2.4. PK Studies

The solutions of 1 mg/mL and 2 mg/mL of Ewha-18278 in two different formulations were prepared immediately prior to intravenous (IV) injection and oral administration (PO), respectively. The DMSO-based formulations were comprised of 10% DMSO and 90% saline-based solution (25% Cremophor^® EL, 5% polyethylene glycol 400, 0.5% Tween^® and 69.5% saline, v/v/v/v). The diazepam injection-based formulation applicable to humans was composed of 40% propylene glycol, 10% dehydrated ethanol, and 50% water (v/v/v). In the case of the diazepam injection-based formulations, Ewha-18278 was prepared in two different concentrations, 1 mg/mL and 2 mg/mL for oral administration. These concentrations were utilized to examine the effect of concentration on the PK parameters of the compound.

The rats were divided into five groups on the day of the drug administration. Two groups (n = 6) were administered the DMSO-based formulation: IV injection of 2 mg/kg and oral administration of 20 mg/kg. Three groups (n = 5–6) were treated with the diazepam injection-based formulation: IV injection of 2 mg/kg and oral administration of 20 mg/kg in two different concentrations (1 mg/mL and 2 mg/mL). The common carotid artery was catheterized for blood sampling. Blood sampling (0.2 mL) was conducted at 0, 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 6, 10, and 24 h for the IV group and 0, 0.083, 0.25, 0.5, 1, 2, 4, 6, 10, 24, and 34 h for PO groups [8,9]. Plasma samples mixed with IS (Ewha-18212) were analyzed by the HPLC system.

WinNonlin^® Professional version 5.2 software (Pharsight Corporation, Mountain View, CA, USA) was used to estimate the PK parameters following intravenous or oral administration of Ewha-18278 to rats. Non-compartmental analysis was performed using the plasma Ewha-18278 concentration-time profiles to obtain the following PK parameters: initial plasma concentration (C₀), area under the plasma concentration-time curve from zero time to infinity (AUC_inf), elimination half-life (t_1/2), apparent volume of distribution (V_d), total clearance (Cl_t), apparent volume of distribution following oral administration (V_d/F), and oral clearance (Cl_t/F). The maximum plasma concentration (C_max) and the time required to reach C_max (T_max) were directly measured from the plasma Ewha-18278 concentration-time curve. The absolute BA (F, %) of Ewha-18278 was calculated by using the equation: