Artikel Penelitian

Bioequivalence Study of Two

Amlodipine Tablet Formulations

Effi Setiawati, Sukmayadi, Danang Agung Yunaidi, Lucia Rat Handayani,

Gunawan Harinanto, Iwan Dwi Santoso, Siti Hawa Deniati, Asri Purnomo

PT Equilab International, Bioavailability and Bioequivalence Laboratory, Jakarta

Abstract: The present study was conducted to find out whether the bioavailability of amlodipine
besylate tablet equivalent to 10 mg amlodipine tablet (Lopiten) produced by PT Guardian
Pharmatama was equivalent to that produced by the innovator. This was a cross-over, random-
ized, single-blind study which included 12 adult male and female volunteers. The participating
volunteers were required to have an overnight fast and in the next morning were given orally 1
tablet of the test drug (Lopiten, produced by PT Guardian Pharmatama) or 1 tablet of the
reference drug (Norvask, produced by the innovator). Blood samples were drawn immediately
before taking the drug (control), and at 3, 5, 6, 7, 8, 9, 10, 11, 12, 15, 24, 48, 96, and 144 hours
after drug administration. Two weeks after the first drug administration (washout period), the
procedure was repeated using the alternate drug. Plasma concentrations of the drug were
determined by high performance liquid chromatographic method with mass spectrometer detec-
tor (LC-MS). Using Wilcoxon matched-pairs test on the original data, there was no statistically
significant difference found between the test and the reference drug products for tmax values. It
was concluded that the amlodipine besylate tablet equivalent to 10 mg amlodipine tablet
(Lopiten) produced by PT Guardian Pharmatama was bioequivalent to that produced by the
innovator.
Keywords: amlodipine liquid chromatography mass spectrometer bioequivalent

Maj Kedokt Indon, Volum: 58, Nomor: 2, Pebruari 2008 41

Bioequivalence Study of Two Amlodipine Tablet Formulations

Uji Bioecivalensi Dua Formula Tablet Amlodipine

Effi Setiawati, Sukmayadi, Danang Agung Yunaidi, Lucia Rat Handayani,

Gunawan Harinanto, Iwan Dwi Santoso, Siti Hawa Deniati, Asri Purnomo

PT Equilab International, Bioavailability and Bioequivalence Laboratory, Jakarta

Abstrak: Penelitian ini dilakukan untuk mengetahui apakah bioavailabilitas tablet amlodipine
besylate yang setara dengan tablet amlodipine 10 mg (Lopiten) produksi PT Guardian
Pharmatama sebanding dengan bioavailabilitas produk yang sama yang dibuat oleh pabrik
inovatornya. Penelitian ini menggunakan desain menyilang, acak, dan tersamar tunggal yang
mengikutsertakan 12 sukarelawan laki-laki dan perempuan dewasa. Sukarelawan dipuasakan
semalam dan keesokan harinya diberi 1 tablet obat uji (Lopiten, produk PT Guardian
Pharmatama) atau 1 tablet obat pembanding (Norvask, produk inovator) per oral. Contoh
darah diambil pada saat sebelum minum obat (kontrol), dan pada 3, 5, 6, 7, 8, 9, 10, 11, 12, 15,
24, 48, 96, dan 144 jam setelah minum obat. Dua minggu setelah pemberian obat pertama
(periode washout), prosedur yang sama diulang dengan memberikan obat pembandingnya.
Kadar obat ditentukan secara kromatografi cair dengan detektor spektrometer massa (LC-MS).
Dengan menggunakan uji Wilcoxon berpasangan, tidak ditemukan perbedaan yang bermakna
secara statistik antara nilai tmax dari obat uji dan obat pembanding. Disimpulkan bahwa tablet
amlodipine besylate yang setara dengan tablet amlodipine 10 mg (Lopiten) produksi PT Guard-
ian Pharmatama bioekuivalen dengan produk yang sama yang dibuat oleh innovator.
Kata kunci: amlodipine kromatografi cair spektrometer massa bioekivalen

Introduction Amlodipine is a second generation dihydropyridine

Amlodipine besylate is a besylate salt of amlodipine, a
long-acting calcium channel blocker. Amlodipine besylate
(CAS 111470-99-6) is chemically described as (RS)3-ethyl-5-
methyl-2-(2-aminoethoxymethyl)4-(2-chlorophenyl)-1,4-
dihydro-6-methyl-3,5-pyridine carboxylate benzenesulpho-
nate. Its empirical formula is C 20H25ClN 2O 5.C 6H6O 3S.
Amlodipine besylate is a white crystalline powder with a
molecular weight of 567.1. It is slightly soluble in water and
sparingly soluble in ethanol. The structural formula is:
H Amlodipine is a peripheral arterial vasodilator that acts on
H3C N CH2OCH 2CH 2NH2
CH3OOC COOC2H5
H .C6H5S O 3H
Cl
Figure 1. Molecular Structure of Amlodipine Besylate
calcium antagonist. It is used as an anti-hypertensive and
anti-anginal agent. Amlodipine is a dihydropyridine calcium
antagonist (or calcium-channel blocker) that inhibits the trans-
membrane influx of calcium ions into vascular smooth muscle
and cardiac muscle. Experimental data suggest that amlodipine
binds to both dihydropyridine and nondihydropyridine bind-
ing sites. The contractile processes of cardiac muscle and
vascular smooth muscle are dependent upon the movement
of extracellular calcium ions into these cells through specific
ion channels. Amlodipine inhibits calcium ion influx across
these cell membranes selectively, with a greater effect on
vascular smooth muscle cells than on cardiac muscle cells.
vascular smooth muscle to cause a reduction in peripheral
vascular resistance and consequently a reduction in blood
pressure.1
The most common side effects caused by Ca2+-channel
antagonists, particularly the hydropyridines, are due to ex-
cessive vasodilation. These effects may be expressed as diz-
ziness, hypotension, headache, flushing, and ankle edema1.
In general, treatment with amlodipine was well-tolerated
at doses up to 10 mg daily. Most adverse reactions reported
during therapy were of mild or moderate severity. In con-
trolled clinical trials directly comparing amlodipine (N=1730)
in doses up to 10 mg to placebo (N=1250), discontinuation of

42 Maj Kedokt Indon, Volum: 58, Nomor: 2, Pebruari 2008

 Bioequivalence Study of Two Amlodipine Tablet Formulations
amlodipine due to adverse reactions was required in only
about 1.5% of patients and was not significantly different
from placebo (about 1%). The most common side effects are
headache and edema. For several adverse experiences that
appear to be drug- and dose-related, there was a greater
incidence in women than in men associated with amlodipine
treatment.2
Following oral administration, amlodipine is slowly ab-
sorbed. Its peak plasma concentration is reached between 6
and 12 hours. The drug has a high oral bioavailability (ap-
proximately 64-90%) which is not altered by the presence of
food. Amlodipine is extensively (about 90%) converted to
inactive metabolites via hepatic metabolism with 10% parent
compound and 60% of the metabolites excreted in the urine.2
The maximum concentration (Cmax) and the area under the
concentration versus time curve extrapolated to infinite time
(AUC0- ) of amlodipine in serum after 10 mg oral administra-
tion is around 7.281.67 ng.mL-1 and 402.16109.95 ng.h.mL-
1
respectively.
Elimination from plasma is biphasic with a terminal half-
life (t1/2) of 30-50 hours. Due to its long half-life, the steady
state plasma levels are reached after 7-8 days of consecutive
daily dosing. In patients with hypertension, once daily dos-
ing provides a satisfactory reduction in blood pressure over
24 hours. Moreover, due to its slow rate of absorption, it
produces gradual vasodilatation, and consequently reduces
side effects such as acute hypotension, reflex tachycardia
and headache.3
Nowadays bioequivalence studies are a pivotal part of
registration dossiers. These studies measure the bioavai-
labilities of two (or more) formulations of the same active
ingredient. The purpose of the study is to show that the
bioavailabilities of the formulations under investigation are
similar. Based on that conclusion, one may subsequently
claim that the therapeutic quality of these formulations is
essentially the same. The latter means that both the benefi-
cial and side effects are essentially the same and hence the
formulations are interchangeable.
The objective of this study is to investigate the phar-
macokinetics and bioavailability of two different oral
amlodipine formulations following single dosing in healthy
volunteers in order to prove bioequivalence between both
preparations.
Methods
Subject and Study Design
The study was performed at PT Equilab International
(Jakarta, Indonesia) and was conducted according to the
Declaration of Helsinki and its amendments and to the rel-
evant Good Clinical Practice Guidelines and in agreement
with the local Ethics Committee. The study protocol was
reviewed and approved by the Committee of The Medical
Research of The Faculty of Medicine, University of
Maj Kedokt Indon, Volum: 58, Nomor: 2, Pebruari 2008
Indonesia.4
Design of this study were randomized, single-blind (in-
vestigator blind), cross-over study, and 2-sequence with two-
week washout period.5
In this cross-over study, each subject is given the test
drug (T) and the reference drug (R), in the different sequence
(TR or RT). The sequence of each subject was determined by
randomization based on Dixon WJ & Massey FJ - Random
Numbers Table.6
The test preparation was Lopiten (10 mg amlodipine
tablets, batch number T061109, manufacturer: PT Guardian
Pharmatama, Jakarta, Indonesia). The reference formulation
(Norvask) was purchased from a local pharmacy.
Selection of the volunteer was based on the inclusion
and exclusion criteria. They are assessed for physical and
laboratory examination within 10 days prior to their first dos-
ing day.
Participating subjects have to fulfil the following crite-
ria:
1. Healthy male and female volunteers as determined by
the screening assessments.
2. Aged 18 55 years inclusive.
3. A body mass index in the range of 18-25.
4. Able to participate, communicate well with the investi-
gators and willing to give informed consent.
5. Vital signs (after 10 minutes resting) must be within the
following ranges:
- Systolic blood pressure: 110 - 135 mm Hg
- Diastolic blood pressure : 75 - 90 mm Hg
- Pulse rate : 60 80 bpm
Twelve healthy adult male and female volunteers aged
between 20-40 years, body weight within normal range (BMI
= 18.67-23.15 kg/m2), blood pressure within normal range (110-
130 mmHg for systolic, and 7090 mmHg for diastolic blood
pressure), pulse rate between 72-80 bpm, and had signed the
informed consent were included in the study.
Subjects who met the following exclusion criteria were
excluded from this study:
1. Pregnant women, nursing mothers, women of childbear-
ing potential without adequate contraception
2. Subjects with known contraindications or hypersensi-
tivity to amlodipine
3. Chronic gastrointestinal problems
4. Liver dysfunction or renal insufficiency or clinically sig-
nificant haematology abnormalities
5. Clinically significant ECG abnormalities
6. Positive test result of HBsAg or Hepatitis C or HIV
7. Participation with an intake of any prescription drug
within 14 days of this studys first dosing day, intake of
any non-prescription drug, food supplement or herbal
medicine within 7 days of this studys first dosing day
8. A donation or loss of 500 ml (or more) of blood within 3
43
Bioequivalence Study of Two Amlodipine Tablet Formulations
months before this studys first dosing day
9. History of drug or alcohol abuse within 12 months prior
to this screening
10. Participation in a previous study within 3 months of this
studys first dosing day.
Twelve adult male and female volunteers signed the
informed consent were included in the study.
Treatment Phase and Blood Sampling
Volunteers attended the study unit in the morning (ap-
proximately 6:00 a.m.) of the dosing day (day 1) after an
overnight fast, i.e., they were requested to fast from any
food and drink except mineral water from 9:00 p.m. the night
before. A pre-dose pharmacokinetic blood sample was taken.
The study drug (one tablets of Lopiten or the Norvask)
was given at 7.00 a.m. with 200 mL of water.
Ten milliliters of blood samples were drawn immediately
before taking the drug (control), and 10 mL each at 3, 5, 6, 7,
8, 9, 10, 11, 12, 15, 24, 48, 96, and 144 hours after drug admin-
istration. Plasma was separated from the blood samples by
centrifuging at 3000 rpm for 25 minutes, and kept in the freezer
at 20C until analysis.
The date and the time of taking each sample were re-
corded in the Case Report Form (CRF). Lunch and dinner
were provided 4 hours and 10 hours after drug administra-
tion. On days-2 and 3, breakfast were served at time points
+24 and +48 hours. On day 2 lunch and dinner were served
at the same time as on day 1. All meals and fluids taken by
the subjects should be standardized with regards to the type,
the amount and the time of administration during the sam-
pling period. Two weeks after the first drug administration
(washout period), the same procedure was repeated with the
alternate drug.
One physician and two nurses with sufficient qualifica-
tions and training were present at dosing time and stayed at
the site until the last volunteer left the study unit; thereafter
they were reachable by mobile telephone.
Analysis of Drug Concentration
Method of Analysis
The amlodipine concentrations in plasma were assayed
using a fully validated high performance liquid chromatog-
raphy with mass spectrometry detection method, with re-
spect to adequate sensitivity, specificity, linearity, recovery,
accuracy and precision (both within and between days). Sta-
bility of the samples under frozen conditions, at room tem-
perature, and during freeze-thaw cycle were also determined.
The following data were taken from the validation report:
calibration curve for amlodipine ranged from 0.2 to 20.02
ng.mL-1; linear relationship between concentration and sig-
nal intensity were obtained (r) = 0.9998; the limit of
quantitation (LOQ) was 0.2 ng.mL-1; precision: intra assay
coefficient of variation were 2.71%, 3.66%, and 4.22%, and
44
assay coefficient of variation were 6.92%, 6.24%, and 4.32%,
both at low, medium, and high concentrations; accuracy:
intra assay (% diff) ranged from -6.09% to -0.81% for the low
concentration, -9.10% to -0.38% for the medium concentra-
tion, and -11.65% to -1.28% for the high concentration; inter
assay (% diff) ranged from -13.95% to +9.57% for the low
concentration, -12.66% to +10.94% for the medium concen-
tration, and 12.99% to +1.20% for the high concentration.
Assay Procedure
The procedures described were applied for the extrac-
tion of subject samples, calibration and quality control stan-
dards. Serum sample was dispensed in an appropriate tube,
then internal standard solution (bisoprolol 1 mg.mL-1), NaOH,
and ethyl acetate were added. The content of the tube was
vortexed and centrifuged, and the organic phase was trans-
ferred to another set of clean glass tubes and evaporated to
dryness at a temperature of 500C under nitrogen stream. The
residue was reconstituted with methanol. Then, the solution
was transferred to a vial and an aliquot was injected into the
HPLC-MS system.7,8
The HPLC system consisted of a binary pump, mobile
phase vacuum degassing unit, autosampler, and Agilent LC-
mass spectrometric detector (MSD). SunFire C18 (4.6 x 150
mm) column was used.
The mobile phase was 1% acetic acid:methanol (35:65)
with total run time of 3 minutes. The flow rate was 0.8 mL/
min. MS condition: ionization mode API-ES; gas temperature
350C; polarity signal positive; nebulizer 45 psig; drying
gas flow 11.0 L/min. The detector was set to monitor m/z =
348 and m/z = 431 for amlodipine and bisoprolol., API-ES
ionization mode.
All chromatograms in the same batch were processed
automatically by a software using the same processing pa-
rameters such as integration, peak-to-peak amplitude and
peak detection. Manual integration was performed only when
necessary.
Pharmacokinetic Evaluation
The non-compartmental pharmacokinetic analysis
method was employed to determine the pharmacokinetic
parameters of amlodipine. Maximum serum concentration
(Cmax, ng.mL-1) and the time to reach the peak concentration
(tmax, h) were obtained directly from the observed data. The
area under the concentration-time curve from time zero to the
last measurable concentration time t (AUCt) was calculated
by the trapezoidal method. The area under cocentartion-time
curve from time zero extrapolated to infinite time (AUCinf)
was calculated as AUCt + Ct/ke, where Ct was the last quanti-
fiable concentration, ke was the terminal elimination rate con-
stant and was determined by least-squares regression analy-
sis during the terminal log-linear phase of the concentra-
tiontime curve. The terminal phase half-life time (t1/2, h) was
Maj Kedokt Indon, Volum: 58, Nomor: 2, Pebruari 2008
 Bioequivalence Study of Two Amlodipine Tablet Formulations

calculated as 0.693/ke. tions.

Data Analysis
EquivTest version 2.0 (Statistical Solution Ltd., Saugus,
MA, USA) was used to perform the statistical analyses of
AUCt, AUCinf, and Cmax using analysis of variance (ANOVA)
after transformation of the data to their logarithmic (ln) val-
ues. Using the error variance (S2) obtained from the ANOVA,
the 90% confidence intervals (CIs) were calculated from the
Mean plasma concentration-time profiles of amlodipine
in 12 healthy volunteers after administration of both formu-
lations are shown in Fig.1. The mean plasma concentration-
time curves of the test product and the reference drug were
comparable.
2.50

following equation:
90% CI = ( - )t \S2 x n2 2.00

Plasma concentration (ng/mL)

T R 0.1(v)

- T
, R : the means of the ln transformed values for the test 1.50

product (T) and the reference product (R)

- S2: the error variance obtained from ANOVA 1.00

- n: the number of subjects

- t0.1 : the t value for 90% CI
0.50

- v: the degree of freedom of the error variance from the

0.00
ANOVA. 0 12 24 36 48 60 72 84 96 108 120 132 144

Time (hour)

The anti ln of the above confidence intervals were the Lopiten Norvask

90% Cls of the ratios of the test/the reference geometric

means.
The criteria for bioequivalence are that the 90% Cls of
the geometric mean ratios 0.80 1.25 for the AUC and 0.75
1.33 for the Cmax.5
The tmax difference was analyzed non- parametrically
on the original data using Wilcoxon matched-pairs test.
Results
A total of 14 volunteers were invited to participate in
this study, 2 volunteers were extras to anticipate the possi-
bility of dropout cases. The demographic data of subject
tabulated in Table 1.
Fig 1. Mean Serum Concentration- Time Profiles of Amlo-
dipine in 12 Volunteers After Oral Administration of
10 mg Amlodipine Tablet Produced by PT Guardian
Pharmatama (Test product = Lopiten) and That Pro
duced by the Innovator (Norvask)
X
The individual pharmacokinetic parameters (AUCt,
AUCinf , and Cmax) are tabulated in Table 2. The %CV of AUCt
obtained from the ANOVA was 13.38%.
Table 2. Pharmacokinetic Parameters of Amlodipine After
10 mg Amlodipine Single Dose Administration of Test
and Reference Product in 12 Volunteers

Parameter Mean ratio (90% CI)

Table 1. Demographic, B lood Pressure and Heart Rate Data
of Subjects in Amlodipine Bioequivalence Study AUCt 95.50 (86.49 - 105.44)
AUCinf 96.68 (89.38 - 104.58)
Male (n=9) Female (n=3) Cmax 97.89 (86.12 - 111.09)

Age 28 (20 to 33) 38 (34 to 39)

Body Weight (kg) 55 (48 to 71) 48 (42 to 54)
Height (cm) 164 (157 to 176) 153 (150 to 156)
The pharmacokinetic parameters AUCt, AUCinf, Cmax, and
BMI (kg/m2 ) 21.4 (19.0 to 22.9) 19.7 (18.7 to 23.1)
Systolic blood pressure 120 (110 to 130) 110 tmax were used for bioequivalence evaluation of the drug
(mm Hg ) products studied. The geometric mean ratio (90% confidence
Diastolic blood pressure 90 (70 to 90) 70 (70 to 80) intervals) of the AUCt, AUCinf, and Cmax after administration
(mm Hg )
of both amlodipine dosage forms are shown in Table 3.
Heart rate per minute 80 (76 to 80) 80 (72 to 80)
The 90% convidence of intervals for geometric mean of
Data presented in median (range) test/reference ratios for AUCt, AUCinf, and Cmax were within
the acceptable limits of bioequivalence which implies that
There was no dropout case during the study. Twelve the bio-equivalence criteria were met. Using Wilcoxon
volunteers were required for the pharmacokinetic evalua- matched-pairs test on the original data, there was no statisti-
tion, therefore the blood samples from only 12 subjects were cally significant difference found between the two drug prod-
analyzed for determination of amlodipine plasma concentra- ucts for tmax values.

Maj Kedokt Indon, Volum: 58, Nomor: 2, Pebruari 2008 45

Bioequivalence Study of Two Amlodipine Tablet Formulations
Table 3. Statistical Comparison of Pharmacokinetic Parame-
ters of Amlodipine After 10 mg Amlodipine Single
Dose Administration of Test and Reference Product
in 12 Volunteers
Parameter Test product Reference product
(Lopiten) (Norvask)
Cmax (ng.mL-1) 1.79 (0.47) 8.0 (5.0 15.0)
t max (h) 78.04 (24.01) 95.83 (25.95)
AUCt (ng.h.mL-1) 51.69 (17.49) 1.86 (0.58)
AUCinf(ng.h.mL-1) 7.0 (5.0-12.0) 79.57 (18.57)
t (h) 97.25 (19.33) 52.44 (10.77)
Discussion
The aim of the study was to evaluate the bioavailability
of the test amlodipine tablet produced by PT Guardian
Pharmatama (Lopiten) and the reference amlodipine tablet
(Norvask) administered as 10 mg single oral dose.
The formulations were administered to overnight fast-
ing volunteers in order to eliminate the influence of food on
drug absorption.
For this study, AUCt, AUCinf, and Cmax were defined as
the main parameters in order to assess possible bio-equiva-
lence between both preparations. Based on standard
bioequivalence guidelines, the criteria for bioequivalence are
the 90% confidence interval of the test/reference-ratio in the
range of 80 to 125% for AUC and between 75 to 133% for
Cmax.5
The results of the present study showed that the 90%
confidence intervals of the test/reference AUCt-ratio, AUCinf
ratio, and Cmax-ratio were within the acceptable range for
bioequivalence.
The t range values of amlodipine based on the litera-
ture is 30 50 hours. Since the guidelines for bioequivalence
study recommend that sampling time should be continued
for at least 3 times the t, therefore the sampling time in this
study was continued until 144 hours.
In the present study the intrasubject coefficient of varia-
tion (%CV) obtained from the ANOVA for the AUCt was
13.38%, it means that the study only require a sample size of
less than twelve subjects.5,9 Therefore this study has ad-
equate power to confirm a statistical conclusion.
There was no adverse event encountered during this
study.
46
Conclusion
Based on the results shown above, that the 90% confi-
dence interval of the test/reference AUC-ratios were within
the acceptance range for bioequivalence, it was concluded
that the amlodipine besylate tablet equivalent to 10 mg
amlodipine tablet (Lopiten) produced by PT Guardian
Pharmatama was bioequivalent to that produced by the in-
novator (Norvask).
Acknowledgments
We thank the volunteers for their participation in this
study, and we appreciate PT Guardian Pharmatama as a spon-
sor for funding the study.
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EV/MS
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