Twenty-one healthy Korean male volunteers aged 20 to 45 years were enrolled in the study. Subjects underwent screening examinations including a medical history, physical examination, and laboratory tests (hematology, clinical chemistry, and urinalysis). Exclusion criteria included medically documented conditions, including cardiovascular, respiratory, renal, hepatic, or gastrointestinal disorders; a chronic disorder that might influence the absorption, disposition, metabolism, or excretion of valsartan; a history of disturbances of organ or acute illness; a history of hematologic disorder or blood donation within 2 months; a history of blood fraction donation within 1 month; a history of psychiatric disorder; a history of drug hypersensitivity; a history of alcohol or drug abuse; participation in another clinical trial within 3 months; consumption of over 5 glasses daily of beverages containing xanthine derivatives; smoking > 20 cigarettes daily; and use of any over-the-counter drug medication having the potential to affect the study results. The study purpose and procedures were explained before the study, and written informed consent was obtained from all subjects.

The study was a single group, two-sequence, open label, pre- and post-comparison, with a 1-week washout between study days. The study protocol was reviewed by the institutional review board of Kyung Hee University Hospital. All study procedures were conducted in accordance with the principles of the Declaration of Helsinki and the Korean Good Clinical Practice guidelines. Subjects were assigned to free of medication in the first period; in the second period, they received the valsartan 80 mg. In both periods, subjects were admitted to the Kyung Hee University Clinical Research Center at 5 p.m. on the day before administration of valsartan or study initiation. They received a standardized dinner, and no food was permitted after 8 p.m. The next day, subjects received no drug (baseline) or valsartan (treatment) along with 240 mL of tap water. Food and water were prohibited during the first 4 hours after dosing. At 4 hours after the oral administration, all subjects were given standardized meals. The subjects were not allowed to remain in a supine position or to sleep until 8 hours after the oral administration. Blood samples (7 mL) for valsartan concentration determination were obtained through a 22-guage indwelling catheter in a forearm vein or by direct venipuncture before dosing and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, and 24-hour after dosing in each period. Each sample was collected in heparinized tubes. The blood samples were centrifuged immediately (3,000 rpm, 10 minutes), and were frozen at -80℃ until UPLC-MS/MS analysis.

To determine the valsartan plasma concentration, ACQUITY UPLC (Waters, Milford, MA, USA)-QTRAP 5500 (AB SCIEX, Foster City, CA, USA) were used as UPLC-ESI-MS/MS method.11) The chromatographic separation was performed on a 2.1 × 50 mm, 3.5 µm xBridge C18 column (Waters) maintained at 25℃ using a mobile phase of methanol-10 mM ammonium acetate (50:50, v/v), and the separation was under isocratic conditions with a flow rate of 0.3 mL/min. Valsartan form m/z 434.2 (Q1) to m/z 179.1 (Q3) and valsartan-D3 (IS) m/z 437.2 (Q1) to m/z 179.1 (Q3) were detected by multiple reaction monitoring with electrospray interface in the negative ion mode. The stock solution of valsartan was prepared in acetonitrile and calibration curve was linear over range in a concentration of 5, 10, 50, 200, 1,000, and 5,000 ng/mL. Concentrations at 20, 500, and 4,000 ng/mL for valsartan was prepared as quality control samples. Internal standard solution of a concentration that 150 ng/mL was prepared in acetonitrile. Human plasma sample were thawed at room temperature, and 300 µL of internal standard solution was added to a 100 µL aliquot of plasma sample for precipitate proteins. One minute vortex mixing and 10 minutes at 14,000 rpm centrifuging were conducted, and the upper clear solution layer was collected and a 3 µL aliquot of solution was injected into the UPLC-MS/MS system.

BA-CALC 2008 program ver. 1.0.0 (Korea Food and Drug Administration, Cheongwon, Korea) that supplied by the KFDA was used to assessed individual pharmacokinetic parameters which include AUC_t, AUC_inf, C_max, and T_max.

Twenty four hours blood pressure changes were monitored by ABPM. ABPM can provide a 24-hour blood pressure profile and blood pressure information over 24-hour as well as during specific periods, such as daytime, nighttime, and early morning.7) The ABPM measurement was done according to "Guideline for blood pressure monitoring" established by Korean Society of Hypertension.8) Total, daytime and nighttime systolic blood pressure (SBP), diastolic blood pressure, and heart rate of each participant were automatically measured every 20 minutes during the 24 hours (8 AM to next day 8 AM) with a validated device (TM 2430 ambulatory blood pressure monitor; A&D Medical, San Jose, CA, USA). Subjects were studied by ABPM under baseline conditions at 1st period when they were free of medication and assessed the effects of valsartan at 2nd period.

The blood pressure patterns were classified as non-dipper, dipper, extreme-dipper, and reverse-dipper (Table 1). Subjects that diurnal blood pressure pattern changed from reverse dipper to non-dipper or dipper and changed from non-dipper to dipper or extreme dipper were assigned to responder group; subjects that have no change in diurnal blood pressure pattern were assigned to non-responder group; subjects that diurnal blood pressure pattern changed from non-dipper to reverse dipper and changed from dipper to non-dipper were assigned to reverse-responder group (Table 2).

Adverse events (AEs) were monitored throughout the study based on spontaneous reports by volunteers, questioning by investigators, and clinical examinations. The investigators assessed all clinical adverse effects in terms of intensity (mild, moderate, or severe), duration, outcome, and relationship to the study drug.

Pre- and post-dose blood pressure changes matched to pharmacokinetic parameters were analyzed by Pearson correlation analysis using SPSS ver. 20.0 (SPSS Inc., Chicago, IL, USA). Pharmacokinetic parameters were analyzed between responder, non-responder and reverse-responder groups according to pre- and post-dose changes in ABPM pattern. SPSS ver. 20.0 was used to conduct the one-way analysis of variance to compare the differences of mean AUC_t, AUC_inf, and C_max between three groups. All analysis were performed at an α level of 0.05.

Twenty-one healthy Korean male volunteers were participated. The mean (standard deviation, SD) age, height, and weight of subjects were 25.3 ± 3.5 years, 175.0 ± 3.9 cm, and 68.5 ± 7.8 kg, respectively. Twenty-four hours blood pressure monitoring data was summarized in Table 3.

The ABPM results and pharmacokinetic parameters of each individual were summarized in Table 3. Although all subjects were participated entire trial, ABPM data of post-dose from three subjects (R02, R07, and R16) was missed for various reasons. After finished 2nd period, 15 subjects showed decrease in mean SBP and 3 subjects showed increase in mean SBP. To associate pharmacokinetic parameters and changes in ABPM data, Pearson correlation analysis was conducted between ABPM data and pharmacokinetic parameters(C_max, AUC_t, and AUC_inf). There were no significant associations between changes of ABPM data and pharmacokinetic parameters (Table 4).

The ABPM patterns and pharmacokinetic parameters of each individual were summarized in Table 5. Ten, four, and seven subjects showed dipper, non-dipper, and reverse dipper pattern in 1st period, respectively. After finished 2nd period, eleven, one, and four subjects showed dipper, non-dipper, and reverse dipper pattern in 1st period, respectively. The mean AUC_t (mean ± SD), total amount of absorbed valsartan was 25,808 ± 6,863.0 ng·hr/mL, 20,683 ± 8,782.7 ng·hr/mL, and 12,502 ± 5,566.6 ng·hr/mL in responder, non-responder, and reverse responder, respectively. There were significant differences between mean AUC_t of three groups (p = 0.041), and mean AUC_t of responder group was significant higher than reverse group (p = 0.035, Turkey honestly significant difference [HSD]). The mean AUC_inf (mean ± SD), total amount of absorbed valsartan was 26,710 ± 7,024.4 ng·hr/mL, 22,895 ± 8,344.0 ng·hr/mL, and 12,942 ± 5,589.2 ng·hr/mL in responder, non-responder, and reverse responder, respectively. There were significant differences between mean AUC_inf of three groups (p = 0.025), and mean AUC_t of responder group was significant higher than reverse group (p = 0.025, Turkey HSD). C_max was 4,314 ± 1,522.6 ng/mL, 2,588 ± 1,273.9 ng/mL, and 2,056 ± 1,075.5 ng/mL in responder, non-responder, and reverse responder, respectively. There were significant differences between mean C_max of three groups (p = 0.034), and mean C_max of responder group was significant higher than reverse group (p = 0.037, Turkey HSD). In Tmax, time required to reach maximum plasma concentration was 3 ± 0.8 hours, 4 ± 0.7 hours, and 3 ± 1.3 hours in responder, non-responder, and reverse responder, respectively. There were no significant differences between mean T_max of three group (p = 0.239). The mean plasma concentration versus time profiles of valsartan in responder, non-responder, and reverse-responder group were shown in Fig. 1.

No serious AEs were reported, and no subjects discontinued the study due to AEs. But three subjects were failure to obtain ABPM data at the second period, and total 18 subjects were enrolled the statistical analysis.