original papers - Advances in Clinical and Experimental Medicine

original papers - Advances in Clinical and Experimental Medicine

orIginal papers
Adv Clin Exp Med 2011, 20, 5, 605–611
ISSN 1230-025X
© Copyright by Wroclaw Medical University
Shujaat A. Khan1, Mahmood Ahmad1, Rozina Kousar1, Ghulam Murtaza2
Nimesulide-Serratiopeptidase Sustained Release
Microparticles – Combined Formulation
and In Vitro Characterization
Nimesulid i serratiopeptydaza – preparat złożony w postaci
mikrocząsteczek o przedłużonym uwalnianiu – charakterystyka in vitro
1
2
Faculty of Pharmacy & Alternative Medicine, The Islamia University of Bahawalpur, Pakistan
Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad, Pakistan
Abstract
Objectives. To develop a combined once-daily sustained release microencapsulated dosage form of nimesulide
(NSD) and serratiopeptidase (SPD) with different physicochemical properties using ethyl cellulose (ETC) as the
release-controlling factor and to evaluate the drug release parameters in accordance with various kinetic release
models.
Material and Methods. In order to achieve the intended sustained-release profile, microparticles were prepared
using a non-solvent addition coacervation technique. An in-vitro dissolution analysis was conducted using a USP
dissolution apparatus type II. The temperature of the dissolution medium (phosphate buffer pH 6.8 plus sodium
lauryl sulphate 1%, 900 mL) was maintained at 37oC ± 1oC with a stirring rate of 75 rpm.
Results. The physico-chemical parameters of the formulated microparticles were assessed and the results were
found to be within acceptable limits. Various kinetic models were used to analyze the dissolution data to investigate
the mode of drug release. The dissolution behavior was found to be a complex anomalous pattern of drug release,
best explained by the Higuchi expression. ETC had no significant chemical interaction with the drugs. All the
batches of microcapsules showed good stability and reproducibility.
Conclusions. The non-solvent addition coacervation technique is a promising method to develop a combined oncedaily sustained release microencapsulated dosage form of nimesulide and serratiopeptidase with different physicochemical properties using ethyl cellulose as release-controlling factor (Adv Clin Exp Med 2011, 20, 5, 605–611).
Key words: nimesulide; serratiopeptidase; ethyl cellulose; microencapsulation.
Streszczenie
Cel pracy. Opracowanie złożonej, podawanej raz dziennie, o przedłużonym uwalnianiu, w mikrokapsułkach postaci nimesulidu (NSD) i serratiopeptydazy (SPD) o różnych właściwościach fizykochemicznych, z zastosowaniem
etylocelulozy (ETC) jako czynnika kontrolującego uwalnianie i ocenę wskaźników uwalniania leku według różnych
kinetycznych modeli uwalniania.
Materiał i metody. W celu osiągnięcia zamierzonego profilu o przedłużonym uwalnianiu, przygotowano mikrokapsułki za pomocą techniki koacerwacji bez dodatku rozpuszczalnika. Analizę rozpuszczania in vitro przeprowadzono z użyciem urządzenia do rozpuszczania USP typu II. W środowisku uwalniania (bufor fosforanowy o pH 6,8
oraz laurylosiarczan sodu 1%, 900 mL) utrzymywano temperaturę 37°C ± 1ºC z szybkością mieszania 75 rpm.
Wyniki. Oceniono wskaźniki fizykochemiczne powstałych mikrokapsułek. Wyniki zawierały się w dopuszczalnych granicach. Do analizy danych dotyczących rozpuszczania wykorzystano różne kinetyczne modele uwalniania
w celu zbadania sposobu uwalniania leku. Zaobserwowano złożony nietypowy wzorzec uwalniania leku, który
najlepiej opisuje wyrażenie Higuchiego. ETC nie miała istotnych chemicznych interakcji z lekiem. Wszystkie partie
mikrokapsułek wykazały dobrą stabilność i powtarzalność.
Wnioski. Technika koacerwacji bez dodatku rozpuszczalnika jest obiecującą metodą opracowania złożonej, podawanej raz dziennie, o przedłużonym uwalnianiu, w mikrokapsułkach, postaci nimesulidu i serratiopeptydazy o różnych właściwościach fizykochemicznych z użyciem etylocelulozy (ETC) jako czynnika kontrolującego uwalnianie
(Adv Clin Exp Med 2011, 20, 5, 605–611).
Słowa kluczowe: nimesulid, serratiopeptydaza, etyloceluloza, mikrokapsułkowanie.
606
Many different peroral modified release (MR)
formulations have been developed in the last twenty years. The aim of MR formulations is to deliver
the drugs at an established rate, in order to minimize dose frequency, improve patient compliance
and achieve more uniform circulatory drug levels,
thus improving the therapeutic effectiveness of the
medication [1, 2]. A variety of polymers have been
introduced for developing MR formulations. One
of these is ethyl cellulose (ETC), a lipophilic nonbiodegradable polymer which is increasingly used
in the fabrication MR products due to its attractive
properties: excellent compressibility and a compatible physico-chemical nature offering minimal
toxicity [3, 4]. ETC was therefore selected for the
formation of a matrix to control drug release.
Nimesulide (NSD) is a nonsteroidal anti-inflammatory drug. It is acidic in nature due to the
presence of a sulfonanilide group [5]. NSD is able
to inhibit the activity of cyclo-oxygenase-2, which
ultimately limits the production of harmful prostaglandins and enhances the availability of useful
prostaglandins [6]. NSD is categorized as a class-II
drug in the biopharmaceutic classification scheme,
since it exhibits very sparing solubility (0.01 mg of
NSD is soluble in 1 ml of water) along with high
permeability [7], due to which formulators consider NSD a problematic drug that is difficult to
formulate as an oral drug delivery product with
high bioavailability.
Current research trends in pharmaceutical
technology are focused on the development of
multi-unit MR formulations like nano- and micro-particles. Multi-unit formulations are capable
of distributing the drug more evenly in the alimentary canal for more even availability of drug to the
systemic circulation [3–4, 8–9].
Proteolytic enzymes such as serratiopeptidase (SPD) have been proposed as good analgesic
and anti-inflammatory agents that are rapidly absorbed in the intestinal region [10–13]. However,
the acidic contents of stomach cause SPD to biodegrade, which results in its very low oral bioavailability [14]. This situation necessitates the development of formulations that deliver the drugs in
the intestine, such as enteric-coated preparations.
The mode of action of SPD involves the thinning of body fluids that accumulate around injured regions, thus enhancing fluid drainage. SPD
also promotes the repair of damaged tissues, followed by a reduction in pain, which is believed to
be due to the blocking of amines. This enzyme also
has a characteristic capability to dissolve dead and
damaged tissue, which is a by-product of healing
activity and causes no harm to living tissue. Serratiopeptidase can also modify cell-surface adhesion
molecules that lead inflamed cells to their target
S.A. Khan et al.
sites and contribute to the development of arthritis
and other autoimmune disorders [3].
Due to the different modes of action of NSD
and SPD, the therapeutic spectrum can be increased
by administering them simultaneously. The present study is focussed on efforts to fabricate an oral
microparticulate MR formulation containing serratiopeptidase and nimesulide.
Moreover, the resulting microparticles were
not compressed into tablets, but were enclosed in
hard gelatin capsules so that the duration of their
stay in the stomach is reduced. As soon as a capsule reaches the stomach, it releases its contents,
which stay in stomach for a very short time, since
the cardiac sphincter is less resistant to very fine
multi-unit dosage forms than to macro-size entities such as tablets, and the denaturing of SPD
in the acidic environment of the stomach can be
avoided.
In the study, NSD and SPD loaded ETC microparticles were prepared for oral delivery in
combined form, filled in hard gelatin capsules
and subjected to various quality control tests in
triplicate. A survey of the literature showed no
sustained-release formulation for SPD or for combined NSD-SPD. Thus, the present study may be
considered a pioneer achievement.
Material and Methods
Material
The nimesulide and serratiopeptidase samples used in the study were received as a gift from
PharmEvo Pharmaceuticals, Pakistan. Ethyl cellulose (22 cp) was procured from Sigma, USA. Dichloromethane was purchased from BDH Chemicals Limited, UK. Methanol and n-hexane were
purchased from Merck, Germany. Mineral oil was
obtained from Acros Organics, USA. All the materials were of analytical grade and were used as
purchased.
Preparation
of the Microparticles
The non-solvent addition coacervation technique was used to prepare SPD-ETC and NMDETC microparticles, employing relatively safe
solvents and mediums such as dichloromethane
(DCM) and mineral oil as the solvent and non-solvent, respectively. First, 1 g of ETC was dissolved in
15 ml of DCM with continuous magnetic stirring
(Velp, Italy) at 700 rpm and then 1 g of the drug
is dispersed in the ETC solution. Phase separation
607
Nimesulide-Serratiopeptidase Sustained Release Microparticles
was achieved by adding mineral oil, and ultimately
a microparticulate suspension was achieved with
a milky color. This was followed by triple washing
with n-hexane, air- and oven-drying for 48 hours
at 45ºC, and then storage in tightly closed containers until the characterizations were done. The
dried microparticles were filled into hard gelatin
capsules (size 000) so that each capsule contained
200 mg of NMD and 20 mg of SPD, which are the
maximum daily doses of the drugs. For stability
studies, equal-weight quantities of microparticles
were placed in nine air-tight amber bottles and
stored at 25ºC ± 0.1 (in an oven, Memmert, Germany), 40ºC ± 0.1 (in an oven) and 40ºC ± 0.1/75%
relative humidity (in an oven). Every month, one
bottle was taken, analyzed for the drug contents
and used for the dissolution study.
In Vitro Release Study
The in vitro dissolution study was conducted
using a USP dissolution apparatus type II. The
temperature of the dissolution medium (phosphate buffer pH 6.8 plus sodium lauryl sulphate
1%, 900 mL) was maintained at 37oC ± 1, with
a stirring rate of 75 rpm. One capsule filled with
microparticles of NSD and SPD was placed in each
dissolution vessel (n = 6). At 0, 0.5, 1, 1.5, 2, 3, 4,
5, 6, 8, 10, 12 and 24 hours, 5 mL of samples were
withdrawn with an automatic sample collector
(Pharma Test, Germany). The volume of the dissolution fluid was adjusted every time to 900 mL.
Samples were assayed spectrophotometrically at
λmax = 404 nm (NSD) and λmax = 230 nm (SPD)
in a double beam UV-visible spectrophotometer
(Shimadzu UV-1601, Japan) [15]. The drug concentration was calculated using a standard calibration curve on a range of 5–30 μg/mL for both
drugs. The regression coefficients for NSD and
SPD were 0.992 and 0.984, respectively.
Determination of Production
Yield and Encapsulation
Efficiency
The production yield (PY %) was calculated
by weighing the raw materials (WR) and the microparticles obtained (WM) and using following
formula:
PY% = WM × 100.
WR
Encapsulation efficiency was assessed by dissolving the weighed quantities of microparticles
from each batch in 10 ml of methanol to dissolve
the ethyl cellulose coating. Then an equal volume
of phosphate buffer pH 6.8 was added and moderately heated to evaporate the methanol. Phosphate buffer pH 6.8 was added in sufficient quantity to bring the final volume up to 900 mL; this
was followed by filtration and analysis at 230 and
404 nm for SPD and NSD, respectively. Encapsulation efficiency was calculated using equation
1 and 2 [16]:
DL% = AD × 100,
AM
(1)
where DL %, AD and AM are drug loading, the
amount of drug in microspheres, and the amount
of microspheres, respectively.
EE% = AL × 100,
TL
(2)
where EE %, AL and TL represent encapsulation
efficiency (%), actual drug loading (%) and theoretical drug loading (%), respectively.
Micromeritic Properties
of the Microparticles
To assess the flowability characteristics of the
microparticles, the angle of repose was evaluated.
It has been proposed that particles exhibit good
flow characteristics if the angle of repose is under
50, and vice versa [17]. The angle of repose was
assessed by passing the microparticles through
a sintered glass funnel with an internal diameter of
27 mm, resulting in the formation of a heap. The
height (h) of this heap and the radius (r) of the
cone base were measured. The following equation
was used to assess the angle of repose (θ):
θ = tan−1 (h/r).
The microparticles were evaluated by assessing their particle size and micromeritic properties,
including tapped bulk density, percentage compressibility index (% CI: a crucial parameter used
for the assessment of flowability) and true density
(d). The diameter of dried microparticles was measured from the scanning electron micrographs. The
density of the microparticles was determined by
immersing them for three days in a 0.02% Tween
80 solution placed in a metal mesh basket, then
using the submerged particles to determine density by the displacement method, using n-hexane
as a non-solvent [4]. Tapped densities and % CI
were evaluated by the tapping method, using the
following equations:
Tapped density = Mass of microparticles/Volume of microparticles after tapping
Compressibility index (%) = [1–V/Vo] × 100,
608
S.A. Khan et al.
where V and Vo are, respectively, the volumes of
the sample after and before the standard tapping.
A 10 mL measuring cylinder was used for the tapping. The initial volume of the microparticles was
noted, followed by continuous tapping at a rate
of 100 taps/min on a hard surface; tapping was
stopped when no further change in the volume
was observed.
Table 1. Physico-chemical characterization of the
microparticles (experiments performed in triplicate)
Tabela 1. Fizykochemiczne właściwości mikrokapsułek
(eksperyment przeprowadzony 3 razy)
Interaction Study
The drug-polymer interaction was studied by
FTIR spectroscopy (MIDAC M2000, USA). Active
SPD and NSD and their microparticles were evaluated by the KBr disc method. The background
spectrum was recorded before studying each sample. The FTIR spectrum of each sample was taken
in the range of 500–4500 cm–1 [16].
The X-ray powder diffractometric analysis of
the microparticles and their individual components was conducted using a D8 Discover (Bruker,
Germany) to detect any possible alteration in the
crystallinity of NMS during microencapsulation.
The sample scanning in a diffraction angle range
from 10° to 70° was conducted using the following experimental conditions: Cu-K∞ radiation
1.5406 A° (source), 4°/min scan speed, scintillation detector, primary slit 1 mm, secondary slit
0.6 mm [16].
Results and Discussion
The non-solvent addition coacervation technique was used to prepare NSD and SPD microparticles. NSD and SPD were dispersed in dichloromethane (the solvent) and mineral oil was
used as a non-solvent to induce coacervation. The
election of ETC as a coating material was made on
the basis of its safety, stability, hydrophobicity and
compactness among hydrophobic polymers [18].
The microparticles of NSD were light yellow,
free flowing and spherical in shape when observed
under a scanning electron microscope, while the
SPD microparticles were white in color. However,
the microparticles of both drugs were aggregated.
The mean particle size for NSD and SPD microparticles was 32 μm and 43 μm, respectively. The
entrapment efficiency of the microparticles was
89% and 85% for NSD and SPD, respectively. The
rest of the micromeritic characteristics are given
in Table 1. Weight variation of the capsule shells
filled with microparticles was within the permitted
compendial limits.
The full drug release profiles (Fig. 1) were evaluated kinetically, and the goodness of fit with various models was investigated: the zero order mod-
Formulations (Właściwości)
NSD
SPD
Bulk density (g/mL)
0.231
0.243
Tapped density (g/mL)
0.243
0.261
Hausner’s ratio
1.052
1.075
5
7
Angle of repose
23.47o
21.78o
Packing rate (C)
0.13
0.15
Yield (%)
96
94
Entrapment efficiency a (%)
89
85
Particle size b (μm)
32
43
t 60% (M ± S.D) (hrs)
5
4
Compressibility index (%)
a
b
a
b
Average of three batches ± SD.
Average of one hundred microcapsules ± SD.
Średnia trzech partii ± SD.
Średnia 100 mikrokapsułek ± SD.
el, the first order model, the Higuchi model, the
Hixson-Crowell model and the Korsmeyer-Peppas
model. The one with the highest co-efficient of determination R2 was deemed the appropriate model.
The release profiles of all the microparticles were
best explained by the Higuchi model, with the
highest linearity (R2 = 0.97 and 0.98 respectively
for SPD and NSD), followed by the zero order
model (R2 = 0.88 and 0.94 respectively) and the
first order model (R2 = 0.49 and 0.52 respectively).
This indicates that the drug release is controlled
by diffusion through pores in the coating and not
through the swollen polymer [19]. The n-values,
calculated by the Korsmeyer-Peppas model, were
in the range of 0.75–0.62, which confirmed the release of NMS from ETC microparticles via nonFickian/anomalous diffusion. The erosion seems
to be the result of drug release. Judging from the
structure of the microparticles, diffusion seems to
be the predominant mechanism (Fig. 2).
There were no significant differences in the
release profiles of the various batches of microcapsules, which indicates that the processes used
in their manufacture are reliable and reproducible
(p > 0.05, f1 < 0.28, f2 > 99.67). It was also noted
there was no alteration in the release behavior for
up to three months of storage. In addition, there
were no changes in the physical characteristics of
the microparticles, indicating that NSD and SPD
were stable in the ETC microparticles for up to
three months.
609
Nimesulide-Serratiopeptidase Sustained Release Microparticles
Table 2. Goodness of Fit (using R2) for the mathematical models used to determine the drug release rate from NSD and SPD
microparticles [Y-equation (Y = aX + b), determination co-efficient (R2), and release exponent (n)] (experiments performed
in triplicate)
Tabela 2. Dobroć dopasowania (z użyciem R2) dla modeli matematycznych stosowanych w celu określenia tempa uwalniania
leku z mikrokapsułek NSD i SPD [równanie (Y = aX + b), określenie współczynnika (R2), a następnie wykładnika uwalniania
(n)] (doświadczenie przeprowadzone 3 razy)
Model
Parameters (Wskaźniki)
Zero Order
First Order
Higuchi
Korsmeyer-Peppa
Microparticles (Mikrokapsułki)
NSD
SPD
Y-equation
6.61x + 14.65
6.77x + 19.36
R2
0.94
0.88
Y-equation
0.19x + 2.52
0.19x + 2.63
R
0.52
0.49
Y-equation
27.53x – 6.28
29.09x – 3.81
R
0.98
0.97
Y-equation
0.62x + 2.98
0.75x + 2.97
R
0.99
0.99
0.62
0.75
2
2
2
n (release exponent)
drug release
125
%
100
75
50
NSD
25
SPD
0
0
6
12
18
time Hrs
The FTIR spectra of the formulations showed
all the characteristic peaks of the drugs, including
C-H aromatic, S=O, C-O-C ether linkage, CH3
C-H bending, NO2, CH3 C-H stretching and N-H
(Fig. 3). Thus, the spectroscopy results confirmed
that there was no strong interaction of ETC with
these drugs when they were encapsulated in ETC
coating.
The X-ray diffractometry showed the amorphous and crystalline nature of nimesulide in both
its encapsulated and free forms (Fig. 4). There was,
however, a decrease in the signal intensity (i.e. the
crystallinity) of the drugs in microparticle form as
compared to the pure components, which is an indication of a slight shift from a crystalline nature
to an amorphous one. This change increases the
24
Fig. 1. Dissolution profile of NSD and SPD
microparticles (experiments performed in
triplicate)
Ryc. 1. Profil rozpuszczania mikrokapsułek NSD i SPD (doświadczenie przeprowadzone 3 razy)
solubility of the drugs, which ultimately increases
their bioavailability [16].
This study showed that the non-solvent addition coacervation technique employing dichloromethane and mineral oil as solvent and nonsolvent, respectively, is an appropriate method
for microencapsulate drugs with different physicochemical properties (such as SPD and NSD)
in ETC coating. The variations observed in the
entrapment efficiency, production yield, mean
particle size and the drug release behavior of the
formulations can be attributed to the nature of
the drugs. This suggests the potential of ethyl cellulose microparticles as a suitable multi-unit sustained release drug delivery system, because it does
not affect the nature of the drugs and there is no
610
Fig. 2. Scanning electron microscope images of the
external structure of NSD and SPD microparticles
Ryc. 2. Zewnętrzna struktura mikrokapsułek NSD
i SPD – obraz ze skaningowego mikroskopu elektronowego
S.A. Khan et al.
Fig. 4. X-ray diffractometer analysis of active nimesulide (A), active serratiopeptidase (B), ethylcellulose (C),
microparticles of nimesulide (D), microparticles of serratiopeptidase (E)
Ryc. 4. Rentgenowska analiza dyfrakcyjna aktywnego
nimesulidu (A), aktywnej serratiopeptydazy (B),
etylocelulozy (C), mikrokapsułek nimesulidu (D)
i mikrokapsułek serratiopeptydazy (E)
Fig. 3. FTIR spectra of active nimesulide (A),
active serratiopeptidase (B), ethylcellulose
(C), microparticles of nimesulide (D) and
microparticles of serratiopeptidase (E)
Ryc. 3. Widmo IR aktywnego nimesulidu
(A), aktywnej serratiopeptydazy (B), etylocelulozy (C), mikrokapsułek nimesulidu (D)
i mikrokapsułek serratiopeptydazy (E)
Nimesulide-Serratiopeptidase Sustained Release Microparticles
chemical bonding between these drugs and ethyl
cellulose during the microencapsulation method.
The in vitro characterization confirms this pioneer
formulation as a suitable combined-drug delivery
611
system for NSD and SPD. At the time of writing
an in vivo characterization is in progress in the authors’ laboratory, and it will be presented after its
completion.
Acknowledgement. The authors are very grateful to PharmEvo Pharma for generously providing the sample of nimesulide and serratiopeptidase. We also thank the Research Foundation of the Higher Education Commission (HEC),
Pakistan, for the financial support given to this study.
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Address for correspondence:
Ghulam Murtaza
Department of Pharmaceutical Sciences
COMSATS Institute of Information Technology
Abbottabad
Pakistan
Tel.: 0092 0314 2082826
E-mail: [email protected]
Conflict of interest: None declared
Received: 10.01.2011
Revised: 22.08.2011
Accepted: 5.10.2011