Analgesics Anti-inflammatories Antiphlogistics Antirheumatic Drugs

Comparative in vitro dissolution and in vivo

bioequivalence of two diclofenac enteric coated
formulations
Saeed Basmenji\ Hadi Valizadeh
2

3
, Parvin Zakeri-Milani
3

4
1
Daana Pharmaceutical Company, Tabriz, Iran
2
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran
3
Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
4
Liver and Gastrointestinal Diseases Research Center, Tabriz University of Medical Scien ces, Tabriz, Iran
Correspondence to: Parvin Zakeri -Milani, PhD, Associate Professor of Pharmaceutics, Faculty of Pharmacy,
Tabriz University of Medical Sciences, Tabriz, 51664, Iran; e-mail: pzakeri@tb=ed.ac. ir
Abstract
The aim of this study was the comparison
of in vitro dissolution and in vivo bioa-
vailability of two different brands of di-
clofenac sodium (CAS 15307-86-5) enteric
coated tablets in healthy male Iranian
volunteers in a single-dose, randomized,
open-label, single blind study, which was
conducted according to a crossover
design in healthy volunteers. A washout
interval of two weeks was selected
between administrations to each subject
in this study. Serial venous blood samples
over 10 h after each administration to
measure diclofenac sodium concentra-
tion in serum were obtained, and placed
into tubes containing sodium heparin.
Then the plasma was separated and kept
frozen at -20 oc for subsequent analysis
with a modified HPLC method with UV
detection. In addition, the in vitro
dissolution study was performed on the
1. Introduction
brands. For the test and reference
formulation, mean Cmax values were
2257.3 (ng/rnl) and 2156 (ng/rnl), respec-
tively. The mean U G ~ and AUGi)' were
5726.1 (ng h/rnl) and 5917.8 (ng h/rnl)
for the test and 5689.9 (ng h/rnl) and
5967.4 (ng h/rnl) for the reference for-
mulation, respectively. Results show that
the 90 % confidence intervals for the ratio
of test and reference products in Cmax
(101.4-114.9 %) , U G ~ (96.3 -109.1 %)
and AUGO' (94.7-107.3 %) were all within
the 80-125% interval proposed by the
FDA and EMA. Both formulations re-
leased > 80 % of drug within 30 min in
buffer pH = 6.8 medium. Therefore the
diclofenac sodium enteric coated tablets
of the test and reference formulations are
bioequivalent in terms of rate and extent
of absorption.
Keywords
Bioavailability
Bioequivalence
Diclofenac
Dissolution
Arzneimittelforschung
2011;61(10):566-570
Non-steroidal anti-inflammatory drugs (NSAIDs) are the
drugs most commonly used to reduce inflammation and
pain. NSAIDs inhibit cyclooxygenase-2 at the inflamma-
tion, but unfortunately most of them inhibit gastric mu-
cous cyclooxygenase-1, which produces gastric damage.
Diclofenac sodium or, sodium [o-(2,6-dichloro-phenyl)-
amino-phenyl] acetate (CAS 15307-86-5) is a non-selec-
tive cyclooxygenase 1 and 2 inhibitor when tested in vi-
tro, but a slightly preferential cyclooxygenase-2 inhibi-
tor when tested ex vivo [1, 2]. It is approved for long
term therapy in patients with osteoarthritis, rheumatoid
arthritis, ankylosing spondylitis, acute gout, following
some surgical procedures and soft-tissue inflammation
in dosages ranging from 75 to 150 mg/day, depending
on the indication. Diclofenac sodium is well absorbed
orally and dissolves in the intestinal fluid [3]. It is gener-
ally known that the drug gets into blood within 30 min
and reaches the maximum blood concentration CCmax)
within 1.5- 2.5 h following oral administration of an en-
teric coated tablet. The oral bioavailability is around
60 % with an excretion half-life between 1.1 and 1.8 h
[4]. Diclofenac sodium is characterized by rapid sys-
temic clearance, which necessitates two or three daily
doses [5]. There are substantial evidences that the meth-
od of manufacture and the final formulation of the drug
can markedly affect the bioavailability of the drug. On
the other hand there are several drug products contain-
ing the same amount of diclofenac sodium marketed by
different pharmaceutical manufacturers. In fact the
World Health Organization (WHO) and all drug regula-
566 Basmenji eta!. - Diclofenac
Arzneimittelforschung 2011;61(10):566-570
ECV Editio Cantor Verlag, Aulendorf (Germany)
tory agencies support commercialization of generic
medicines because they control costs and are irreplace-
able therapeutic options in countries lacking the inno-
vator product [6]. However, it is necessary to examine
brand drug products in regard to in vitro dissolution
and in vivo bioequivalence, and interchangeable uses
with the innovator product. Therefore in the present
study in vitro dissolution behavior and also the rate
and extent of absorption of two commercial brands of
enteric coated tablets of diclofenac sodium were deter-
mined and compared following oral administration in
healthy Iranian volunteers.
2. Methods
2.1 In vitro dissolution study
A comparative in vitro dissolution study was conducted ahead of
the in vivo bioequivalence studies according to the procedure
described in the US Pharmacopeia. It was ensured that the in vi-
tro dissolution data were acceptable as per the regulatory guide-
lines for conducting bioequivalence studies. The dissolution
study was carried out on 12 units each of the test and reference
formulations. The paddle rotation speed was maintained at 100
rotations per minute at 37 0.5 C. The test was carried out using
900 mL of 0.1 N HCl for 1 h followed by pH 6.8 phosphate buffer
for the next hour as dissolution media. Samples of 5 ml were
withdrawn at predetermined time intervals (30, 60, 65, 70, 75,
90, 105 and 120 min and replaced with the same volume of fresh
buffer. Each sample solution was filtered, diluted and the absor-
bance reading determined at 276 nm using a spectrophotometer
(Shimadzu 160, Kyoto, Japan) against the blank. The concentra-
tions were thereafter determined from the calibration curve. The
mean dissolution values at each time interval were used to calcu-
late the difference factor (f
1
) and similarity factor (fzl using the
standard mathematical equations [7,8]:
f
2
=50 x log {[1 + (1/n)

(R, - T,)
2
r
05
x 100}
f1 = {[L,';,!IRr - Tdl I R,]} X 100
where n is the number of dissolution sample times, R, and T,
are the individual or mean percent dissolved at each time
point, and t stands for the reference and test dissolution pro-
files, respectively.
2.2 Subjects and study design
In this investigation 24 male healthy volunteers were recruited
with informed consent in an open label, single-dose, rando-
mized study with a crossover design and a washout period of
two weeks between the two phases in accordance with the
guidelines of the Declaration of Helsinki (World Medical As-
sembly 1964) as last revised in Seoul (2008). The study protocol
was approved by the local ethics committee of Tabriz Univer-
sity of Medical Sciences, Iran. Subject selection was according
to certain established criteria for inclusion into or exclusion
from the study. For examples any volunteers being smokers,
having allergies to the drug, being overweight or taking any
other medications prior to the study were excluded. Two
brands of diclofenac sodium enteric coated tablets were se-
lected as a reference (batch no. UOOll) and test (Exir Pharma-
ceutical Co, Boroujerd, Iran, batch no. 059-01 06) formulations.
Subjects were all Iranians and their average age and weight
were 23.2 1.6 years and 67.3 8.6 kg, respectively. They were
Arzneimittelforschung 2011;61(10):566-570
ECV . Editio Cantor Verlag, Aulendorf (Germany)
Analgesics . Anti-inflammatories Antiphlogistics Antirheumatic Drugs
examined for their health conditions. Two tablets (equivalent
to 25 mg diclofenac sodium) of the test and the reference for-
mulations were randomly given to the volunteers. Seven milli-
liters of blood taken from subject's forearm veins serially at 0,
0.5, 1, 1.5, 2, 3, 4, 6, 8, and 10 h after each administration. All
blood samples were collected into heparinized tubes and plas-
ma samples were separated and kept frozen at a temperature
below - 20 oc for subsequent analysis.
2.3 Plasma sample analysis
In different studies many methods for determination of diclofe-
nac sodium plasma concentrations were utilized. Among them
validated high-performance liquid chromatography (HPLC)
either with UV [1, 9], fluorimetric [2], or electro-chemical detec-
tion [1 , 3, 5, 10-17]; gas chromatography- mass spectrometry
[ll] and thin-layer chromatography (TLC) [5] have been re-
ported. In this study the analytical procedure for determination
of diclofenac sodium in plasma was adopted from the method of
El-Sayed and co-workers and validated for specificity, accuracy,
precision and sensitivity [18]. For extraction of diclofenac so-
dium, on 1 ml of plasma 50 JlL of the internal standard (200 Jlgl
m1 ofnaproxen) and 200 JlL ofHCI1 Nwas added, then all sam-
ples were extracted with 5 ml hexane/ isopropyl alcohol (99 I 1) by
vortexing for 5 min. After centrifugation for 5 min at 1000 g, the
upper organic phase was transferred to a 7 ml glass evaporation
tube. The tubes were placed in a 45 oc water bath. After dryness
the residue reconstituted with 200 JlL of the mobile phase by vor-
tex mixing for 20 s. 150 JlL of the resulting solution was injected
onto the HPLC column. The mobile phase was a mixture of acet-
onitrile and water (45: 55% v/v), pH= 3.3 adjusted with acetic
acid. The analytical column used for chromatographic separa-
tions was Shim pack CLC C18, 5 Jlm (150 x 4.6 mm) with a Shim-
pack CLC-C18, 5 Jlm 4.6 x 20 mm guard column (Shimadzu,
Kyoto, Japan) . The flow rate was 2 ml/min and the detector wa-
velength was set at 282 nm. Under these conditions the retention
times for diclofenac sodium and the internal standard (naprox-
en) were 4.2 and 2.3 min respectively. All plasma samples were
measured in the same chromatographic run. Each run had a se-
parate daily calibration. Four quality control samples with con-
centrations within the calibration range were used in triplicates
(n = 3) to determine the accuracy and precision of the method
[ 19, 20]. Calibration curves were obtained by plotting the diclofe-
nac sodium to naproxen peak area ratio against the concentra-
tions of the standard solutions.
2.4 Data analysis
The plasma concentration-time data for each volunteer was
presented in a graphical form. For oral dosage form, the bioa-
vailability of the drug is most often described by measuring the
area under the plasma drug concentration-time curve (AUC),
the time for peak drug concentration, Tmro<> and the peak drug
concentration, Cmax The area under the plasma concentration-
time curve from time zero tot (AUCb), AUCO' and (t112l
were calculated as previously described [21 -28] .
3. Results and discussion
3.1 In vitro dissolution study
Dissolution curves are shown in Fig. 1. From the disso-
lution profiles, it is clear that there were almost no drug
release from both enteric coated formulations in the
acidic medium as expected. However, in the phosphate
buffer medium (pH 6.8), more than 80 % of drug was re-
Basmenji et al. - Diclofenac 567
Analgesics Anti-inflammatories . Antiphlogistics Antirheumatic Drugs
HCI0.1N Phosphate bufferpH=6.8
100
80

"'
60
.,
>

V>
i:5
40
20
0
0 20 40 60 80 100 120
Time(min)
Fig. 1: Dissolution profiles of diclofenac sodium. Open and
solid circles are the data for reference and test formulations,
respectively.
leased within the first 30 min. Moreover the difference
factor (f
1
) of 10.8 (acceptable limit 0- 15) and the simi-
larity factor (f
2
) of 57.7 (acceptable limit 50-100) were
calculated [8]. A comparative in vitro dissolution study
provides a basis for predicting the likelihood of achiev-
ing a successful in vivo bioequivalence performance.
This in vitro dissolution study showed that the test and
reference formulations were comparable, indicating in-
terchangeability of the two brands.
3.2 Method validation
This analytical method was linear in the 10 to 4000 ng/
ml range; with a coefficient of correlation (r) greater
0.4
0.3
0. 3
1
0.2
.,.

=
0.2
.,
0


0
.,.

A
0.2
0 g

u
2
...,
!2


{/)
i5
....;
.;!
0. 1

.
I

i5
Q
I
: I
0.0
0.0


0.0
I
15 20 25 30
;a
than 0.99 and sensitivity CLOD) of 5 ng/ml of diclofenac
sodium in plasma. The recovery of diclofenac sodium
was 81.54, 88.5, and 90.1% at a concentration of 20,
200, and 1000 ng/ml, respectively. Recovery values for
the extraction procedure were calculated by comparing
chromatographic responses obtained from spiked ex-
tracted plasma and drug free plasma samples spiked
with the same concentration immediately after extrac-
tion. Recovery of the internal standard was 92 %. More-
over the intra-assay accuracy of the method ranged
from 97.6 to 99 %, while the inter-assay accuracy ranged
from 100.4 to 104.7%. A representative chromatogram
of different diclofenac sodium standard concentrations
in plasma samples is shown in Fig. 2.
3.3 Pharmacokinetic study
The mean concentration-time plots after administration
of reference and test formulations to 24 healthy volun-
teers are shown in Fig. 3. In Table 1, the averages of
pharmacokinetic parameters after administration of
both formulations to 24 subjects are reported. The mean
maximum serum concentrations of 2257.3 (ng/ml) and
2156.4 (ng/ml) were obtained for the test and reference
formulation, respectively. In only three volunteers a sec-
ond peak phenomenon was observed which can be at-
tributed to diclofenac transformation to the tetrahydrate
with lower solubility [29]. The respective values for T max
were 2.13 hand 2.21 h, and the amount of the AUC& and
AUC
0
for the test formulation were 5723.1 (ng h/ml)
and 5917.8 (ng h/ml) respectively, the obtained values
for the reference formulation were 5689.9 (ng h/ml)
and 5967.4 (ng h/ml), respectively.
.40

.36

0
0
0
M
.32
)l
g

=:
i.
u .28

Q
0

1
<>
0

.24
"'
I
.20
;a
;a
!2
.16
.12

.08
.04
.00
35 40 45 50
Minutes
Fig. 2: Representative chromatogram of different diclofenac sodium standard concentrations in plasma samples.
568 Basmenji et a!. - Diclofenac
Arzneimittelforschung 2011;61(10):566-570
ECV Editio Cantor Verlag, Aulendorf (Germany)
Analgesics Anti-inflammatories Antiph logistics Antirheumatic Drugs
Fig. 3: Average( SD) plasma concentration of diclofenac sodium versus time plots
of 24 subjects after administration of 25 mg diclofenac sodium tablets of test and
reference formulations. Top: linear scale, bottom: log-linear scale.
Table 1: Diclofenac Tmax, Cmroo A U C ~ A U C ~ T
112
values following administration
of test and reference formulations to 24 healthy volunteers.
Test formulation Reference formulation
Parameters
Mean (SD) %CV Mean (SD) %CV
Cmax (ng ml 2257.3 ( 254.5) 11.3 2156.4 ( 433.2) 20.1
Tmax (h) 2.13 ( 0.34) 15.9 2.21 ( 0.41) 18.8
AUCb (ng h/ rnl) 5723.1( 1106.2) 19.3 5689.9 ( 1198.8) 21.1
AUCQ' (ng h/ml) 5911.8 <= 1176.6) 19.9 5967.4 ( 1228.7) 20.6
T112 (h) 1.88 <= 0.49) 26. 0 2.17 ( 0.51) 23.7
Table 2: Analysis of variance (AN OVA) for the assessment of the product, group and
period effects, and 90 % confidence intervals (90% Cl) for the ratio of Cmroo A U C ~
and U C ~ values for the test and reference formulations, after administration of
reference and test products to 24 healthy volunteers (a= 0.05).
ANOVA (P-value)
Pharmacokinetic
Variation source
90 % CI for the
parameter ratios
Product Group Period
Cmax 0.3094 0.7176 0. 4783 101.4- 114.9
AUCb 0.8884 0.4269 0.0903 96.3- 109. 1
AUCQ' 0.8347 0.4347 0.456 94.7- 107.3
Arzneimittelforschung 2011;61 (10) :56&-570
ECV Editio Cantor Verlag, Aulendorf (Germany)
3.4 Statistical analyses
Because of the variability inherent in
human subjects, pharmacokinetic
parameters were analyzed statisti-
cally. On the other hand a statistical
confidence interval was used to pro-
vide an estimated range that is likely
to contain the mean pharmacoki-
netic value if the drug was given to
the entire population [30, 31]. There-
fore in the present study, bioequiva-
lence between the formulations was
determined by calculating 90% con-
fidence intervals (90% C. I.) for the ra-
tio of Cmax A U C ~ and AUC
0
values
for the test and reference formula-
tions [24, 32] . The multivariate analy-
sis, accomplished through analysis of
variance (ANOVA), was used to assess
group and period effects, and values
are shown in Table 2. These were all
inside the acceptance limits for bio-
equivalence (80 %-125 %) and there-
fore the results supported the bio-
equivalence. On the basis of FDA
and EMA guidelines, the recently ac-
cepted criterion for bioequivalence
for most dosage forms requires that
the mean pharmacokinetic param-
eters of the test dosage form should
be within 80% to 125% of the refer-
ence dosage form using the 90% con-
fidence interval. Thus, from the re-
sults obtained it is evident that the
diclofenac sodium enteric coated ta-
blets of test and reference formula-
tions are bioequivalent in terms of
rate and extent of absorption. There-
fore they are expected to have the
same safety and efficacy profiles
when administered under the condi-
tions listed in the product labeling.
4. Conclusion
The in vitro dissolution study indi-
cated suitability of the test formula-
tion for use in the in vivo bioequiva-
lence studies. The in vivo studies in
healthy human subjects demon-
strated that the generic enteric
coated test tablet, diclofenac so-
dium 25 mg, is bioequivalent to the
reference formula in terms of rate
and extent of absorption.
Conflict of Interest
This study was sponsored by Exir Phar-
maceutical Co, Boroujerd, Iran. The
authors report no conflicts of interest in
this work.
Basmenji et al . - Diclofenac 569
Analgesics Anti -inflammatories Antiphlogistics Antirheumatic Drugs
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