**1. Introduction**

The incidences of diabetic mellitus and hypertension are rapidly growing worldwide, and hypertension is an important cardiovascular risk factor in patients with type 2 diabetes mellitus (T2DM) [1–3]. The dramatically increased (two- to four-fold) risk of cardiovascular diseases in T2DM patients is their leading cause of mortality [4–6]. Insulin resistance and compensatory hyperinsulinemia are also frequent findings in hypertensive patients [7,8]. Since blood pressure-lowering treatment is important to reduce the risk of developing cardiovascular complications in T2DM patients, a combination of anti-diabetic and anti-hypertension drugs is needed and increasingly used [9].

Metformin, an oral biguanide antihyperglycemic agent, increases hepatic and skeletal muscle insulin sensitivity and decreases hepatic glucose production without causing hypoglycemia [10–12]. Metformin also reduces cardiovascular disease complications such as high blood pressure in patients with type 2 diabetic mellitus (T2DM) [3,13,14]. Metformin pharmacokinetics (PK) and pharmacodynamics (PD) rely on the organic cation transporter (OCT) 1 in the sinusoidal membrane of hepatocytes to transport metformin into the liver, its pharmacological target site. The metformin concentration in the liver determines the metformin's effect on inhibiting glucose production [15–17]. OCT2 in the basolateral membrane of the renal proximal tubules mediates the renal excretion of metformin as ~70% of the metformin dose [16,18]. Reduced renal excretion of metformin via renal

OCT2 inhibition causes an increase of systemic exposure (i.e., plasma concentration of metformin), which can result in lactic acidosis [19]. Previous reports that metformin suppresses P-glycoprotein (P-gp) and pregnane X receptor (PXR)-regulated transactivation of the cytochrome P450 (CYP) 3A4 gene [20,21] suggest that the potential for a PK-based drug-drug interaction (DDI) occurrence between metformin and verapamil.

Verapamil, as a calcium channel blocker, is recently emerged as a combination drug in DM patients with hypertension, because verapamil lowers fasting blood glucose levels and enhances insulin secretion in T2DM patients [22,23]. In particular, it is an important issue to regulate insulin secretion as a compensatory mechanism of hyperinsulinemia in hypertensive patients [7,8]. Verapamil has been popularly used for treatment of hypertension and supraventricular tachyarrhythmias [22–27]. Orally administered verapamil is rapidly and well-absorbed, and then it is eliminated via extensive cytochrome P450 (CYP)-mediated hepatic metabolism [25–29]. An *N*-methylated metabolite, norverapamil, has been shown to have a vasodilator effect in vitro [13]. Verapamil also has an inhibitory effect on P-gp and OCT2 in in vitro and clinical studies [16,30,31]. Although it was reported that verapamil reduced the maximum blood glucose concentration and area under the blood glucose concentration-time curve in healthy adults orally administered with metformin and verapamil together [32], there was no direct measurement of metformin concentration in liver, which may be a substantial evidence on its glucose tolerance activity. Moreover, there has been no report to explain whether transporter and/or metabolic enzyme-mediated PK changes of metformin and verapamil in their combination. In the meantime, new findings regarding positive effect of verapamil for the treatment of hypertension in DM patients have been introduced [22,23], there is a need to investigate how the plasma and tissue concentrations of metformin and verapamil change in DDI events, especially at preclinical levels [33,34]. Since changes in plasma and tissue concentrations of these drugs in combination are associated with their efficacy and toxicity [35,36], the PK interactions of metformin and verapamil were evaluated in rats based on their plasma and tissue concentration changes.
