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Bioekivalent PDF
Bioekivalent PDF
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, randomized, 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 detector (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
41
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 Guardian Pharmatama bioekuivalen dengan produk yang sama yang dibuat oleh innovator.
Kata kunci: amlodipine kromatografi cair spektrometer massa bioekivalen
Introduction
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-5methyl-2-(2-aminoethoxymethyl)4-(2-chlorophenyl)-1,4dihydro-6-methyl-3,5-pyridine carboxylate benzenesulphonate. 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:
H3C
CH3OOC
H
N
COOC2H5
.C6H5S O 3H
Cl
42
Indonesia.4
Design of this study were randomized, single-blind (investigator blind), cross-over study, and 2-sequence with twoweek 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 dosing day.
Participating subjects have to fulfil the following criteria:
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 investigators 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 (110130 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 childbearing potential without adequate contraception
2. Subjects with known contraindications or hypersensitivity to amlodipine
3. Chronic gastrointestinal problems
4. Liver dysfunction or renal insufficiency or clinically significant 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
Assay Procedure
The procedures described were applied for the extraction of subject samples, calibration and quality control standards. 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 transferred 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 LCmass 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 parameters 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 quantifiable concentration, ke was the terminal elimination rate constant and was determined by least-squares regression analysis during the terminal log-linear phase of the concentrationtime curve. The terminal phase half-life time (t1/2, h) was
44
tions.
Mean plasma concentration-time profiles of amlodipine
in 12 healthy volunteers after administration of both formulations are shown in Fig.1. The mean plasma concentrationtime curves of the test product and the reference drug were
comparable.
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) values. Using the error variance (S2) obtained from the ANOVA,
the 90% confidence intervals (CIs) were calculated from the
following equation:
S2 x n2
- )t
90% CI = (
-
0.1(v)
2.50
2.00
1.50
1.00
0.50
0.00
0
12
24
36
48
60
72
84
96
108
120
132
144
Time (hour)
Female (n=3)
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)
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
(mm Hg )
Diastolic blood pressure 90 (70 to 90)
70 (70 to 80)
(mm Hg )
Heart rate per minute
80 (76 to 80)
80 (72 to 80)
Data presented in median (range)
Lopiten
Norvask
Fig 1. Mean Serum Concentration- Time Profiles of Amlodipine 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)
AUCt
AUCinf
Cmax
Test product
(Lopiten)
Cmax (ng.mL-1)
t max (h)
AUCt (ng.h.mL-1)
AUCinf(ng.h.mL-1)
t (h)
1.79 (0.47)
78.04 (24.01)
51.69 (17.49)
7.0 (5.0-12.0)
97.25 (19.33)
Reference product
(Norvask)
8.0 (5.0 15.0)
95.83 (25.95)
1.86 (0.58)
79.57 (18.57)
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 fasting 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-equivalence 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 literature 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 variation (%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 adequate power to confirm a statistical conclusion.
There was no adverse event encountered during this
study.
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Conclusion
Based on the results shown above, that the 90% confidence 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 innovator (Norvask).
Acknowledgments
We thank the volunteers for their participation in this
study, and we appreciate PT Guardian Pharmatama as a sponsor for funding the study.
References
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EV/MS