A Bioequivalence study of two Azithromycin tablet formulations in Indonesian healthy subjects

Article (PDF Available)inJournal of Bioequivalence & Bioavailability 4(5):048-051 · May 2012with677 Reads
DOI: 10.4172/jbb.1000111
Abstract
Abstract Aim: To compare the bioavailability of two Azithromycin tablet formulations 500 mg Azivol® tablets as test formulation and 500 mg Zithromax® tablets as reference formulation. Methods: 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 120 hours after dosing. Plasma concentration of Azithromycin was determined using liquid chromatography – tandem mass spectrometry method with TurboIon Spray 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. Results: The point estimates and 90% confidence intervals (CI) for AUC0-t, AUC0-∞, and Cmax for Azithromycin were 94.63% (86.27-103.81%), 95.35% (87.15-104.31%), 94.16% (80.31-110.41%) respectively. Conclusion: These results indicated that the two formulations of Azithromycin were bioequivalent and thus may be prescribed interchangeably.
Volume 4(5): 048-051 (2012) - 048
J Bioequiv Availab
ISSN:0975-0851 JBB, an open access journal
Research Article Open Access
Harahap et al., J Bioequiv Availab 2012, 4:5
http://dx.doi.org/10.4172/jbb.1000111
Research Article
Open Access
Bioequivalence & Bioavailability
Keywords: Azithromycin; Antibiotic; Bioequivalence and
Bioavailability; LC-MS/MS
Introduction
Azithromycin, 9-Deoxo-9a-aza-9a-methyl-9a-homoerythromycin
a dihydrate, is a semi-synthetic 15-membered azalide antibiotics
derived from erythromycin. Its chemical structure diers from that of
erythromycin by the insertion of methyl-substituted nitrogen at position
9a in the lactone ring. is modication results in the improved acid
stability associated with more reliable and greater oral bioavailability,
more extensive tissue penetration, and signicantly longer elimination
half-life, which exhibits an extensive spectrum of activity compared
with erythromycin. Azithromycin is eective against gram-positive
and gram-negative pathogens. Due to its extensive tissue penetration
and distribution, Azithromycin appears to be suitable antibiotic for the
treatment and prophylaxis of respiratory tract infection, skin and so-
tissue infection, and sexually transmitted diseases [1,2].
Azithromycin given orally is rapidly absorbed from gastrointestinal
track but is inhibited by food. Its absolute oral bioavailability is about
37%. Peak plasma concentrations are achieved 2 to 3 hours aer a
dose, but Azithromycin is extensively distributed to the tissues, and
tissue concentrations subsequently remain much higher than those
in the blood. Small amounts of Azithromycin are demethylated in
the liver, and it is excreted in bile as unchanged drug and metabolites.
Azithromycin metabolites are thought to possess no signicant
antimicrobial activity. About 6% of an oral dose (representing about
20% of the amount in the systemic circulation) is excreted in the urine.
e terminal elimination half-life is about 68 hours [2-4].
As for erythromycin, gastrointestinal disturbances are the
most frequent adverse eect but are usually mild and less frequent
than with erythromycin. e central and peripheral nervous
system, predominantly headache and dizziness may occur. Severe
hypersensitivity reactions occur rarely but may be prolonged [2].
ere are many generic products of Azithromycin in Indonesia
and it must also go through the bioequivalence study in order to assure
the ecacy, safety, and quality. e present study was conducted
to investigate the pharmacokinetics and bioavailability of two
Azithromycin tablet formulations in order to prove bioequivalence
between both formulations.
Subjects and Methods
e protocol study was reviewed by the Committee of e 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). is 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
aer they had been informed of the nature and details of the study in
accordance with Indonesian Guidelines for Bioequivalence Studies
[5,6].
e 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. e test
preparation was 500 mg of Azivol
®
tablets, manufactured by PT. Novell
*Corresponding author: Yahdiana Harahap, Faculty of Mathematic and
Natural Sciences, University of Indonesia, Depok, West Java Indonesia, E-mail:
yahdiana03@yahoo.com
Received April 18, 2012; Accepted May 24, 2012; Published May 26, 2012
Citation: Harahap Y, Prasaja B, Lusthom W, Hardiyanti, Ginting MB, et al. (2012)
A Bioequivalence Study of Two Azithromycin Tablet Formulations in Indonesian
Healthy Subjects. J Bioequiv Availab 4: 048-051. doi:10.4172/jbb.1000111
Copyright: © 2012 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.
Abstract
Aim: To compare the bioavailability of two Azithromycin tablet formulations 500 mg Azivol® tablets as test
formulation and 500 mg Zithromax® tablets as reference formulation.
Methods: 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 120 hours after dosing. Plasma concentration of Azithromycin was determined
using liquid chromatography tandem mass spectrometry method with TurboIon Spray mode. Pharmacokinetic
parameters AUC
0-t
, AUC
0-∞
and C
max
were tested for bioequivalence after log-transformation of data and ratios of t
max
were evaluated non-parametrically.
Results: The point estimates and 90% condence intervals (CI) for AUC
0-t
, AUC
0-∞,
and C
max
for Azithromycin
were 94.63% (86.27-103.81%), 95.35% (87.15-104.31%), 94.16% (80.31-110.41%) respectively.
Conclusion: These results indicated that the two formulations of Azithromycin were bioequivalent and thus may
be prescribed interchangeably.
A Bioequivalence Study of Two Azithromycin Tablet Formulations in
Indonesian Healthy Subjects
Yahdiana Harahap
1
*, Budi Prasaja
2
, Windy Lusthom
2
, Hardiyanti
2
, Mena Bertony Ginting
2
and Lipin
2
1
Faculty of Mathematic and Natural Sciences, University of Indonesia, Depok, Indonesia
2
PT. Clinisindo Laboratories, Jakarta, Indonesia
Citation: Harahap Y, Prasaja B, Lusthom W, Hardiyanti, Ginting MB, et al. (2012) A Bioequivalence Study of Two Azithromycin Tablet Formulations
in Indonesian Healthy Subjects. J Bioequiv Availab 4: 048-051. doi:10.4172/jbb.1000111
Volume 4(5): 048-051 (2012) - 049
J Bioequiv Availab
ISSN:0975-0851 JBB, an open access journal
Pharmaceutical Laboratories, Indonesia (Batch no. 11D183) and the
reference formulation was 500 mg Zithromax
®
tablets, produced by
Pzer Australia Pty Ltd., (Batch no. B914640151). e sample size
n = 24 subjects was sucient 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% [7].
A total of 24 subjects (18 males and 6 females) were selected among
Indonesia residents and participated in this study. e demographic
data of twenty-four volunteers are shown in Table 1.
Subjects were selected aer passing a clinical screening procedure
including a physical examination, ECG and clinical laboratory tests
(hemoglobin, hematocrit, WBC, platelets, WBC dierential, blood
urea nitrogen, sGPT, sGOT, alkaline phosphatase, total bilirubin, total
protein, fasting glucose, albumin, 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 any
illness of the hepatic, renal and cardiovascular system, took alcohol or
other medications for a long period of time, had hypersensitivity to
Azithromycin, 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.
All subjects were avoided using other drugs for at least two weeks
prior to the study and aer its completion. ey were also refrained
from ingesting alcohol, caeine, chocolate, tea or coke containing
beverages at least 48 hours before each dosing and until the collection
of the last blood sample. Subjects were conned 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 aer dose administration.
Water intake was allowed 2 hours aer the dose. Standard meals were
served at 4 and 11 hours aer drug administration. Snack was served
at 8 hours aer drug administration. Subjects were remained upright
(sitting or standing) for the rst 4 hours. Subjects were conned at
clinical unit of Clinisindo Laboratories for 24 hours aer dosing and
were 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, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 48, 72, 96 and
120 hours aer drug administration in the heparinized tubes. Aer
blood separation, plasma was frozen at -20ºC until analysis. Aer 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.
Safety Evaluation
Analysis of safety-related data was considered using the more
common adverse events which occurred aer initiation of study
treatment and supported by the following more detailed tabulations
and analysis (Table 2).
LC-MS/MS assay of Azithromycin in plasma
e concentration of Azithromycin in plasma was determined
using LC-MS/MS method with TurboIon Spray mode. Propranolol was
used as the internal standard. e method has already been validated
in terms of selectivity, sensitivity, linearity, accuracy and precision,
recovery, stability, and also has been veried just before being used in
study. e limit of quantication for Azithromycin was 2.0 ng/mL. e
standard calibration curves for Azithromycin were ranged from 2-500
ng/mL. e best linear t and least-squares residual for the calibration
curve were achieved with 1/x2 weighing factor. e recoveries of
Azithromycin were 84.54-87.91%. e analytical separation was
performed on a Synergi 4 μ POLAR- RP-80A, 50 × 2.00 mm, 4 μm
(Phenomenex
®
, USA) and protected by guard column AQ C18, 4 × 2.0
mm (Phenomenex®, USA). e mobile phase used gradient of 0.1%
formic acid in acetonitrile and 0.1% formic acid in water, pumped
0.7 mL/min for 4.0 min run time. e column temperature was
maintained at 40°C. Briey, a 250 μL of human plasma in microtube
was added with 20 μL of internal standard (10 ppm). Aer mixing,
250 μL of acetonitrile was added and vortex mixed for 30 seconds.
e mixture was centrifuged at 3000 rpm for 10 min. A volume of 5
μL supernantant was injected into LC-MS/MS system. e retention
time for Azithromycin and propranolol were 0.95 min and 1.10 min,
respectively.
Pharmacokinetic and statistical analysis
e bioequivalence was determined using the primary parameters,
AUC
0-t
, AUC
0-∞
, C
max
. C
max
and t
max
were obtained directly by inspection
of the individual drug plasma concentration time data, and were used
as measures of rate of absorption. AUC
0-t
was calculated using the
trapezoidal rule. e elimination rate constant (Kel) was calculated
by the technique of least-squares regression from the data of the last
3-5 points of each plasma concentration data curve. e AUC
0-∞
values
were determined by adding the quotient of Ct and the appropriate Kel
to the corresponding AUC
0-t,
that is:
AUC
0-∞
= AUC
0-t
+ Ct / Kel
e apparent elimination half-life (t½) of Azithromycin in plasma
was calculated by using the following equation:
t ½= (ln 2) / Kel
For the parameters of AUC
0-t,
AUC
0-∞
and C
max
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 AUC
0-t
,
Mean (± SD) Value Range
Age (years) 28.6 (4.9) 19-39
Weight (kg) 57.7 (9.3) 41-73
Height (m) 162.4 (7.6) 142.5-174
Body mass index (kg m-2) 21.8 (2.7) 18-25
Table 1: Demographic data for Azithromycin bioequivalence study in 24
volunteers.
Cause relation to study drug Events Total
Related Abdominal discomfort 15
Dizziness 7
Somnolence 2
Nausea 1
Probable Weakness 2
Myalgia 1
Total 28
Table 2: Disposition of adverse events.
Citation: Harahap Y, Prasaja B, Lusthom W, Hardiyanti, Ginting MB, et al. (2012) A Bioequivalence Study of Two Azithromycin Tablet Formulations
in Indonesian Healthy Subjects. J Bioequiv Availab 4: 048-051. doi:10.4172/jbb.1000111
Volume 4(5): 048-051 (2012) - 050
J Bioequiv Availab
ISSN:0975-0851 JBB, an open access journal
AUC
0-∞
and C
max
were calculated assuming a multiplicative model.
e accepted bioequivalence range for these parameters was 80-125%.
All statistical analyses were performed using Equiv Test version 2.0
soware (Statistical Solution, Cork, Ireland).
Results and Discussion
Both Azithromycin formulations were well-tolerated at the
administered dose and no signicant adverse clinical events were
observed. All adverse events were of mild intensity and recovered
without concomitant medication. ere were no serious adverse
events. However, all events resolved completely. e disposition of
adverse events is shown in Table 2.
e number register of this clinical trial is NCT 01602055. A total of
24 subjects participated in this study and all the subjects were available
for pharmacokinetic evaluation. e Azithromycin concentration
versus time proles of twenty four subjects for both formulations
are shown in Supplementary Figure 1 and the mean Azithromycin
concentration versus time proles for both formulations are shown
in Figure 1. e pharmacokinetic parameters that are used to assess
the bioequivalence of the test formulation versus the reference were
AUC
0-t,
AUC
0-∞
for the extent of the absorption and C
max
and t
max
for
the rate of absorption. Descriptive statistics of the pharmacokinetic
parameter for Azithromycin test and reference are summarized in
Table 3 where the geometric mean values and the range for the AUC
0-t
,
AUC
0-∞
, C
max
and values obtained for each formulation are shown.
e pharmacokinetic characteristic t
max
was presented as mean values.
e mean obtained values for test and reference formulations were
586.64 and 623.51 ng/mL for C
max
; 4712.31 and 5016.39 ng.h/mL for
AUC
0-t
; 5370.66 and 5711.19 ng.h/mL for AUC
0-∞
. e median t
max
for
test and reference formulations were 1.75 h and 2.25 h.
e results of the bioequivalence analysis for Azithromycin are
given in Table 4. e intra-subject variability of Azithromycin in
the AUC
0-t
, AUC
0-∞
, C
max
, and estimates from the coecient of
variables as determined by ANOVA were 18.65%, 18.11%, 32.09%, and
11.53%, respectively. As shown in Table 4, 90% condence intervals
(CI) of AUC
t
, AUC
0-∞,
C
max
, and t
½
log-transformed individual ratios
of Azithromycin were included into the range of bioequivalence, i.e.
80-125% when analyzed by parametric statistics. In the same way,
individual t
max
dierence was not statistically dierent between the two
formulations. e mean ratio of AUC
0-t
/AUC
0-∞
for all individuals and
for both products was around 12%, indicate an adequate sampling time
since the extrapolated portion of the total AUC is less than 20%. e
results for t
½
in the present study (50.50 ± 7.33 h for test product and
47.89 ± 7.23 h for reference product) were consistent with the results
reported in the literatures (~ 40-50 h) [4,8,9].
In this research the variability of C
max
is high but from the previous
research in healthy volunteers also showed that the intra-subject
variability of C
max
can be as high as 34.7%. Azithromycin can therefore
be considered as a highly variable drug. Highly variable drugs can
therefore pose a problem in bioequivalence assessment using standard
0.8 1.25 approach. It is therefore justied from that point of view to
use wider C
max
acceptance limits [10].
In conclusion, the application of either parametric or non-
parametric statistics reveals the presence of bioequivalence between
Azivol
®
FC tablet produced by PT. Novell Pharmaceutical Laboratories
and Zithromax
®
FC tablet produced by Pzer Australia Pty, Ltd
for the rate and extent of absorption. us, it can be assumed that
the two formulations are therapeutically equivalent and therefore
interchangeable.
Acknowledgment
This study was supported by PT. Novell Pharmaceutical Laboratories, Jakarta,
Indonesia.
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-100.00
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
0 6 12 18 24 30 36 42 48 54 60 66 72
Time (h)
Concentration (ng/mL)
Azivol®
Zithromax®
Figure 1: Mean Plasma Concentration Time Proles of Azithromycin Following
the Administration of Each Product.
Parameter
Test
Formulation
Reference Formulation
Geometric mean
C max (ng/mL)
range
534.67
193.87 – 1423.04
567.82
298.80 – 1585.02
Geometric mean
AUC
0-t
ngxh/ml)
Range
4443.48
2178.76 – 8148.69
4695.61
2452.39 – 8203.54
Geometric mean
AUC
0-∞
(ngxh/ml)
Range
5075.61
2615.24 – 9154.81
5323.31
2722.78 – 9340.76
Geometric mean
t
1/2
(h)
range
49.99
36.29 – 65.73
47.40
36.63 – 63.81
Median
t
max
(h)
range
1.75
1.00 – 6.00
2.25
1.00 – 4.00
Table 3: Mean pharmacokinetic characteristic for Azithromycin after Administration
for the two formulations.
Parameter T/R Point Estimate
Condence
Limits
Geometric mean
C
max
(ng/mL)
range
94.16 80.31 – 110.41
Geometric mean
AUC
0-t
ngxh/ml)
Range
94.63 86.27 – 103.81
Geometric mean
AUC
0-∞
(ngxh/ml)
Range
95.35 87.15 – 104.31
Geometric mean
t
1/2
(h)
range
105.46 99.59 – 111.67
Table 4: Parametric 90% condence interval for the mean pharmacokinetic
characteristic of Azithromycin formulations.
Citation: Harahap Y, Prasaja B, Lusthom W, Hardiyanti, Ginting MB, et al. (2012) A Bioequivalence Study of Two Azithromycin Tablet Formulations
in Indonesian Healthy Subjects. J Bioequiv Availab 4: 048-051. doi:10.4172/jbb.1000111
Volume 4(5): 048-051 (2012) - 051
J Bioequiv Availab
ISSN:0975-0851 JBB, an open access journal
Bioavalaibility and pharmacokinetics comparison between generic and branded
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Azithromycin 250 mg Capsules PL 20176/0007.
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    • "Consequently, a low therapeutic concentration can be set in blood to establish a dosing interval. In this study it is possible to set 0.1 µg/mL, derived from the pilot study here performed and complies well with the minimum plasma therapeutic concentration adopted from human studies (Harahap et al., 2012). This value allows a 48 h dosing interval. "
    [Show abstract] [Hide abstract] ABSTRACT: The pharmacokinetics (PK) of a long acting 20% solution of azithromycin (AZ-LA) in pigs after a single peri-caudal injection at 10 and 20 mg/kg body weight was determined, using an aqueous formulation of the drug administered IV as reference, HPLC was used as analytical method. C-max was 1.4 and 2.5 mu g/mL, and T-max was 3.1 and 3.4 h, respectively for 10 and 20 mg/kg. Both preparations had 99.5% purity. Values for MRT were longer in the 10 mg/kg dose group (18.2 h) as compared to the 20 mg/kg group (16.5 h). Yet T1/2 beta was comparable between both dose levels (8.5 h vs. 7.2 h, for 10 and 20 mg/kg, respectively). No linear increments linked to dose were found in variables such as relative bioavailability, C-max and apparent volume of distribution. Values for relative bioavailability (Fr) of the higher dose as compared to the lower were 86.81% and absolute bioavailability (F) was 110.5 for the lower dose and 115.9% for the higher. Best PK/PD ratios for clinical efficacy of azithromycin describe this antibacterial drug as time-dependent. Considering pig's serum concentrations of the AZ-LA preparation here studied after its peri-caudal injection and based on a theoretical minimal therapeutic concentration in plasma of 0.1 mu g/mL based on bacteriological testing, a dose-interval of up to 48 h can be achieved. The suitability of this preparation to treat pig respiratory diseases is addressed. (C) 2014 PVJ. All rights reserved
    Full-text · Article · Jan 2015