oral gavage. After 24 h of the last rifampin treatment, the dual cocktail mixture was administered as previously described to avoid the direct inhibitory effect of rifampin on drug transporters [42]. Blood samples were collected via the femoral artery at 0, 0.25, 0.5, 1, 2, 4, 8, and 24 h following the oral probe substrate administration. After the centrifugation of blood samples at 8000× g for 1 min, 50 μL aliquots of plasma samples were stored at −80 °C until the analysis of probe substrates and their metabolites.

For the dual cocktail application (Figure 1), the rats were randomly divided into the control (n = 7) and multiple RGE treatment (1.4 g/kg for 7 days) groups (n = 7). The rats in the multiple RGE group received RGE suspension (1.4 g/mL/kg/day) for 7 days orally via oral gavage. The control group received water (1 mL/kg) for 7 days by oral gavage. After 1 h of the last RGE treatment, the rats received the oral 5 CYP cocktail solution and simultaneously received the intravenous 5 transporter cocktail mixture at the same dose and method previously described. Blood samples were collected via the femoral artery at 0, 0.25, 0.5, 1, 2, 4, 8, and 24 h following the probe substrate administration. After the centrifugation of blood samples at 8000× g for 1 min, 50 μL aliquots of plasma samples were stored at −80 °C until the analysis of probe substrates and their metabolites.

The samples were prepared using protein precipitation. The samples (50 μL) were then added to 200 μL of internal standard (IS) working solution (berberine 2 ng/mL in methanol). After vortex mixing of the sample mixture for 15 min and centrifugation at 16,000× g for 5 min, 5 μL of the supernatant was injected into the LC-MS/MS system.