Key words
atenolol - bioequivalence study - volunteers
Introduction
Atenolol is a selective β1 receptor antagonist without intrinsic partial agonist or membrane-stabilizing activities.
Atenolol is commercially available as a racemic mixture. The (–)S-form is the active
isomer, whereas the R(+)-isomer has no significant pharmacological activity [1]
[2]. Importantly, it is widely used to treat hypertension, angina acute myocardial infarction,
supraventricular tachycardia, ventricular tachycardia, and congestive heart failure
[3].
The onset of atenolol starts 2–4 h after oral administration. The absorption of atenolol
has been shown to be rapid and consistent but incomplete, with a bioavailability of
50% [4]. Approximately 6–16% of atenolol is bound to plasma protein [3], and very little atenolol is metabolized by the liver. Instead, approximately 40–50%
of non-metabolized atenolol is excreted in urine after administration [3]
[5].
The objective of this study was to compare the pharmacokinetics and bioequivalence
of 50 mg atenolol test drug manufactured by Hana Pharmaceuticals (Seoul, Korea; Hana
Atenolol®) with 50 mg atenolol reference drug manufactured by Hyundai Pharmaceuticals (Seoul,
Korea; Tenormin®) in 24 healthy Korean male volunteers.
Materials and Methods
Test and reference medications
The test medication (Hana Atenolol, 50 mg, lot No. 6003, Hana Pharmaceuticals) and
the reference medication (Hyundai Tenormin, 50 mg, lot No. 66042, Hyundai Pharmaceuticals)
were supplied as tablets.
Subjects and methods
This study was a single-dose, randomized, open-label, 2 period crossover study. The
study was conducted at Bestian Hospital in Seoul, Korea. The protocol of this study
was approved by the Research Institute of Pharmaceutical Sciences, College of Pharmacy,
Seoul National University (Seoul, Korea) and performed according to the rules of Good
Clinical Practice and in accordance with the Declaration of Hensinki. All participants
provided a written informed consent after they had been informed of the nature and
details of the study in accordance with Korean Guidelines for Bioequivalence Tests
[KGBT 2006].
24 healthy male Korean volunteers, ranging in age from 20 to 28 years (median: 24),
weight from 58 to 95 kg (71.0±9.15 kg), and height from 168 to 183 cm (176±4.17 cm)
completed the study. Volunteers were selected after passing a clinical screening procedure.
The screen consisted of a physical examination and laboratory tests, including a blood
analysis of hemoglobin, hematocrit, white blood cell (WBC), platelets, differential
counting of WBC, blood urea nitrogen, serum creatinine, total protein, albumin, alanine
aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, cholesterol,
glucose fasting, and alkaline phosphatase. In addition, a urine analysis was conducted
for specific gravity, color, pH, glucose, protein, bilirubin, red blood cells (RBC),
and WBC assessment. Volunteers were excluded if they were possibly sensitive to this
type of medication, had a history of any illness of hepatic, renal, or cardiovascular
systems, or had taken alcohol or other medications that may affect drug metabolism
over a long period of time. These exclusion criteria were used to ensure that any
illness or other medication would not induce variation in assessment. All subjects
avoided using other drugs for at least 1 month before the study and until study completion.
Patients were also required to refrain from consuming alcoholic beverages and xanthine-containing
foods and beverages 48 h prior to each dosing and until collection of the last blood
sample. The volunteers were randomly assigned to 2 groups and received an oral dose
of 50 mg of atenolol in a standard 2×2 crossover model. A 1 week washout period was
included between doses.
Subjects were notified at 8 p.m. on the day prior to the study initiation and fasted
12 h before drug administration and 4 h after. The brachial vein was cannulated with
a heparin lock catheter and 1 mL heparinized normal saline solution (20 units/mL)
was flushed into the cannula to prevent blood clots from forming prior to administration.
At 9:00 a.m. on the day of the study, the subjects were given 50 mg atenolol per oral
(p. o.) with 240 mL of tap water. At 4 and 9 h after the administration, all subjects
were given standardized meals. The subjects were not allowed to be in a supine position
or sleep for any period of time during the entire blood collection period. Approximately
10 mL of blood was collected through the cannula at the following times: predose,
30 min, and 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, and 24 h after administration of atenolol.
The heparin lock was flushed with 1 mL heparinized normal saline after each blood
sampling. The blood sample was immediately centrifuged after collection and the plasma
sample was frozen at − 70°C for subsequent high performance liquid chromatography
(HPLC) analysis.
HPLC assay of atenolol in plasma
The concentrations of atenolol in plasma were analyzed by HPLC using a slight modification
[4]. A 50 μL aliquot of internal standard (7.5 μg/mL metoprolol), 500 μL aliquot of
0.5 N NaOH, and 5 mL aliquot of ether:dichloromethane (70:30 v/v) were added to a
1 mL aliquot of plasma sample. After 10 min of extraction, the tubes were centrifuged
at 5 400×g for 10 min. The organic layer was then collected and dried under a gentle
stream of nitrogen gas at 40 °C. A 125 μL aliquot of the mobile phase was added to
reconstitute the residue and a 50 μL aliquot was injected directly onto a reverse
phase HPLC column. The mobile phase consisted of acetonitrile:methanol:10 mM KH2PO4 (pH 3.0) (15:15:70, v/v) and was processed at a flow rate of 1.0 mL/min. The column
effluent was monitored using a fluorescence detector with excitation and emission
wavelengths set at 222 nm and 300 nm, respectively.
Pharmacokinetic analysis
Standard methods [6] were used to calculate the pharmacokinetic parameters using a non-compartmental
analysis (WinNonlin®; Pharsight Corporation, Mountain View, CA). The Cmax and Tmax were compiled from the concentration-time data. The area under the curve (AUC)0–24 h was calculated using a linear-log trapezoidal formula up to the last measured time
at 24 h in plasma, and the AUC0–∞ was calculated by extrapolation [7]. The terminal elimination rate constant, ke, was defined as the terminal elimination rate constant estimated by log-linear regression
analysis on data visually assessed to be a terminal log-linear phase. The apparent
terminal elimination t1/2 was calculated as follows:·
t1/2=0.693/ke
Statistical analysis of data
Analysis of variance (ANOVA) was performed using logarithmically transformed AUC0–24 h, and Cmax was used to assess group or sequence, period, subjects per group, and drug effects.
The Schuirmann’s 3-sided t-tests were conducted to test the bioequivalence of the pharmacokinetic characteristics
of the 2 formulations [8]. The range of bioequivalence for parametric analysis was set to the commonly accepted
80–125% of the test/reference ratios of AUC0–24 h and Cmax according to Korean Guidelines for Bioequivalence Tests [KGBT 2006]. All statistical
comparisons were made using the Equiv test and confirmed with the K-BE test program
[9].
Results
Both atenolol formulations were well-tolerated by the subjects of this study. Clinically
relevant drug-related side effects were not observed in any of the 24 volunteers,
and no volunteer withdrew from the study.
A representative chromatogram of blank plasma spiked with internal standard (metoprolol)
and the chromatogram of a plasma sample are shown in [Fig. 1]. In this HPLC method, no interference from endogenous substances was observed in
human plasma. This method, with slight modification, was able to determine the atenolol
plasma concentration within 5 min for each plasma sample. The retention times for
atenolol and internal standard were approximately 0.7 and 2.8 min, respectively. The
detection limit for atenolol in human plasma was 20 ng/mL based on a signal-to-noise
ratio of 10. The standard curve was achieved from the least squared regression equation:
y=0.0007x + 0.0068 (r2=0.9983). The intra- and inter-day precision coefficients of variation for human plasma
were 3.839–9.274% and 7.475–10.19%, respectively, and the intra-and inter-day accuracy
coefficients of variation for human plasma were 95.66–105.2% and 97.91–108.8%, respectively,
from atenolol plasma concentrations in the range of 20–1 000 ng/mL.
Fig. 1 Chromatogram obtained from plasma of a volunteer following oral administration of
atenolol (50 mg): atenolol (370.3 ng/mL) + internal standard (propranolol 7.5 μg/mL).
The mean plasma atenolol concentration-time profiles are shown in [Fig. 2]. We found that the mean plasma atenolol concentration profiles of the 2 formulations
were almost identical ([Fig. 2]). The mean extrapolated section of AUC from the last sampling time to time infinity
was 14.78%. The mean pharmacokinetic parameters, such as Cmax, Tmax, AUC0–24 h, AUC0–∞, ke, and t1/2 after the administration of test and reference atenolol formulations are listed in
[Table 1]. The 90% confidence intervals of AUC0–24 h and Cmax are presented in [Table 2]. The parametric point estimates for the mean ratios of test drug/reference drugs
for AUC0–24 h and Cmax were 1.026 and 1.048, respectively ([Table 2]). The 90% confidence intervals for AUC0–24 h and Cmax were 0.9037 ~ 1.166 and 0.9169 ~ 1.1987, respectively ([Table 2]). The test/reference ratio of Tmax was 0.8860. The arithmetic mean±standard deviation of Tmax for the test and reference drugs was 1.794 ~ 3.706 and 2.051 ~ 4.157 h, respectively
([Table 1]).
Fig. 2 Plasma concentration-time curve of test drug and reference drug of atenolol 50 mg
(n=24, Bars represent standard deviation).
Table 1 Pharmacokinetic parameters of atenolol 50 mg after a single oral dose of test and
reference formulations of atenolol 50 mg tablets in healthy Korean male volunteers.
|
Parameter
|
Test drug*
|
Reference drug**
|
|
Data are expressed as the mean(standard deviation(SD))
|
|
*atenolol 50 mg test drug manufactured by Hana Pharmaceuticals (Seoul, Korea) (Hana
Atenolol®); **atenolol 50 mg reference drug manufactured by Hyundai Pharmaceuticals (Seoul,
Korea) (Tenormin®) in 24 healthy Korean male volunteers
|
|
Cmax (ng/mL)
|
268.4 (78.96)
|
256.9 (79.34)
|
|
Tmax (h)
|
2.750 (0.9555)
|
3.104 (1.053)
|
|
AUC0–24h (ng/mL×h)
|
1 981 (729.2)
|
1 872 (604.8)
|
|
AUC0–∞ (ng/mL×h)
|
2 228 (697.1)
|
2 187 (628.5)
|
|
ke (h − 1)
|
0.1332 (0.02748)
|
0.1421 (0.04223)
|
|
t1/2 (h)
|
5.419 (1.110)
|
5.442 (2.357)
|
Table 2 Standard bioequivalence analysis for log-transformed AUC0–24h (ng/mL×h) and log-transformed Cmax (ng/mL) based on multiplicative model after a single-dose administration of two 50 mg
atenolol tablet formulations in healthy Korean male volunteers. AUC0–24 h (ng/mL×h) and Cmax (ng/mL) were also analyzed based on additive model for completeness.
|
Multiplicative statistical method
|
Test/Reference Point estimate
|
90% Confidence Interval
|
|
AUC0–24 h
(ng/mL×h)
|
1.026
|
0.9037–1.166
|
|
Cmax
(ng/mL)
|
1.048
|
0.9169–1.1987
|
Discussion
In this study, we slightly modified the HPLC method described in a previous study
to determine plasma atenolol levels in healthy volunteers [4]. The mobile phase, which consisted of acetonitrile:methanol:0.02 M sodium phosphate
buffer containing 0.1% sodium dodecyl sulfate (SDS) 35:15:50 (v/v), was changed to
acetonitrile:methanol:10 mM KH2PO4 (pH 3.0) (15:15:70, v/v). Using this modified method, the retention times of atenolol
and metoprolol became much shorter from 5.5 and 8 min to 0.7 and 2.8 min, respectively.
De Abreu et al. [5] demonstrated that although only 500 μL of plasma per sample was necessary, the retention
times remained 3.1 and 6 min for atenolol and metanolol, respectively. However, in
this study, the chromatographic run time per sample was only 5 min. In addition, our
analysis of atenolol was very precise, effective, and economical, since retention
times were shorter and the assay did not require solid-phase extractions.
The pharmacokinetic parameters, including Cmax, Tmax, AUC0–24 h, AUC0–∞, ke, and t1/2 were comparable between the 2 atenolol formulations and no statistical difference
was found. Importantly, our findings in this study were similar to data previously
reported [4]
[5]
[10].
The ANOVA test showed that the F-test values were lower than the F-test table in the
group or sequence, period, and drug. These results indicate that the crossover design
was properly performed and there were no group or sequence and drug effects. The AUC0–24 h, Cmax, and Tmax were similar for both test and reference drugs. In addition, the 90% confidence intervals
of AUC0–24 h and Cmax were within the accepted bioequivalence range of 0.80–1.25, which satisfied the bioequivalence
criteria of the European Committee for Proprietary Medicinal Products and the US Food
and Drug Administration guidelines.
In conclusion, the results from this study indicate that the 2 50 mg atenolol formulations
are bioequivalent. Therefore, they may be prescribed interchangeably.
