*2.7. Rat Plasma Protein Binding of Metformin and Verapamil Using Equilibrium Dialysis*

Protein binding values of metformin and verapamil with and without each other were measured in the fresh plasma of control rats using equilibrium dialysis (*n* = 4 for each; [17]). A 1 mL aliquot of the plasma was dialyzed against 1 mL of isotonic Sørensen phosphate buffer (pH 7.4) containing 3% dextran (*w*/*v*) in a dialysis cell (Spectrum Medical Industries, Laguna Hills, CA, USA) using a Spectra/Por 4 membrane (mol. wt. cutoff 12–14 KDa; Spectrum Medical Industries, USA). After 24 h incubation, two 50 µL aliquots were collected from each compartment, and the samples were stored at −80 ◦C.

#### *2.8. Analytical Methods for Metformin, Verapamil and Norverapamil*

In the HPL-UV system, the metformin concentration in the sample was determined using the analytical method developed by Choi et al. [17]. The quantitation limits of metformin in rat plasma, urine and GI samples were 0.05, 0.1 and 0.1 µg/mL, respectively. The inter- and intra-day coefficients of variation were below 8.94%. Additionally, the metformin concentration in the sample from the metformin uptake study was determined by LC-MS/MS analysis [33,34]. The quantitation limits of metformin in rat plasma, urine and GI samples were 0.01, 0.02 and 0.02 µg/mL, respectively. The inter- and intra-day coefficients of variation were below 9.53%. Verapamil and norverapamil concentrations in the sample were also determined by the HPLC-UV analytical method of Hong et al. [29]. The quantitation limits of verapamil in rat plasma, urine and GI samples were 0.01, 0.05 and 0.05 µg/mL, respectively. The corresponding values of norverapamil in all biological samples was 0.02 µg/mL; the inter- and intra-day coefficients of variation were below 12.9%.

#### *2.9. Pharmacokinetic Analysis*

Standard methods [41] were used to calculate the following pharmacokinetic parameters using non-compartmental analysis (WinNonlin; Pharsight Corporation, Mountain View, CA, USA): the total area under the plasma concentration–time curve from time zero to infinity (AUC), terminal half-life, time-averaged total body, renal, and non-renal clearances (CL, CL<sup>R</sup> and CLNR, respectively), and apparent volume of distribution at a steady state (Vss). The peak plasma concentration (*C*max) and time to reach *C*max (*T*max) were directly read from the experimental data.

### *2.10. Statistical Analysis*

A *p*-value < 0.05 was deemed to be statistically significant using a Student's *t*-test between the two means for the unpaired data. All results are expressed as mean ± standard deviation (S.D.) except the medians (ranges) for *T*max.

#### **3. Results**

### *3.1. E*ff*ect of Verapamil on Metformin Pharmacokinetics*

The mean arterial plasma concentration–time profiles and relevant pharmacokinetic parameters of metformin after its intravenous administration with and without verapamil are shown in Figure 1 and Table 1, respectively. After intravenous administration of metformin with verapamil, the AUC was significantly greater (by 66.7%); CL and CL<sup>R</sup> were significantly slower (by 50.8 and 70.7%, respectively); and Ae0–24 h was significantly smaller (by 43.0%) than those without verapamil.

After oral administration of metformin with verapamil, the AUC was significantly greater (by 73.5%), *C*max was significantly higher (by 60.5%), CL<sup>R</sup> was significantly slower (by 68.4%) and Ae0–24 h was significantly smaller (by 46.0%) than those without verapamil.


**Table 1.** Mean (± S.D.) pharmacokinetic parameters of metformin after its intravenous and oral administration with and without verapamil to rats. Doses of metformin and verapamil were 30 and 20 mg/kg, respectively.

AUC, total area under the plasma concentration−time curve from time zero to infinity; MRT, mean residence time; Vss, apparent volume of distribution at steady state; CL, time-averaged total body clearance; CLNR, time-averaged non-renal clearance; CLR, time-averaged renal clearance; Ae0–24 h, percentage of the dose excreted in the urine up to 24 h; GI24 h, percentage of the dose recovered from the gastrointestinal tract (including its contents and feces) at 24 h; *C*max, peak plasma concentration of docetaxel; *T*max, time to reach *C*max; *F*, extent of absolute oral bioavailability. a Significantly different (*p*<0.01) from with verapamil. b Significantly different (*p*<0.001) from with verapamil. c Significantly different (*p*<0.05) from with verapamil.

– ○ or without (●) **Figure 1.** Mean (± S.D.) arterial plasma concentration–time profiles of metformin after intravenous (**A**; *n* = 6 for each) and oral (**B**; *n* = 5 for each) administration of metformin with (#) or without (•) verapamil to rats. Doses of metformin and verapamil were 30 and 20 mg/kg, respectively.
