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Introduction
Hypertension is a major risk factor for cardiovascular diseases, so blood pressure (BP) control is important in preventing relevant complications.[1] According to the hypertension management guideline of the Korean Society of Hypertension, if the systolic blood pressure (SBP)/diastolic blood pressure (DBP) exceeds 160/100 mmHg, or is 20/10 mmHg higher than the target BP, combination therapy with two antihypertensive agents of different classes is recommended.[2] Especially, it is well known that the concomitant use of a calcium channel blocker (CCB) with an angiotensin II receptor blocker (ARB) is more effective than doubling the dose of one single drug.[3456]
Fimasartan, an ARB, is rapidly absorbed, reaching its maximum plasma concentration (Cmax) in 0.5–3.0 hours, and has a terminal elimination half-life of 9.0–16.0 hours. More than 90% of fimasartan in the plasma presents as the parent drug, and its relatively very small portion undergoes metabolism, mainly by CYP3A4.[7] Additionally, fimasartan is known as a highly variable drug (HVD), in that its intra-subject variability for Cmax is larger than 30%.[8910] Amlodipine, a CCB, reaches at Cmax within 6.0–8.0 hours and has a terminal elimination half-life of 40–60 hours,[11] and it is extensively metabolized by CYP3A4.[12] In a previous drug-drug interaction study, there was no clinically relevant pharmacokinetic (PK) interaction between fimasartan and amlodipine.[7]
Fixed-dose combination (FDC) is known to improve patients' compliance and reduce medical costs, and it may be more effective in controlling BP in some patients.[13141516] Referring to these points, an FDC tablet of fimasartan/amlodipine 60/10 mg was developed by Boryung Pharmaceutical Co., Ltd. (Seoul, Republic of Korea).
According to the bioequivalence study guidelines of the regulatory agencies, including the Korea Ministry of Food and Drug Safety (MFDS), a replicated crossover design can be used for bioequivalence studies of an HVD, and a widened bioequivalence range can be accepted.[171819] Since fimasartan is an HVD, a full or partial replicated crossover design can be selected for the bioequivalence study between FDC of fimasartan/amlodipine 60/10 mg and the corresponding loose combination.
The aim of this study was to compare the PK characteristics and evaluate the bioequivalence between FDC of fimasartan/amlodipine 60/10 mg and the corresponding loose combination in healthy male subjects.
Methods
Subjects and study design
The study protocol was approved by the institutional review board of Seoul National University Hospital (Seoul, Republic of Korea) and MFDS (NCT02920047). All the procedures were performed in compliance with the Korean Good Clinical Practice guidelines and tenets of the Declaration of Helsinki. All the subjects provided written informed consent prior to any procedures related to the study.
This study included healthy male subjects between 19 and 50 years of age, weighing ≥ 55 kg and with a body mass index ranging from 18.0 to 27.0 kg/m2. All subjects had no clinically significant abnormalities based on their medical histories, vital signs, physical examination, clinical laboratory tests, and 12-lead electrocardiogram (ECG). Subjects with any hypersensitivity to drugs such as fimasartan and amlodipine were excluded from the study. Additionally, subjects having SBP ≤ 100 mmHg or ≥ 140 mmHg, or DBP ≤ 65 mmHg or ≥ 90 mmHg were excluded from the study at the screening.
This study was designed as a randomized, open-label, two-treatment, three-period, three-sequence, partial replicated crossover study with 14-days washout between periods. The enrolled subjects were randomly assigned to one of the three sequences, and received a single oral dose of an FDC tablet of fimasartan/amlodipine 60/10 mg (Boryung Pharmaceutical Co., Ltd., Seoul, Republic of Korea) as the test drug, or a loose combination of fimasartan 60 mg (Kanarb® tablet 60 mg, Boryung Pharmaceutical Co., Ltd.) and amlodipine 10 mg (Norvasc® tablet 10 mg, Pfizer Inc., Seoul, Republic of Korea) as the reference drug in each period. Each sequence consisted of a single oral administration of the test drug in one period and the reference drug in the other two periods (Sequence A: Reference → Reference → Test; Sequence B: Reference → Test → Reference; Sequence C: Test → Reference → Reference).
Blood samples for PK analysis of fimasartan were collected at 0 (pre-dose), 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 12, 24, and 48 h post-dose. For amlodipine, blood samples were collected at 0 (pre-dose), 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 48, 72, 96, and 144 h post-dose. Approximately 5 or 8 mL of the blood sample was collected in a heparinized tube for each blood sampling point and subsequently centrifuged at 3,000 rpm for 10 minutes at 4℃. The supernatants were then transferred to three Eppendorf tubes and stored at −70℃ until analysis.
Determination of plasma fimasartan and amlodipine concentrations
Plasma concentrations of fimasartan and amlodipine were analyzed at Kyung Hee Drug Analysis Center of Kyung Hee University (Seoul, Republic of Korea).
Plasma concentrations of fimasartan were determined by a validated high-performance liquid chromatography (HPLC, Agilent 1200 series, Agilent Technologies, USA) coupled with tandem mass spectrometry method (MS/MS, The Applied Biosystems MDS SCIEX API 4000 triple quadrupole mass spectrometer, Applied Biosystems, Canada). In the HPLC system, a Luna C18 column (50 × 2.0 mm, 3.0 µm, Phenomenex, USA) was used for the chromatographic separation of fimasartan and BR-A-563 (Internal standard; IS) under gradient conditions. The MS/MS system was operated in the ionization mode using positive ion electrospray and the multiple reaction monitoring (MRM) mode. The MRM mode was monitored based on an m/z transition of 502.4 → 207.1 for fimasartan and 526.5 → 207.2 for BR-A-563 (IS).
Plasma concentrations of amlodipine were determined by a validated HPLC (Agilent 1100 series, Agilent Technologies, USA) coupled with MS/MS method (The Applied Biosystems MDS SCIEX API 2000 triple quadrupole mass spectrometer, Applied Biosystems, Canada). A Luna C18 column (50 × 2.0 mm, 3.0 µm, Phenomenex, USA) was used for the chromatographic separation of amlodipine and amlodipine-d-4 (IS) under gradient conditions. The MS/MS system was operated in the ionization mode using positive ion electrospray and the MRM mode. The MRM mode was monitored based on an m/z transition of 409.0 → 238.0 for amlodipine and 413.1 → 238.0 for amlodipine-d-4 (IS).
The calibration curves were linear in the range of 1–1000 ng/mL for fimasartan and 0.2–20 ng/mL for amlodipine (r2 ≥ 0.9955 for fimasartan and ≥ 0.9983 for amlodipine). The intra- and inter-batch accuracy ranges were 90.2–106.3% and 99.8–100.6% for fimasartan, and 93.1–105.3% and 99.2–100.0% for amlodipine, respectively. The intra- and inter-batch precision coefficient of variation (CV)% were < 10.0% and < 5.2% for fimasartan, and < 5.5% and < 4.3% for amlodipine, respectively.
PK analysis
The following PK parameters were calculated by noncompartmental methods using WinNonlin® software, version 7.1 (Pharsight, Mountain View, CA, USA). Cmax and time to reach Cmax (Tmax) were determined from the observed plasma concentration-time profiles. The area under the concentration-time curve from 0 to last measurable time point (AUClast) was calculated using the linear trapezoidal rule for the ascending concentrations and log trapezoidal rule for the descending concentrations. The area under the concentration-time curve from 0 to infinity (AUCinf) was calculated using the following formula: AUCinf = AUClast + Clast/λz, where Clast is the last measurable concentration, and λz is the terminal elimination rate constant. The terminal elimination half-life (t1/2) was calculated as 0.693/λz.
Statistical analysis
A minimum sample size of 45 subjects was estimated to achieve the widened bioequivalence range of the HVD with 80% statistical power at a 5% level of significance, assuming that the highest intra-subject variability of fimasartan was 62%.[1719] After considering the dropout rate, the total number of 60 subjects were chosen to enroll in this study.
The statistical analyses were performed using SAS® software version 9.4 (SAS Institute, Cary, NC, USA). Analysis of variance (ANOVA) was performed to compare the treatments, considering period, sequence, and the group as fixed effects, and subject nested within the sequence as a random effect. Geometric mean ratios (GMRs) and its 90% confidence intervals (CIs) for Cmax and AUClast variables were estimated. The scaled bioequivalence criteria for the Cmax of fimasartan was calculated using exp [±0.760*(SWR)], where SWR is the intra-subject standard deviation of the log-transformed values of Cmax of the reference drug estimated by this study results.[1719] The bioequivalence between the two treatments was assessed by using the scaled bioequivalence criteria for the Cmax of fimasartan and the conventional bioequivalence criteria for the other PK variables of fimasartan and amlodipine. We used Cochran's Q test to evaluate whether the incidence of adverse events (AEs) is different between the treatments.
Blood pressure monitoring
SBP and DBP were measured at 0 (pre-dose), 4, 8, 12, 24, 48, 72, 96, and 144 h post-dose in each period.
Safety and tolerability assessments
Safety and tolerability were evaluated by AE monitoring, clinical laboratory tests, 12-lead ECG, physical examination, and vital signs. All the AEs were coded according to the Medical Dictionary for Regulatory Activities ver.19.1 and summarized by treatment, severity, and relationships with treatments.
Results
Demographic characteristics
A total of 60 healthy Korean male subjects were enrolled and randomized, and 57 subjects completed the study since three subjects withdrew their consent before the second period. Age, height, weight, and body mass index of the enrolled subjects were 30.6 ± 6.6 (mean ± standard deviation) years, 173.1 ± 5.5 cm, 70.4 ± 7.6 kg, and 23.5 ± 2.0 kg/m2, respectively. There was no significant difference among the sequences in demographic characteristics. Safety and tolerability were assessed in all the enrolled subjects, and the PK characteristics were analyzed in 56 subjects who had completed the study without major deviation; one subject was excluded from the PK evaluation due to inclusion criteria violation, whose weight was 54 kg at the screening.
Pharmacokinetics
The mean plasma concentration-time profiles and PK characteristics of fimasartan were similar between the FDC and loose combination (Fig. 1A, Table 1). For fimasartan, the majority of the subjects exhibited double-peak plasma concentration-time profiles. Unlike the individual AUClast values of fimasartan, the individual Cmax values of fimasartan were highly variable between two treatments (Fig. 2A, 2B). The GMRs (90% CIs) of the FDC to the loose combination for Cmax and AUClast of fimasartan were 1.0440 (0.9202–1.1844) and 1.0412 (0.9775–1.1090), respectively. Since the intra-subject CV% for the Cmax of fimasartan was 48.51%, the expanded bioequivalence range for the Cmax of fimasartan was 0.7051–1.4182.[1719] The GMR and its 90% CI for Cmax of fimasartan fell not only within the expended bioequivalence range but also within the conventional bioequivalence criteria of 0.80–1.25. The corresponding values for the AUClast of fimasartan were also included in the conventional bioequivalence criteria (Table 2).
The mean plasma concentration-time profiles and PK parameters of amlodipine were comparable between the FDC and loose combination (Fig. 1B, Table 1). The individual values of Cmax and AUClast of amlodipine showed no significant variations between the two treatments (Fig. 2C, 2D). The GMRs (90% CIs) of the FDC to the loose combination for Cmax and AUClast of amlodipine were 1.0430 (1.0156–1.0711) and 1.0339 (1.0055–1.0631), respectively. All the GMRs and their 90% CIs for Cmax and AUClast of amlodipine were within the conventional bioequivalence criteria of 0.80–1.25 (Table 2).
Effect on blood pressure
The reductions in SBP and DBP were similar between the FDC and loose combination (Fig. 3); In the FDC and loose combination groups, the lowest mean ± standard deviation values of SBP were 101.7 ± 8.9 mmHg and 101.4 ± 8.8 mmHg, respectively, and the corresponding values of DBP were 56.8 ± 5.9 mmHg and 57.1 ± 6.0 mmHg, respectively.
Safety and tolerability assessments
No clinically significant changes were observed in clinical laboratory tests, 12-lead ECG, physical examination, and vital signs. During the study, a total of 44 treatment-emergent AEs (TEAEs) were reported in 24 subjects. Among them, 15 TEAEs occurred in 11 subjects who received the FDC, and 29 TEAEs occurred in 18 subjects who received the loose combination. There was no significant difference in the TEAEs between the two treatments (p-value = 0.8338).
Discussion
This study compared the PK properties and evaluated the bioequivalence between FDC of fimasartan/amlodipine 60/10 mg and the corresponding loose combination. The FDC and loose combination showed similar PK characteristics in healthy male subjects. The GMR and its 90% CI for Cmax of fimasartan were included in the scaled bioequivalence criteria, which was 0.7051–1.4182. Also, the GMRs and their 90% CIs for the other PK variables of drugs were within the conventional bioequivalence criteria of 0.80–1.25. These results indicated that the FDC was bioequivalent to the loose combination when administered to healthy male subjects.
Conventional 2 × 2 crossover bioequivalence studies with HVDs have the disadvantage of requiring large sample sizes for attaining sufficient statistical power. According to the guidelines of the regulatory agencies, a replicated crossover design can be used for bioequivalence studies with HVDs,[171819] and it is helpful for reducing the number of subjects needed to demonstrate bioequivalence by up to about 50%.[20] Based on the highest observed intra-subject CV% for the Cmax of fimasartan (62%), approximately 114 subjects would be required for detecting a 20% difference between the two treatments with 80% statistical power at a 5% level of significance under the conventional 2 × 2 crossover design, while this study could reduce the number of subjects by up to 60 subjects through the partial replicated crossover design by widening the bioequivalence range. Using the intra-subject CV% for the Cmax of fimasartan calculated in this study (48.51%), about 57 subjects are enough to achieve the conventional bioequivalence criteria with 80% statistical power at a 5% level of significance under partial replicated design. Therefore, a sample size of 60 subjects chosen in this study was sufficient to assess the conventional bioequivalence as well as the scaled bioequivalence through partial replicated design between the FDC of fimasartan/amlodipine 60/10 mg and the corresponding loose combination.
Although the subjects had normal BP, BP was monitored as a safety assessment. After a single administration of the FDC or the loose combination, the maximal decreases in SBP/DBP were 11.41/12.12 mmHg and 12.56/12.21 mmHg, respectively. Although BP evaluation was not the primary aim of this study, these results suggest that FDC of fimasartan/amlodipine 60/10 mg will show similar BP-lowering effects compared to the corresponding loose combination.
In conclusion, FDC of fimasartan/amlodipine 60/10 mg showed similar PK profiles with the corresponding loose combination. The GMRs and their 90% CIs for Cmax and AUClast of fimasartan and amlodipine fell not only within the scaled bioequivalence criteria but also within the conventional bioequivalence criteria, indicating the bioequivalence between the FDC and loose combination.
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