*3.2. E*ff*ect of Verapamil on OCT1- or OCT2-Mediated Metformin Uptake in HEK-293 Cells Overexpressing OCT1 or OCT2*

To examine the verapamil effect on OCT1 and OCT2 activities, the changes of metformin uptake with and without verapamil were compared in HEK-293 cells overexpressing OCT1 or OCT2. As shown in Figure 2, verapamil considerably inhibited metformin uptake in HEK-293 cells overexpressing either OCT1 or OCT2. The IC<sup>50</sup> values of verapamil for inhibiting OCT1- and OCT2-mediated metformin uptake were 51.9 ± 1.09 and 19.3 ± 0.220 µM, respectively. 0.220 μM, respectively.

**Figure 2.** Mean (± S.D.) inhibitory effect of verapamil on metformin uptake in HEK293 cells overexpressing OCT1 (**A**, *n* = 2) or OCT2 (**B**, *n* = 2).

### *3.3. E*ff*ect of Verapamil on Metformin Concentrations in the Liver and Kidneys*

After intravenous and oral administration of metformin with and without verapamil, metformin concentrations in the liver and kidneys are shown in Figure 3 and Table 2, respectively. After intravenous and oral administration of both drugs together, verapamil did not cause any change of the metformin concentration in the liver, but the metformin concentration in the kidneys was significantly decreased (by 38.6 and 48.7% at 1 and 3 h in the intravenous study and 42.9, 41.1 and 30.2% at 1, 3 and 6 h in the oral study, respectively) compared to metformin alone. Metformin concentrations in the plasma after intravenous and oral co-administration of metformin and verapamil were significantly lower than those after administration of metformin alone, showing similar patterns to the pharmacokinetic study.

○ or without (●) **Figure 3.** Mean (± S.D.) concentrations of metformin in the plasma, liver and kidneys after its intravenous (**A**, *n* = 4 for each) or oral (**B**, *n* = 4 for each) administration with (#) or without (•) verapamil. The dose of metformin and verapamil were 30 and 20 mg/kg, respectively.


μ μ **Table 2.** Mean (± S.D.) concentrations (µg/mL or µg/g tissue) of metformin in the plasma, liver and kidneys after intravenous or oral administration of metformin with and without verapamil.

<sup>a</sup> Significantly different (*p* < 0.01) from metformin. <sup>b</sup> Significantly different (*p* < 0.05) from metformin.

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