| Research Article |
Open Access |
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| Comparative Bioavailability Study of Two Ramipril Tablet
Formulations in Indonesian Healthy Volunteers |
| Yahdiana Harahap1, Lucy Sasongko2, Budi Prasaja3*,
Windy Lusthom3, Evy C. Setiawan3, Raria S. Meliala3, Lipin3 |
| 1Department of Pharmacy, Faculty of Mathematic and Science, University of Indonesia, Depok (Indonesia) |
| 2School of Pharmacy, Bandung Institute of Technology, Bandung (Indonesia) |
| 3Clinisindo Laboratories, Jakarta (Indonesia) |
| *Corresponding author: |
Dr. Budi Prasaja, S.Si., Apt, MM,
Clinisindo Laboratories,
Jl. Ulujami Raya 12, Jakarta 12250 (Indonesia),
Tel : +6221
73889918 ,
Fax : +6221 73885453 ,
E-mail : budi.prasaja@clinisindo.com |
|
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| Received November 05, 2009; Accepted December 13, 2009; Published December 13, 2009 |
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| Citation: Harahap Y, Sasongko L, Prasaja B, Lusthom W, Setiawan
EC, et al. (2009) Comparative Bioavailability Study of Two Ramipril Tablet
Formulations in Indonesian Healthy Volunteers. J Bioanal Biomed 1:
017-021. doi:10.4172/1948-593X.1000004 |
| |
| Copyright:© 2009 Harahap Y, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited. |
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| Abstract |
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| Aim |
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| To compare the bioavailability of two ramipril tablet formulations– 10 mg Prohytens® tablets as test formulation
and 10 mg Triatec® tablets as reference formulation. |
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| Methods |
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| A single-dosed, open-label, randomized two-way crossover
design under fasting period with two weeks wash
out period was evaluated in 24 subjects. For the analysis
of pharmacokinetic properties, the blood samples were
drawn taken up to 72 hours after dosing. Plasma concentration
of ramipril and ramiprilat were determined using
liquid chromatography – tandem mass spectrometry
method with TurboIonSpray mode. Pharmacokinetic parameters
AUC0-t, AUC0-∞ and Cmax were tested for
bioequivalence after log-transformation of data and ratios
of tmax were evaluated non-parametrically. |
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| Results |
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| The point estimates and 90% confidence intervals (CI)
for AUC0-t, AUC0-∞ and Cmax for ramipril were 93.21%
(85.67-101.41%), 93.45% (85.88-101.69%), 94.02%
(80.09-110.38%) and for ramiprilat were 92.26% (87.76-
96.99%), 94.59% (89.71-99.73%) and 91.55% (84.88-
98.74%). |
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| Conclusion |
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| These results indicated that the two formulations of
ramipril were bioequivalent and thus may be prescribed
interchangeably. |
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| Keywords |
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| Angiotensin-coverting enzyme inhibitor (ACEi);
Bioequivalence and bioavailability; Ramipril; Ramiprilat |
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| Introduction |
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| Ramipril, 2-[N-[(S)-1-(ethoxycarbonyl)-3-phenylpropyl)]-Lalanyl]-(
1S,3S,5S)-2-azabicyclo (3-3-0) octane-3-carboxylic acid
(Figure 1), is a potent angiotensin-converting enzyme inhibitor
(ACEi) that is used in the treatment of hypertension, heart failure,
in patients after myocardial infarction (MI), in the prevention
of diabetic and non-diabetic nephropathy, and in the prevention
of MI in high-risk patients. In general, ramipril exerts its
effects through actions in renin-angiotensin-aldosterone system
(RAS). It inhibits the conversion of angiotensin I to angiotensin II through actions of ACE, thus decreasing the vasoconstriction,
sympathetic activation, and trophic changes in the heart and blood
vessels that angiotensin produces (Smith and Ball, 2000; Vuong
and Annis, 2003). |
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|
Figure 1: Chemical structure of Ramipril and Ramiprilat. |
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Ramipril acts as a prodrug of the diacid ramiprilat, its active
metabolite. After oral doses at least 44 to 66% is absorbed. Absorption
is not influenced by silmutaneously intake of food.
Ramipril is metabolized in the liver to ramiprilat; other metabolites
are inactive. Peak plasma levels of ramipril are reached
within one hour of administration with peak levels of ramiprilat
are achieved 2 to 4 hours after an oral dose of ramipril (van
et al., 1995). The Cmax for ramipril is about 12 ng/mL
and the AUC is about 15 ng.h/mL after the single oral administration
of 5 mg ramipril (Mendes et al., 2006). Ramiprilat is about
56% bound to plasma proteins. After oral administration, 60%
of the dose is found in the urine and 40% is found in the feces.
The effective half-life for accumulation of ramiprilat is 13 to 17
hours after daily doses of ramipril 5 to 10 mg (Smith and Ball,
2000; Vuong and Annis, 2003). |
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| The present study was conducted to investigate the pharmacokinetics
and bioavailability of two ramipril tablet formulations
in order to prove bioequivalence between both formulations. |
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| Subjects, Materials and Methods |
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| Ethics consideration |
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| The protocol study was reviewed by the Committee of The
Medical Research Ethics of the Faculty of Medicine, University
of Indonesia (Jakarta, Indonesia) and was approved by the National
Agency of Drug and Food Control (Jakarta, Indonesia). |
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This study was conducted in compliance with the ethical principles
of the Declaration of Helsinki for biomedical research
involving human volunteers and Good Clinical Practice (GCP).
All participants signed a written informed consent after they had
been informed of the nature and details of the study in accordance
with Indonesian Guidelines for Bioequivalence Studies |
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| Study design |
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| The study was based on single-dose, open-label, randomized
two-way crossover design under fasting period with two weeks
wash out period. Subjects were randomized to one of the two
sequences to receive the formulations according to randomization
scheme. The test preparation was 10 mg of Prohytens® tablets,
manufactured by PT. Novell Pharmaceutical Laboratories,
Indonesia (Batch no. 10M035) and the reference formulation
was 10 mg Triatec® tablets, manufactured by PT. Aventis Pharma,
Indonesia (Batch no. 065U014). |
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| Based on previous study, the sample size n = 24 subjects was
sufficient to ensure power of 80% for correctly concluding
bioequivalence under the following assumption: a = 0.05, 0.95< μT / μR < 1.05 and an intra-subject variability of 20% (Diletti et
al., 1991; Mendes et al., 2006). |
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| Subjects |
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A total of 26 volunteers were selected among Indonesia residents
and participated in this study. First twenty-four volunteers
(19 males and 5 females) were available for pharmacokinetic
evaluation. If volunteers could not complete the entire study
(dropout/withdrawn), volunteers were replaced with two reversed
volunteers based on the same randomization code. The demographic
data of twenty-four volunteers are shown in Table 1.
Volunteers were selected after passing a clinical screening procedure
including a physical examination, ECG and clinical laboratory
tests (hemoglobin, hematocrit, WBC, platelets, WBC differential,
blood urea nitrogen, sGPT, sGOT, alkaline phosphatase,
total bilirubin, total protein, fasting glucose, albumin, total cholesterol,
creatinine, urine analysis, pregnancy test (for female
subjects) and negative results of HBsAg, anti HBC and anti HIV.
Volunteers were excluded if they had a history of peptic ulcer,
any illness of the hepatic, renal and cardiovascular system, taken
alcohol or other medications for a long period of time, had hypersensitivity
to ramipril or related ACEi, had received any investigation
drug within four weeks (or suitable longer period for
slowly eliminated drugs) of enrollment and donation or loss more than 450 ml of blood within 3 months prior to the screening of
the study. |
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Table 1: Demographic data for ramipril bioequivalence study in 24 volunteers. |
|
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| Drug administration and sampling |
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| All volunteers avoided using other drugs for at least two weeks
prior to the study and until after its completion. They also refrain
from ingesting alcohol, caffeine, chocolate, tea or cokecontaining
beverages at least 48 hours before each dosing and
until the collection of the last blood sample. The grapejuice was
not prohibited (Bailey and Dresser, 2004). |
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| Volunteers were confined to clinical unit of Clinisindo Laboratories
one night before study to assure the fasting condition
(10 hours before drug administration). On the study day, subjects
were given one tablet of either product with 240 ml of water.
No food was allowed until 4 hours after dose administration.
Water intake was allowed 2 hours after the dose. Standard meals
were served at 6 hours (±1008 calories) and 12 hours (±836
calories), snacks were served at 4 hours (±165 calories) and 8
hours (±160 calories) after drug administration. Total calories
were calculated by nutritionist. |
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| Subjects were remained upright (sitting or standing) for the
first 4 hours. Subjects were confined at clinical unit of Clinisindo
Laboratories for 24 hours after dosing and and did not permitted
to take strenuous exercise during the sampling days. Blood pressure,
heart rate, body temperature and adverse events were monitored
during blood sampling. |
| |
| 5 ml of the venous blood were collected at pre dose, 10, 20,
40, 60, 80, 100, 120 minutes, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 24, 36, 48
and 72 hours after drug administration in the heparinized tubes.
After blood separation, plasma was frozen at -20ºC until analysis
(Mendes et al., 2006; van Griensven, 1993). |
| |
| After two weeks wash out period, subjects returned to
Clinisindo Laboratories and the blood sample analysis was repeated
in the second period in the same manner to complete the
crossover design. |
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| Analytical method |
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The concentration of ramipril and ramiprilat in plasma was
determined using LC-MS/MS method with TurboIonSpray mode.
Enalapril (CAS# 75847-73-3, MW 376.4) was used as the internal
standard (Bo Yuan et al., 2000). The method has already
been validated in terms of selectivity, sensitivity, linearity, accuracy
and precision, recovery and also has been verified just before
being used in study. The limit of quantification for ramipril
and ramiprilat was 0.1 ng/mL and 0.2 ng/mL, respectively. The
standard calibration curves for ramipril and ramiprilat were
ranged from 0.1-100 ng/mL and 0.2-100 ng/mL, respectively.
The best linear fit and least-squares residual for the calibration
curve were achieved with 1/x2 weighing factor. The recoveries
of ramipril and ramiprilat were 80.90%, 84.09%, 79.13% and
36.99%, 37.49%, 38.42% at low medium and high QC samples.
The analytical separation was performed on a Synergi 4μ POLAR-
RP-80A, 50 x 2.00 mm, 4 μm (Phenomenex®, USA) and
protected by guard column AQ C18, 4 x 2.0 mm (Phenomenex®,
USA). The mobile phase was used gradient of 0.1 % formic acid
in acetonitrile and 0.1 % formic acid in water, pumped 0.6 mL/
min for 4.0 min run time. The column temperature was maintained
at 40°C. Briefly, a 500 μL of human plasma in polypropylene tube was added with internal standard, 100 μL of 1 M
phosphoric acid and 100 μL of methanol:water (50:50). After
mixing, 3 mL of ethyl acetate was added and vortex mixed for 2
min. The mixture was centrifuged at 3000 rpm for 10 min. The
organic phase was removed and evaporated to dryness under
vacuum at 60°C for 15 min. The residue was reconstituted with
methanol:water (1:1). A volume of 10 μL aliquot was injected
into the LC MS/MS system. The retention time for ramipril and
ramiprilat was 0.41 min and 0.34 min, respectively. |
| |
| Safety evaluation |
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| Analysis of safety-related data was considered using the more
common adverse events which occurred after initiation of study
treatment and supported by the following more detailed tabulations
and analysis. |
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| Pharmacokinetic and statistical analysis |
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| The bioequivalence was determined using the primary parameters,
AUC0-t, AUC0-¥, Cmax. The maximum plasma concentration
(Cmax) and time to reach maximum plasma concentration (tmax)
were obtained directly by inspection of the individual drug plasma
concentration time data, and were used as measures of rate of
absorption. The area under the plasma concentration time curve
up to the last time (t) showing a measurable concentration (Ct)
of the analyte (AUC0-t) was calculated using the trapezoidal rule.
The elimination rate constant (Kel) was calculated by the technique
of least-squares regression from the data of the last 3-8
points of each plasma concentration data curve. The AUC0-¥
values were determined by adding the quotient of Ct and the
appropriate Kel to the corresponding AUC0-t, that is: |
| |
| AUC0-∞ = AUC0-t + Ct / Ket (1) |
| |
| where C is the estimated last plasma concentration. The apparent
elimination half-life (t½) of ramipril and ramiprilat in plasma
was calculated by using the following equation: |
| |
| t1/2= (In 2)/ Ket (2) |
| |
| For the parameters of AUC0-t, AUC0-∞ and Cmax a multiplicative
model was assumed, and analysis of variance (ANOVA) was
applied using the respective ln-transformed data. For estimation
of bioequivalence the 90% CI of the geometric mean ratio test/
reference (T/R) for AUC0-t, AUC0-∞ and Cmax were calculated assuming a multiplicative model. The accepted bioequivalence
range for these parameters was 80-125%. All statistical analyses
were performed using EquivTest version 2.0 software (Statistical
Solution, Cork, Ireland). |
| |
| Results |
| |
| Clinical observation |
| |
| Both ramipril formulations were well-tolerated at the administered
dose and no significant adverse clinical events were observed.
Twenty three out of 26 volunteers experienced 83 adverse
events during the study. All adverse events were of mild
intensity and recovered without concomitant medication. There
were no serious adverse events. However, all events resolved
completely. The disposition of adverse events is shown in Table
2. |
| |
Table 2: Disposition of adverse events. |
|
|
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| Pharmacokinetic evaluation |
| |
A total of 26 volunteers were invited to participate in this study.
First twenty-four volunteers were available for pharmacokinetic
evaluation. The mean ramipril and ramiprilat concentration versus
time profiles for both formulations are shown in Figure 2 and Figure 3. The pharmacokinetic parameters used to assess
the bioequivalence of the test formulation versus the reference
were AUC0-t, AUC0-∞ for the extent of the absorption and Cmax and
tmax for the rate of absorption. Descriptive statistics of the pharmacokinetic
parameter for ramipril and ramiprilat test and reference
are summarized in Table 3 where the geometric mean values
and the range for the AUC0-t, AUC0-∞, Cmax and t½ values obtained
for each formulation are shown. The pharmacokinetic characteristic tmax was presented as mean values. For ramipril,
the mean obtained values for test and reference products were
35.06 and 37.29 ng/mL for Cmax; 21.62 and 23.19 ng.h/mL for
AUC0-t; 21.87 and 23.40 ng.h/mL for AUC0-∞. The median tmax for both formulations was 0.33 h. For ramiprilat, the mean obtained
values for test and reference products were 11.17 and 12.21
ng/mL for Cmax; 109.99 and 119.15 ng.h/mL for AUC0-t; 134.48
and 141.63 ng.h/mL for AUC0-∞. The median tmax was 2.5 h (test)
and 2.75 h (reference). |
| |
|
Figure 2: Arithmetic mean plasma concentration-time profiles of ramipril
after a single dose of two 10 mg ramipril tablets of two different formulations. |
|
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|
Figure 3: Arithmetic mean plasma concentration-time profiles of ramiprilat
after a single dose of two 10 mg ramipril tablets of two different formulations. |
|
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Table 3: Mean pharmacokinetic parameters for ramipril and ramiprilat after
administration of the two formulations to 24 volunteers. |
|
| |
| Statistical evaluation |
| |
| The results of the bioequivalence analysis for ramipril and
ramiprilat are given in Table 4. The intra-subject variability of
ramipril in the AUC0-t, AUC0-∞ , Cmax, t½ estimates from the coefficient of variables as determined by ANOVA were 17.00%,
17.03%, 32.36%, and 36.30%, respectively. The intra-subject
variability of ramiprilat in the AUC0-t, AUC0-∞ , Cmax, t½ estimates
from the coefficient of variables as determined by ANOVA were
10.05%, 10.63%, 15.23%, and 24.23%, respectively. |
| |
Table 4: Statistical evaluation of AUC0-t, AUC0-∞, Cmax and t½ for ramipril and
ramiprilat of two formulations from 24 volunteers. |
|
| |
| The aim of the randomized, single blind, two-period, cross
over study with a washout period of 2 weeks was to evaluate the
bioavailability of the test and the reference ramipril tablet administered
as 10 mg single oral dose each. |
| |
| From the result of the safety evaluation, it was concluded that
both of test and reference preparations were well-tolerated. A
clinically relevant difference to the adverse events stated in the
literature was not detected. |
| |
| In the study the last sampling time for ramipril was 12 hours.
Ramipril was rapidly absorbed and the elimination was fast and
5 hours after administration only 9 subjects showed detectable
ramipril concentration in two period. The formation of ramiprilat
was fast and the elimination was slower than ramipril. |
| |
In literature, ramiprilat displays triphasic elimination kinetics
with half-lives of 2 to 4 hours, 9 to 18 hours, and greater than 50
hours. This triphasic elimination is due to extensive distribution
to all tissues (initial half-life), clearance of free ramiprilat from
plasma (intermediate half-life), and dissociation of ramiprilat
from tissue ACE (terminal half-life) (Brunton et al., 2007). In
this study the half-lives were 40.27 and 35.64 hours for test and
reference and also the reported study by Mendes et al. Thus, the
calculated half-lives may be biased because of their dependence
on the duration of blood sampling (Ruf et al., 1994). |
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| Theoritically, the duration of the study was more than 3 time
of half-life. But for the BE study, a sampling period longer than
72 hours is not considered necessary (CHMP, 2008). |
| |
| According to FDA guidance on ramipril, the analytes that
should be measured were ramipril and ramiprilat. The
bioequivalence data is concluded based on ramipril data while
the metabolite data is submitted as supportive evidence of comparable
therapeutic outcome. (CDER Guidance on Ramipril,
2008). |
| |
| The intra subject variability of AUC0-t for ramipril and
ramiprilat was 17.00% and 10.05%, respectively. Considering
this result the sample size of 24 subjects was sufficient in order
to conclude bioequivalence with the power of 80% at the 5%
nominal level (Diletti et al., 1991). |
| |
As shown in Table 3, the results of the statistical evaluation
for 90% confidence interval of AUC0-t, AUC0-∞
, Cmax, t½ for
ramipril and ramiprilat were entirely included within the
bioequivalence acceptance limit of 80 – 125% (CPMP, 2001). |
| |
| In conclusion, the two ramipril formulations were equivalent
with respect to the rate and extent of absorption and it can be
assumed to be therapeutically equivalent and exchangeable in
clinical practice. |
| |
| Acknowledgements |
| |
| This study was supported by PT. Novell Pharmaceutical Laboratories,
Jakarta, Indonesia. |
| |
|
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