Healthy Korean volunteers aged 19–45 years with body mass index (BMI) of 18.5–25 kg/m² and weight > 55 (for males) or > 50 kg (for females) were screened for enrollment based on medical history, physical examination, and the results of clinical laboratory tests (hematology, blood chemistry, serologic test, urinalysis, and urine drug test). Subjects with a history of gastrointestinal disease or surgery that could affect the absorption of the investigational products (IPs) or those allergic to the IPs were excluded. Additionally, subjects with a history of genetic disorders, such as galactose intolerance or Lapp lactase deficiency, were excluded.

This was a randomized, open-label, multiple-dose, three-treatment, three-period, Williams design crossover study (Fig. 1). The IPs were atorvastatin calcium (40 mg, Lipitor®, Pfizer Korea Ltd.) and ezetimibe (10 mg, Ezetrol®, MSD Korea Ltd.). All subjects were randomly assigned to one of the following six treatment sequences: ABC, ACB, BCA, BAC, CAB, or CBA with an 11-day washout period between treatments A, B, and C. The treatment regimens were once daily administrations of atorvastatin 40 mg (treatment A), ezetimibe 10 mg (treatment B), or co-administration of atorvastatin 40 mg and ezetimibe 10 mg (treatment C) for seven days. The subjects visited the outpatient clinic at 8 am each morning, and were orally administered the assigned IP with 240 mL water at the fasted state for the first six days of each period (D1~D6, D18~D23, and D35~D40). They were hospitalized in the afternoon on the 6^th day of each period for intensive PK sampling and safety assessment. After overnight fasting, the 7^th dose of the IP was orally administered with 240 mL water; all subjects were prohibited from drinking water for 1 h before and after IP administration. After fasting for 4 h post-dose, a standard meal (700–800 kcal and 5–25% fat) was served to all subjects at 4 and 10 h after dosing.

Serial blood samples were collected in heparinized tubes at 0 (pre-dose), 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 36, and 48 h after the last IP administration in each period. Blood samples were also collected to estimate trough plasma concentration at 0 h (pre-dose) before the 5^th and the 6^th doses. For treatment groups B and C, blood samples were additionally collected at 72 and 96 h after the last dosing for the determination of ezetimibe concentrations. The samples were centrifuged (1,800 g, 4℃, 10 min), and the supernatants were separated and stored at −70℃ until the analysis of drug concentrations.

Plasma concentrations of atorvastatin, 2-hydroxyatorvastatin, total ezetimibe (free ezetimibe + ezetimibe-glucuronide), and free ezetimibe were measured by liquid chromatography (Shimadzu Prominence LC, Shimadzu, Kyoto, Japan) coupled with tandem mass spectrometry (API 5000, AB Sciex, Washington D.C., USA) based on validated analytical procedures adopted by the Korean Ministry of Food and Drug Safety. For atorvastatin and its metabolite (2-hydroxyatorvastatin), calibration curves were linear in the range of 0.2–300 ng/mL (correlation coefficient, r>0.9950) with the lower limit of quantification (LLOQ) as 0.2 ng/mL. The assay ranges of free ezetimibe and total ezetimibe were 0.2–200 and 0.5–500 ng/mL, respectively (r>0.9950) with the LLOQ as 0.2 and 0.5 ng/mL, respectively. The accuracy of the assay was within the range of 98.8–103.7% for atorvastatin and its metabolite, 95.0–105.9% for free ezetimibe, and 90.8–100.4% for total ezetimibe; the precision coefficients of variation were <6.9% for all analytes.

Non-compartmental analysis was performed using WinNonlin® Version 6.3 (Pharsight, Mountain View, CA, USA) to determine the following PK parameters of atorvastatin, 2-hydroxyatorvastatin, free ezetimibe, and total ezetimibe: the maximum plasma concentration at steady state (C_max,ss), time to reach C_max (T_max,ss), area under the concentration-time curve within a dosing interval at steady state (AUC_τ,ss), AUC from dosing time extrapolated to infinity at steady state (AUC_inf,ss), terminal elimination half-life (t_1/2), apparent clearance (CL_ss/F), and apparent volume of distribution (Vd_ss/F). Plasma drug concentration-time profiles are presented in linear and log-transformed scales. C_max,ss and T_max,ss were measured, and AUC was calculated by using the linear trapezoidal rule. The t_1/2 was calculated from the ratio of the natural logarithm of 2 and terminal elimination rate constant.

Safety was evaluated by monitoring adverse events (AEs), vital signs, physical examination, clinical laboratory tests (hematology, blood chemistry, urinalysis), and 12-lead electrocardiogram (ECG) throughout the study. All AEs reported by the subjects or observed by the investigators were recorded and analyzed to facilitate causality assessment.

Descriptive statistics were used to summarize baseline demographics, such as age, weight, height, and BMI. PK parameters and safety profiles were also evaluated using descriptive statistics. All statistical analyses were conducted by using SAS version 9.2 (SAS institute, Inc, Cary, NC, USA). Primary PK endpoints (AUC_τ,ss and C_max,ss) were log-transformed to develop a mixed effects model with period, sequence, and treatment as fixed effects and subjects nested within the sequence as a random effect. Geometric mean ratios (GMRs) with 90% confidence intervals (CIs) of the primary PK parameters between two treatment groups (C vs A, or C vs B) were estimated to evaluate PK drug interactions.

Of the 36 subjects enrolled in this study, 32 subjects completed all procedures and provided blood samples for PK analysis; three subjects discontinued the study in the 1^st period, and one subject dropped out in the 3^rd period due to personal requests. All enrolled subjects were male. The demographics of the subjects in each treatment sequence are presented in Table 1.

The mean plasma concentration-time profiles of atorvastatin and 2-hydroxyatorvastatin with or without ezetimibe coadministration are shown in Figure 2, and the calculated PK parameters are summarized in Table 2. The PK profiles of atorvastatin and its metabolite with or without ezetimibe co-administration were similar; their exposures, expressed as C_max,ss and AUC_τ,ss, were not significantly different regardless of ezetimibe co-administration. GMRs with 90% CIs of C_max,ss and AUC_τ,ss for atorvastatin when administered with and without ezetimibe were 1.1087 (0.9799–1.2544) and 1.1154 (1.0079–1.2344), respectively. The corresponding values of 2-hydroxyatorvastatin were 1.1147 (1.0197–1.2184) and 1.0853 (1.0239–1.1504).

The PK parameters and mean plasma concentration-time profiles of total and free ezetimibe with or without atorvastatin co-administration are presented in Table 3 and Figure 3. The T_max of co-administered free ezetimibe (with atorvastatin) was shortened for about 4 h compared with that of ezetimibe alone. GMRs (90% CIs) of C_max,ss and AUC_τ,ss for total ezetimibe when it was administered with and without atorvastatin were 1.0005 (0.9227–1.0849) and 1.0176 (0.9465–1.0941), respectively. The corresponding values for free ezetimibe were 1.7871 (1.5651–2.0406) and 1.2979 (1.1888–1.4170), respectively. The 90% CIs for the C_max,ss and AUC_τ,ss of total ezetimibe were within the range of 0.8–1.25 (Table 3).

In this study, 33 AEs were reported in 19 subjects. All cases were mild (30 events) or moderate (three events) and subjects recovered without complications. Commonly reported AEs were nasopharyngitis (four events) and epistaxis (four events). After causality assessment, 13 AE cases (seven subjects), including four in group A, three in group B, and six in group C, were considered as adverse drug reactions. No clinically significant differences were observed among the treatment groups in terms of vital signs, physical examination, 12-lead ECGs, and clinical laboratory test results.