chromium picolinate loaded superporous hydrogel and...
TRANSCRIPT
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Chromium Picolinate Loaded Superporous Hydrogel and
Superporous Hydrogel Composite as a Controlled Release Device:
In-vitro and In-vivo Evaluation
Sally A. Abdel Halim*, Soad A.Yehia, Mohamed A. El-Nabarawi
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El-
aini street, Cairo 11562, Egypt
*Corresponding Author: [Sally Adel Abdel Halim]
Postal Address: Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo
University, Kasr El-aini street, Cairo 11562, Egypt
Tel: 00201005077279 Email: [email protected]
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Abstract
The aim of this work was to develop chromium picolinate (CP) loaded
gastroretentive device using superporous hydrogel (SPH) and superporous
hydrogel composite (SPHC). The drug was considered as good candidate for
such systems owing to its narrow absorption window. Swelling ratio, apparent
density, scanning electron microscopy (SEM), drug content and drug release in
pH 1.2 were evaluated for hydrogels. SEM of hydrogels showed interconnected
pores with extensive capillary insertion. Swelling ratio for CP- SPH was higher
than that of SPHC while apparent densities were lower. Both SPH and SPHC
retarded drug release as values of half-life attained 3.64 and 2.94h respectively
while plain drug 0.22h. The mechanical strength of SPHC was higher than SPH,
so it was selected for in-vivo studies in dogs. Radiographic examination in dogs
showed that gastric retention persisted for 24h. Percentage relative
bioavailability was 298.8%. SPHC could be thus considered as good
gastroretentive device for CP.
Keywords: Controlled release formulations, chromium picolinate, superporous hydrogel
composite, gastric retention, radiographic examination,
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1- Introduction
Since drug delivery technology (1)
is an equivalent component in drug
development, a successful achievement is design of delivery systems that can
target a candidate drug to its absorption site (2-3)
.
Among the different systems and devices used to control the drug
delivery to the GIT, academic researchers have attracted much attention to
gastroretentive dosage forms (4)
. Those systems are advantageous in case of
drugs characterized by a narrow absorption window. They increase efficacy by
providing a prolonged intimate contact with the absorbing membrane (5)
.
For a successful development of a gastroretentive system, the selected dosage
form, before the normal physiology of the stomach can clear up it to the
intestine, must be able to reside for a time necessary to release the entire drug
included (6)
.
Several attempts have been made to attain gastroretention through
different systems including bioadhesion (7)
or mucoadhesion to gastric mucosa
(8-10), high density systems
(11), floating systems
(12-15), and expandable systems
(16). In our study, we focused specifically on superporous hydrogel systems, as
the fast swelling (17)
highly porous nature (18-20)
of these devices made them
perfect candidate materials for gastroretentive delivery of many drugs (5)
.
Owing to their unique properties, superporous hydrogels swell to a
volume much larger than the opening of the pylorus (21)
, when applied as drug
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carriers; they remains in the stomach for the time necessary to release the
loaded drug within their matrices before they begin to degrade (22)
.
The new technology found extensive pharmaceutical application.
Dorkoosh etal succeeded to prepare superporous hydrogel polymers loaded with
peptide drugs such as buserelin, octreotide and insulin, and proved that these
devices were promising systems for peroral peptide drug delivery (23-24)
. Later
on, Yin etal were able to improve the intestinal absorption of insulin using
superporous hydrogel containing interpenetrating polymer network (IPN) (25)
. A
more recent study achieved by Gümüşderelioğlu etal demonstrated the
superiority of superporous polyacrylate/chitosan interpenetrating network
hydrogels for protein delivery. Bovine serum albumin was taken as a model
protein. Loading was performed by the soaking method before and after IPN
formation (26)
.The method of soaking superporous hydrogels in drug solutions
was also employed in loading rosiglitazone maleate on swelled polymeric
matrix (27)
. Mahmoud etal incorporated a self-nanoemulsifying drug delivery
system into the SPHC matrix (28)
. The incorporation of ranitidine hydrochloride
and release retardant polymers in SPHC through a central hole was
demonstrated by Chavda etal. A piece of SPHC was used to close the hole by
the aid of biodegradable glue. The whole system was used to sustain the
delivery of the drug over 17 hours (29)
.
Chromium (as a mineral) is an essential trace element involved with lipid
and glucose metabolism, circulating insulin levels, and the peripheral activity of
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insulin (30)
. In vitro and in vivo studies suggest that chromium potentiates the
activity of insulin (31)
.
Active transport of minerals in general is an important mechanism of
homeostatic control. The minerals in foods are normally present at low
concentrations. Active transport mechanisms have evolved to ensure their
absorption (32)
.
Our present study deals with the development of a new gastroretentive
release formulation of chromium picolinate (our drug of choice) to enhance its
bioavailability through gastric retention and controlled presentation to intestinal
carriers and to assess the efficiency of the prepared gastric retention devices.
We investigated the possibility of designing a SPHC carrier device loaded with
chromium picolinate. The prepared formulae were evaluated through in-vitro
and in-vivo testing taking dogs as animal model.
2- Materials and Methods
2.1. Materials
2.1.1. Chemicals
Chromium picolinate (Lonza, Germany), kindly supplied by MEPACO Co.,
Egypt. Hydrochloric acid: (Prolabo, France). Acrylic acid (AA), acrylamide
(AM), N-isopropyl acrylamide (NIPAM), hydroxyethylmethacrylate
(HEMA), potassium salt of 3-sulfopropylacrylate (SPAK), (2-(acryloyloxy)
6
ethyl) trimethylammonium methyl sulphate (ATMS), N,N'-
methylenebisacrylamide (Bis), ammonium persulphate (APS), N,N,N',N'-
tetramethylethylenediamine (TEMED), all are Aldrich Chemical Company,
USA). Pluronic® F127 (PF-127) (BASF Corporation, Chemical division,
Parslppany, N. J.,USA). Cross linked carboxymethylcellulose powder (Ac-
Di-Sol®) FMC corp., Pennsylvania, USA). Barium sulphate (El Nasr chemical
company, Egypt). Triton X100 (Sigma Chemicals, USA). Absolute ethyl
alcohol, sodium bicarbonate, hexane, sodium chloride, sodium hydroxide and
Nitric acid (Analytical grade).
2.1.2. Animals
12 mixed – breed dogs – Age (1.5 – 2 years), weight (≈20 kg)
2.2 Methods
Two types of superporous hydrogels were synthesized in this study, these
are superporous hydrogel (SPH) and superporous hydrogel composite (SPHC).
2.2.1.Synthesis of Superporous Hydrogels:
Superporous hydrogels were synthesized using various vinyl
monomers(33)
. Table (I) shows different formulae synthesized in our study.
In general, to make superprous hydrogel, a monomer, crosslinker,
deionized distilled water (DDW) (if necessary), foam stabilizer, acid,
polymerization initiator, initiation catalyst (if any), and foaming agent were
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added sequentially to a test tube (20 mm outer diameter x 150 mm in length).
The test tube was shaken to mix the solution after each ingredient was added.
The pH of the monomer solution was adjusted to 5 using hydrochloric acid
(HCL). For monomers with low pH (such as AA), the monomer solution was
titrated with NaOH to raise the pH to 5-6. When sodium bicarbonate was added
the whole mixture was stirred instantaneously using thin spatula for several
seconds to evenly distribute the generating gas bubbles. Synthesized
superporous hydrogels were removed from test tube after 10 minutes and
allowed to swell in water before drying.
2.2.2. Drying of Superporous Hydrogels:
Superporous hydrogels were dried under two different conditions. Under
first condition (a), swollen superporous hydrogels were dried for one day in an
oven at 60 oC. Under second condition (b), swollen superporous hydrogels were
dehydrated first by applying about 10 ml of absolute ethanol per each gel. After
this initial dehydration step, superporous hydrogels were dehydrated further by
placing them in 50 ml of absolute ethanol several times to ensure replacement
of all the water by ethanol. After the dehydration was completed, the excess
ethanol in dehydrated superporous hydrogels was removed by draining using
filter paper. Then the superporous hydrogels were dried in an oven at 50 oC for
one day.
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2.2.3. Incorporation of Chromium Picolinate into Superporous Hydrogels:
The same procedure for synthesis of superporous hydrogels was
followed. Drug was added to the test tube directly before sodium bicarbonate
and stirred thoroughly using spatula, then just after the addition of sodium
bicarbonate, the whole mixture was stirred using thin spatula for several
seconds to evenly distribute the generating gas bubbles.
Synthesized chromium picolinate superporous hydrogels were removed
from test tube after 10 minutes and allowed to swell in water before drying
using condition (b).
2.2.4. Synthesis of Superporous Hydrogel Composites:
Superporous hydrogel composites were synthesized using various vinyl
monomers (34)
. Table (II) shows different formulae used in our study.
In general, to make superprous hydrogel composite, a monomer,
crosslinker, deionized distilled water (DDW) (if necessary), foam stabilizer,
acid, polymerization initiator, Ac-Di-sol, initiation catalyst, and foaming agent
were added sequentially to a test tube (20 mm outer diameter x 150 mm in
length). The test tube was shaken to mix the solution after each ingredient was
added. The pH of the monomer solution was adjusted to 5 using hydrochloric
acid (HCL). When sodium bicarbonate was added the whole mixture was
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mechanically stirred instantaneously using thin spatula for several seconds to
evenly distribute the generating gas bubbles.
Synthesized superporous hydrogel composites were retrieved from test
tube after 10 min curing time and washed in a 1-litre beaker containing 400 ml
of 0.1N HCL for 24 hr (acidification). The superporous hydrogel composites
were then dried at room temperature for 5 days.
2.2.5. Incorporation of Chromium Picolinate into Superporous Hydrogel Composites:
The same procedure for synthesis of superporous hydrogel composites
was followed. Drug was added to the test tube directly before sodium
bicarbonate and stirred thoroughly using spatula, then just after the addition of
sodium bicarbonate, the whole mixture was stirred using thin spatula for several
seconds to evenly distribute the generating gas bubbles.
Synthesized chromium picolinate superporous hydrogels were retrieved
from test tube after 10 min curing time and washed in a 1-litre beaker containing
400 ml of 0.1N HCL for 24 hr (acidification). The chromium picolinate
superporous hydrogel composites were then dried at room temperature for 5
days.
2.2.6. Wetting of Superporous Hydrogels and Superporous Hydrogel Composites:
Each dried superporous hydrogel or superporous hydrogel composite was
placed in a beaker on the top of a support in desiccator containing saturated
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solution of sodium chloride (relative humidity 75%) at the bottom and well
closed with tight cap at room temperature (33)
.
2.2.7. Evaluation of Superporous Hydrogels and Superporous Hydrogel Composites
2.2.7.1. Swelling Studies:
The dry samples were placed on sieve weighing boat (33)
. The sieve
weighing boat containing the dry sample was immersed in excess deionized
distilled water (DDW) at room temperature. The weighing boat was taken out to
drain the free water from the sieve and a paper towel was used to remove excess
water from underneath the sieve. Then the weight (Electrical balance; Sartorius
GmbH, Gottingen, Germany) of the swollen samples was measured by
subtracting the boat weight from total weight. This method avoided direct
handling of the gel. The weights of hydrating samples were measured at
predetermined time intervals at 37 ºc.
The swelling ratio (Q) (35)
is defined as: Q = Ws / Wd
Where Ws is the weight of swollen sample and Wd is the weight of dried sample.
2.2.7.2. Determination of Apparent Density:
Densities of the dried superporous hydrogels and superporous hydrogel
composites were determined from direct mass and dimensional
measurements(18)
. The density (d) of a dried sample was calculated by dividing
the weight of a dried sample (Wd) with the volume of the dried sample (Vd). The
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volume (Vd) was calculated by a solvent displacing method. Briefly, with the
use of forceps, a dried sample was immersed in a predetermined volume of
hexane in graduated cylinder and the increase in the hexane volume was
measured as the volume of the dried sample.
2.2.7.3. Estimation of Drug loading:
An accurately weighed amount (0.1g) of the dried chromium picolinate
superporous hydrogels and chromium picolinate superporous hydrogel
composites samples was added to a beaker containing 250 ml water and stir for
24 hrs, filter then complete volume to 250ml with water. The absorbance of the
solution was determined after carrying the appropriate dilution at max 265 nm
using water as a blank. All experiments were carried out in triplicates.
2.2.7.4. In-vitro Chromium Picolinate Release Study:
The release of the drug from the prepared dried chromium picolinate SPH
and chromium picolinate SPHC samples was studied using USP dissolution
apparatus type II (Pharma test, Germany). In-vitro release studies were carried
out at 37±0.5 oC in 250 ml of 0.1N HCL for 24 hr. The paddles were rotated at
100 rpm and aliquots each of 3 ml from the release medium were withdrawn at
predetermined time interval. The withdrawn samples were replaced with equal
volumes of the release media. The aliquots were passed through a millipore filter
of 0.22m and assayed spectrophotometrically at 265nm.
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All experiments were carried out in triplicates and the mean results are
illustrated in figures (1A and 1B).
2.2.7.5. Kinetic Study of Release Data of Chromium Picolinate
Superporous Hydrogels and Chromium Picolinate Superporous
Hydrogel Composites:
The data obtained from the release experiments were analyzed by means
of personal computer to find out the mechanism of drug release. The following
linear regression equations were employed:-
A) Ct = Co – kt for zero order kinetics.
B) log Ct = - kt/2.303 + log Co for first-order kinetics.
C) CsCsADtQ )2( for Higuchi-diffusion model (36-38)
Where Q is the amount of drug released per unit area at time t, D is the
drug diffusion coefficient in the matrix, A is the total amount of drug present in
the matrix per unit volume and Cs is the drug solubility in the matrix. This
equation describes drug release as being linear with the square root of time Q =
Kt1/2.
D) Mt/M∞ =K tn Korsmeyer-Peppas model
(39)
Where Mt/M∞ is the fraction of drug released at time t; K a constant
comprising the structural and geometrical characteristics of the system and n;
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the release exponent, is a parameter which depends on the release mechanism
and is thus used to characterize it (40)
. The determination coefficients (r2) were
calculated.
2.2.7.6. Morphological Analysis of Superporous Hydrogels and
Superporous Hydrogel Composites:
The morphology of porous structure of the selected formulae was
examined with scanning electron microscope (SEM) (Joel JXA – 840 A,
Electron probe, Microanalyser). Dried samples were cut to expose inner
structure. The samples were prepared separately on sample holders. The holders
were coated with gold palladium using sputter coater for one minute under
argon gas before electron microscope scanning. Results are illustrated in figures
(2 and 3)
2.2.8. Synthesis of Radio-opaque Chromium Picolinate Superporous
Hydrogel Composites (RO-CP-SPHC):
Radio-opaque superprous hydrogel composite was synthesized by
addition of 1200 l (50% AM ) + 900 l (50% SPAK) as monomer, 450 l (2.5
% Bis) as crosslinker, 90 l (10 % Pluronic F127) as foam stabilizer, 30 l
acrylic acid, 0.5 ml 40% BaSO4 (41)
, 45 l 20 % APS as polymerization
initiator, 270 mg Ac-Di-Sol, 45 l 20% TEMED as initiation catalyst, 0.05 g
chromium picolinate and 100 mg NaHCO3 (foaming agent) were added
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sequentially to a test tube (20 mm outer diameter x 150 mm in length). The test
tube was shaken to mix the solution after each ingredient was added. The pH of
the monomer solution was adjusted to 5 using hydrochloric acid (HCL). When
sodium bicarbonate was added the whole mixture was mechanically stirred
instantaneously using thin spatula for several seconds to evenly distribute the
generating gas bubbles.
Synthesized superporous hydrogel composites were retrieved from test
tube after 10 min curing time and washed in a 1-litre beaker containing 400 ml
of 0.1N HCL for 24 hr (acidification). The superporous hydrogel composites
were then dried at room temperature for 5 days. Then dried superporous
hydrogel composite was placed in a beaker on the top of a support in desiccator
containing saturated solution of sodium chloride (relative humidity 75%) at the
bottom and well closed with tight cap at room temperature. This SPHC became
flexible so can be easily squeezed and packed in capsules (size 000).
2.2.9. In-vitro Evaluation of the Synthesized Radio-opaque Chromium
Picolinate Superporous Hydrogel Composites (RO-CP-SPHC):
Swelling studies, determination of apparent density and estimation of
drug loading were done as previously mentioned. In-vitro chromium picolinate
release study from (RO-CP-SPHC) and kinetic study of release data also done
as before and illustrated in figure (4). Morphological Analysis of Radio-opaque
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Chromium Picolinate SPHC was done as previously mentioned using scanning
electron microscope and illustrated in figure (5)
2.2.10. In-vivo Evaluation of the Prepared Superporous Hydrogel
Composites in Dogs:
2.2.10.1. Animals:
The 12 experimental dogs used in this study were housed in individual
cages. They were fasted for 18 hours with (ad libitum; access to water) before the
experiment (42)
.
2.2.10.2. Experimental design:
2.2.10.2.1. Dosing:
The dogs are divided into two groups; each group consisted of six dogs.
Group (I): Each dog received one capsule containing radio-opaque chromium
picolinate superporous hydrogel composite (RO-CP-SPHC) = 200 g chromium
picolinate.
Group (II): Each dog received one market capsule = 200 g chromium
picolinate.
2.2.10.2.2. Study Schedule:
At the beginning of the experiment all dogs were cannulated for blood
sampling and blood samples were obtained. The cannulas were flushed with at
least 2-3 mls blood, which was discarded.
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Each dog was fed with 450 g canned food then via oral-gastric tube, 400
ml water was administered. Directly after water, capsules are given orally.
Blood samples were collected in acid washed tubes at times (0, 0.5, 1, 2, 3, 4, 6,
8, 12 and 24 h) post administration. Collected blood samples were immediately
centrifuged (Mechanika precyzyn, Poland) and the serum kept frozen pending
analysis. The protocol of the experiment was approved by the institutional
review board of the Research Ethics Committee of Faculty of Pharmacy, Cairo
University, Egypt for the use of animals in scientific experiments. The ethical
approval is firmly based on the protection of the used animals (concerning
housing including the place, food and water intake besides all the needed care)
for the experiment which was carried out by qualified persons.
2.2.10.2.3. Radiography:
Radiographic examinations were performed using Fisher x-ray generating
unit (50 kv, 100 mA, 0.1 sec) (Fisher R 183, Emerald tube 125) to determine the
anatomical location of the gastric retention dosage forms (42)
. For each dog in
group (I), radiographic examinations were performed from two angles, a lateral
view and a dorsoventral view. Radiographs for dogs were exposed at 0 hour (just
before dosing to ensure an empty stomach), at 15 minutes (just after dosing to
ensure that the device was in the stomach) then 1, 2, 3, 4, 6, 8, 12 and 24 h.
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The protocol of the experiment was approved by the institutional review
board of the Research Ethics Committee of Faculty of Pharmacy, Cairo
University, Egypt for the use of animals in scientific experiments.
2.2.10.2.4. Serum Analysis:
Chromium in serum is mainly bound to transferrin and albumin, therefore
chromium was dissociated from these proteins by acid denaturation without
enzymatic solubilization (43)
.
Chromium was measured in 20 l (15 l serum + 5 l 0.1% triton X100)
using Zeeman Atomic Absorption Spectrophotometer with graphite furnace
(AAS-GF) (Perkin – Elmer 4100 ZL, Perkin Elmer, Norwalk, CT). The
concentration of chromium was calculated by linear regression analysis.
Chromium was determined using Perkin Elmer Model 4100 ZL atomic
absorption spectrophotometer equipped with a Zeeman Background corrector,
Graphite furnace, AS-40 autosampler. All signals were monitored at 357.9 nm
with a slit width 0.7 nm (43)
.
2.2.10.2.5. Bioavailability and Pharmacokinetic Studies of Chromium
Picolinate Gastric Retention Delivery Systems:
To assess the bioavailability of chromium picolinate, the serum
concentration–time data were evaluated, and the following pharmacokinetics
parameters were calculated using WinNonlin software:
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1- Cmax (ng/ml): was determined as the highest observed concentration
during the study period.
2- Tmax (hours): was taken as the time at which Cmax occurred.
3- Mean Residence Time (MRT)(hour): was determined from the time of
dosing to the last measurable concentration
4- AUC0-24 (ng.hr/ml): was determined as the area under the plasma
concentration time curve up to the last measured time point calculated
by trapezoidal rule (44)
.
5- AUMC 0-24 (ng.hr2/ml): It is the area under the first moment curve.
6- Relative bioavailability: is calculated as percentage value (45).
RB % = (AUC (o-t) test / AUC (o-t) control) x 100.
Table (III) and figure (7) compiled and illustrated the results of this
bioavailability study.
2.2.10.2.5. Statistical comparison of pharmacokinetic parameters:
The ANOVA test, followed by least significant difference multiple
comparison tests were used to assess the statistical significance of difference
between the results following extravascular mode of administration using social
package for statistical studies (SPSS 17). A P value of less than 0.05 was
considered significant.
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3. Results and Discussion
In this study, porous hydrogels were synthesized with open channels
using the gas blowing (or foaming) technique (46-48)
. Superporous hydrogels
prepared by the gas blowing technique also were called ‘‘hydrogel foams’’ due
to the foaming process used in the preparation(46,47)
. To be practical, the swelling
had to be completed in less than 30 minutes, most preferably in less than 5
minutes. Thus, our efforts have been focused on the synthesis of hydrogels that
swell to equilibrium sizes in less than a few minutes.
3.1. Synthesis of Superporous Hydrogels:
Superporous hydrogels (33)
were prepared by crosslinking polymerization
of monomers in the presence of gas bubbles. Carbon dioxide gas bubbles were
generated by reaction of sodium bicarbonate with acid (Acrylic acid (AA) or
HCL). The foam size was determined by the amount of released gas bubbles,
which in turn, was determined by the amount of acid and NaHCO3. We used
excess amounts of NaHCO3 so that the foam size was controlled by the amount
of the added acid. To make superporous hydrogels with homogeneously
distributed gas bubbles, polymerization and foaming processes had to occur
simultaneously. Thus, control of timing of the two processes was critical. Since
stabilizing foam longer than a few minutes was difficult, the gelling had to start
within a few minutes after the beginning of foaming (e.g., after addition of
NaHCO3 to the monomer mixture). The fast gelling could be achieved by a
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careful choice of monomers (type and concentration), initiators (type and
concentration), temperature, and solvent. Table (I) shows the composition of
superporous hydrogels using various monomers.
Typically, acrylamide (AM), sodium salt of acrylic acid, (2-(acryloyloxy)
ethyl) trimethylammonium methyl sulphate (ATMS), N-isopropyl acrylamide
(NIPAM), potassium salt of 3-sulfopropylacrylate (SPAK), and their copolymers
gelled quite fast in aqueous solution when the ammonium persulphate (APS)/
N,N,N',N' tetramethylethylenediamine (TEMED) pair was used as the initiator.
The monomer concentrations used in our study were higher than 10% to ensure
fast gelling. Some monomers such as hydroxyethylmethacrylate (HEMA))
polymerized too slowly without increasing the temperature to 60°. The
APS/TEMED redox–initiator pair was effective for polymerization of all of the
monomers listed in table (I). They initiated the gelling process within 1–2 min
when used at a concentration of about 1–2% (w/w) of the monomer.
3.2. Polymerization and Foaming Processes:
For making homogeneous superporous hydrogels, the timing of foam
formation and polymerization processes was very critical. The timing for the
addition of the foaming agent and the onset of gelling had to be controlled
carefully. The NaHCO3/acid system used in our study provided a special trigger
system that made controlling the timing rather easy.
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3.3. Drying of Superporous Hydrogel:
To find the optimum drying condition that did not alter the swelling ratio
of dried superporous hydrogels, we examined two different drying conditions.
Air dried (condition (a)) and alcohol dried (condition (b)). During the
dehydration process under condition (b) of drying, the soft and flexible
superporous hydrogels became hard and brittle due to fast evaporation of the
alcohol. This is in accordance with J. Chen et al.(33)
who studied the effect of
drying conditions on superporous hydrogel. Effect of drying condition on
different parameters (e.g swelling ratio and density) will be discussed later.
3.4. Wetting of Superporous Hydrogel:
All formulae were subjected to wetting by water as it was reported that
wetting agents increase the swelling rate of polyacrylate hydrogel particles. (49)
Water itself is a best wetting agent (33)
, and so moisture was absorbed into the
dried superporous hydrogels in a controlled manner using a moistening
chamber. Wetting is also important to make the SPH soft and flexible to be
easily compressed in a capsule.
3.5. Evaluation of Superporous Hydrogels Formulae:
3.5.1. Swelling Studies:
The swelling ratios (Q) for superporous hydrogel formulae dried under
condition (a) are 64.71, 29.79, 12.23, 1.58, 41.56, 44.13, 32.12 and 6.48 for
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F#1, F#2, F#3, F#4, F#5, F#6, F#7 and F#8 respectively. However, the swelling
ratios (Q) for superporous hydrogel formulae dried under condition (b) were
75.33, 35.22, 15.7, 1.64, 51.13, 56.41, 38.93 and 7.94 for F#1, F#2, F#3, F#4,
F#5, F#6, F#7 and F#8 respectively.
It was clear that in plain formulae (F#1 → F#8) there is a marked increase
in swelling ratios when using drying condition (b). When a superporous
hydrogel dried under condition (a) was placed in water, the outer region swelled
to equilibrium only seconds after contact with water. This swelling changed the
outer region from opaque to clear. With the penetration of water, the clear
region gradually expanded towards the center. This penetration step was quite
slow and took most of the swelling time. The center part remained opaque until
water penetrated through. Once water reached the center, the central region
became clear and swelled to the fully swollen state in just a few seconds. The
slow penetration into the center of the dried superporous hydrogels indicated
that the drying under condition (a) somehow disrupted the capillary channels. It
is likely that the removal of water during drying resulted in collapse of polymer
chains and the pores due to the high surface tension of water (33)
. However,
swelling ratio (Q) for formulae dried under condition (b) was high as during
ethanol dehydration the SPH became rigid, probably due to precipitation of
polymer chains in a poor solvent. This rigid structure might have contributed to
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better maintenance of pore structures during drying in ethanol, which has low
surface tension (33)
.
Our study had shown that fast swelling can be achieved by preserving the
capillary channels during drying and by making the surface of pores more
wettable. The swelling ratio (Q) was arranged as follows: F#1 > F#6 > F#5 >
F#7 > F#2 > F#3 >F#8 > F#4 under both drying conditions.
It was concluded that drying SPH under condition (b) is superior to
condition (a) and that F#1, F#6 and F#5 gave the highest swelling ratios.
3.5.2. Determination of Apparent Density:
Solvent displacement method was used to determine the volume of the
sample of SPH. Hexane was used because it is very hydrophobic and superporous
hydrogels do not absorb it.
The apparent densities for superporous hydrogel formulae dried under
condition (a) are 0.31, 0.75, 0.86, 0.85, 0.67, 0.47, 0.7 and 0.86 g/cm3 for F#1,
F#2, F#3, F#4, F#5, F#6, F#7 and F#8 respectively. However, the apparent
densities for superporous hydrogel formulae dried under condition (b) were
0.25, 0.66, 0.74, 0.75, 0.49, 0.43, 0.6 and 0.75 g/cm3 for F#1, F#2, F#3, F#4,
F#5, F#6, F#7 and F#8 respectively. All apparent densities are less than density
of the gastric fluid (≈1.004) (50)
and caused floating of the prepared formulae. So
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their gastric retention may be due to floating together with large volume after
swelling.
The apparent densities for SPH dried under condition (a) were arranged
as follows: F#3 = F#8 > F#4 > F#2 > F#7 > F#5 >F#6 > F#1, while the apparent
densities for SPH dried under condition (b) were arranged as follows: F#4 = F#8
> F#3 > F#2 > F#7 > F#5 >F#6 > F#1.
It was clear that apparent densities of SPH dried under condition (b) are
lower than apparent densities for SPH dried under condition (a). This could be
due to high surface tension of water which resulted in collapse of polymer chains
and the pores during drying. This shrinkage of SPH dried under condition (a)
cause rather high apparent densities. However, low surface tension of ethanol
results in precipitation of polymer chains in a poor solvent which made the SPH
rigid during ethanol dehydration (condition b). This rigid structure results in less
shrinkage of the gel during drying as indicated by lower densities (33)
.
Also, apparent densities of F#1, F#5 and F#6 possessed the lowest
apparent densities and the highest swelling ratio.
3.5.3. Incorporation of Chromium Picolinate into Superporous Hydrogels:
F#1, F#5 and F#6 were chosen for their highest swelling ratio and lowest
apparent densities for further studies. Drying condition (b) was also selected to
be further use.
25
Chromium picolinate was incorporated in the selected SPH formulae by
adding it just before addition of NaHCO3, so as effervescences occurred
promoted efficient mixing of the drug with the rest of the components. F#9,
F#10 and F#11 are medicated SPH as shown in table (I).
The swelling ratio (Q) for chromium picolinate SPH were 69.77, 48.74
and 49.79 while apparent densities were 0.3, 0.51 and 0.49 g/cm3 for F#9, F#10
and F#11 respectively. The decrease in swelling ratios and increase in apparent
densities of medicated SPH than plain SPH may be due to presence of drug
crystals in the capillary channels which could lead to preventing total
penetration of the water in the channels.
3.5.4. Estimation of Drug loading:
The chromium picolinate content of different formulae of superporous
hydrohels was 98.13, 134.28 and 111.45 g per one mg of SPH for F#9, F#10 and
F#11 respectively.
As previously mentioned that SPH prepared from AM (F#1) possessed the
highest swelling ratio, so it is suggested that it possessed the lowest drug content
because it contained more open interconnected capillary channels which cause
escape of the drug during swelling in water after its preparation and before drying.
3.5.5. In-Vitro Chromium Picolinate Release Study from SPH Formulae:
26
Figure (1A) shows the release profile of chromium picolinate from
different formulae of superporous hydrogels using different monomers in pH
1.2 at 37 °C. F#9, F#10 and F#11 showed a flush of 11.85, 2.69 and 8.63 %
respectively during the first 5 minutes.
The initial burst effect may result from rapid dissolution of drug crystals
at or near the surface of the outer matrix of the SPH. After 12 hours of the
release, the SPH formulae released 99.71, 79.26 and 95.32 % for F#9, F#10 and
F#11 respectively. F#9 showed the highest burst effect and the highest
percentage released after 12 hours followed by F#11 then F#10. This result
coincides with swelling results. It is suggested that the most porous formula
released the highest percentage of drug due to rapid and high swelling which
caused good penetration of the dissolution medium.
The release data were kinetically treated, where the computed
determination coefficient (r2) was taken as a criterion for estimation of the order
of chromium picolinate release from its different superporous hydrogel
formulae in pH 1.2 followed by mathematical, statistical and kinetic constants
computation.
The release of chromium picolinate from F#9 and F#11 followed first
order while release from F#10 followed diffusion model (data no shown).
27
Since some of the values of (r2) were very close, differ by the second or
third digit, thus a simple exponential relation was introduced to describe the
general release behavior of the gel as follows (39)
: Mt/M∞ =K t
n
Where Mt/M∞ is the fraction of drug released at time t; K a constant
comprising the structural and geometrical characteristics of the system and n; the
release exponent, is a parameter which depends on the release mechanism and is
thus used to characterize it (40)
. SPH had 0.43 < n values < 1, thus followed
anomalous transport.
For more confirmation of the release order mechanism, the following
equation was applied: Mt/M∞ = K1t1/2
+ K2 t
Where Mt/M∞ is the fraction of drug released in time t, K1 and K2 are
constants describing diffusion controlled and constant rate release respectively.
The K1 and K2 are obtained from non linear regression curve fitting of the release
data. When the ratio K1/K2 is highly more than 1, the release is mainly controlled
by diffusion and when the ratio is highly less than 1, the release is predominantly
controlled by matrix swelling / dissolution, the so called case II transport kinetic
(near zero order). When the ratio is equal 1, the release is controlled by a
combination of diffusion and polymer relaxation (anomalous transport) (51)
.
28
Kinetic analysis of the release data (data not shown) reveals that chromium
picolinate was released from SPH formulae by (case I transport = Fickian
diffusion) mechanism.
The half-life of superporous hydrogel, which were 1.26, 3.64 and 2.51
hours for F#9, F#10 and F#11 respectively.
It was concluded that the three SPH formulae retarded the drug release as
their values of half-life are longer than plain drug (0.22 hour) and possessed
high rate and extent of swelling which favoured their use as gastric retention
formulae but their visual mechanical strength were low. Further strengthening
of these three formulae will be studied.
3.5.6. Morphological Analysis of Superporous Hydrogels:
The morphology of porous structure of the selected formulae was examined
with scanning electron microscope (SEM). To verify the aforementioned results
concerning the drying conditions scanning electron microscope pictures of poly
(AM-co-SPAK) = F#6 (with medium swelling, apparent density and release rate)
superporous hydrogel dried under condition (a) and under condition (b) was done.
Both conditions of drying (a&b) produced SPH with pores connected to
each other to form extensive capillary channels, which help the dried SPH to
swell near equilibrium in a matter of minutes.
29
Figures (2a & 2b) showed the inner structure of superporous hydrogels under
SEM. Figure (2a) showed inner structure of air dried SPH (condition a) where there
were many of the capillary channels closed or partially blocked forming "dead end"
structure. Since a small percentage of closed pores can result in overall poor
capillary action (33)
, the low swelling ratios of these superporous hydrogels was
understandable. Figure (2b) showed inner structure of alcohol dried SPH (condition
b) where the pores remained intact and no sign of pore collapses were seen and this
accounted for the high swelling ratios.
Superporous hydrogels swell rapidly to large sizes. Since most of the
weight of a fully swollen superporous hydrogel is due to water, the gels are
mechanically very weak. For gastric retention applications, the mechanical
strength must be high. This is, first of all, necessary to maintain the fully
swollen superporous hydrogels in the solid form. If the fully swollen hydrogels
behave like a highly viscous solution, they may be emptied from the stomach as
liquid would be emptied. High mechanical strength is also required for
maintaining the superporous hydrogels intact in the stomach by withstanding
the pressure exerted by the gastric contractions. The mechanical strength of
superporous hydrogels was improved substantially by making composites.
Since dried superporous hydrogel composites should swell rapidly, it would be
ideal if the composite materials were also highly hydrophilic. For this reason
Ac-Di-Sol® was the best in promoting the swelling speed
(34).
30
3.6. Mechanism of Synthesis of Superporous Hydrogel Composites:
In the synthesis of SPH composites all aforementioned compounds used
in the synthesis of superporous hydrogels have the same role. Added Ac-Di-Sol
does not contribute to the chemical structure of the polymer, but is applied to
enhance mechanical stability of the polymer.
Role of Ac-Di-Sol in preparation of SPHC:
Ac-Di-Sol appears to have multiple useful functions in making
superporous hydrogel Composites with well structured channels.
First, it helped retain the capillary channels even after air drying of
superporous hydrogel Composites. Ac-Di-Sol exists as stiff fibers in the dry
state with diameters of 10–20 m and lengths of 100–200 m. It is insoluble but
swells in aqueous solution. When a compressed tablet containing Ac-Di-Sol is
placed in aqueous solution, Ac-Di-Sol can quickly absorb water, swell, and
break apart the tablet (52).
Our swelling study showed that the superporous
hydrogel composites indeed swelled faster than those without the composite
material and the effect was related to quantity of the composite material added.
In addition, the swelling action of Ac-Di-Sol can expand and open up closed
capillary channels. This action is similar to that in tablet disintegration. Ac-Di-
Sol exists as long fibers with a hollow lumen (53).
Similar to its action in tablet
disintegration, Ac-Di-Sol not only made a superporous hydrogel composite
31
more hydrophilic (therefore better wettability), but also provided intrafibrous
capillary channels (the hollow lumen of each fiber) that caused a strong wicking
effect. The strong relaxation of cellulose fibers may also facilitate water
penetration by expanding the closed capillary channels (54).
These effects,
causing fast disintegration of tablets, also decreased the swelling time of
squeezed superporous hydrogel composites.
Second, the superporous hydrogel composites could be dried in the air
with their porous structures intact even though they were not washed with
ethanol.
Third, it was also noticed that the presence Ac-Di-Sol resulted in better
stabilization of foams, presumably due to the increase in viscosity, resulting in
easier control of the synthetic process. To make superporous hydrogels with
uniform and interconnected pores, the monomer solution must have good
foaming and foam stabilizing mechanisms. The cell–air interfacial tension must
be lowered and the cell film viscosity must be raised. Both PF127and Ac-Di-
Sol worked together to retain most of the gas bubbles, and resulted in the
production of superporous hydrogel composites with fine pores and uniform
pore distribution.
Fourth, the presence of composite materials makes compression easier
without breaking interconnected capillary channels. This particular property
32
makes it possible to compress, or even fold a superporous hydrogel composite
and place it inside gelatin capsules.
It appears that the best composite materials for fast swelling are those
which are strong enough to prevent collapse of polymer chains during drying by
high water surface tension and at the same time hydrophilic enough to wet very
easily.
The amount of Ac-Di-Sol added was 270 mg which was optimum, as
although increase amount of Ac-Di-Sol causes increase in the physical cross-
linking density of the superporous hydrogel composite but if too much Ac-Di-
Sol is incorporated, due to the increase of solution viscosity, good mixing of all
the ingredients becomes difficult (34)
.
Acidification and drying of superporous hydrogel composites:
The washing step partially converted the anionic SO3¯ group of SPAK for
example into the unionized SO3H group, and it substantially changed the
properties of the superporous hydrogel composites. The acidification of the
SPHC made them much stronger than the SPHC prepared without
acidification(34)
.
Ac-Di-Sol
helped retain the capillary channels even after air drying of
the superporous hydrogel composites. Superporous hydrogels without
composite materials had to be dehydrated with ethanol before drying in the air
33
to preserve their interconnected capillary structures. The superporous hydrogel
composites, however, could be dried in the air with their porous structures intact
even though they were not washed with ethanol.
Wetting of superporous hydrogel composites:
Moisture was absorbed into the dried superporous hydrogel composites in
a controlled manner using a moistening chamber. Wetting is also important to
make the SPHC soft and flexible to be easily compressed in a capsule.
Composition of superporous hydrogel composites of various monomers
were made are shown in table (II).
3.7. Evaluation of Superporous Hydrogel Composite Formulae:
3.7.1 Swelling Studies:
The swelling ratio (Q) values for SPHC formulae are 25, 10.97 and 12.6
for F#12, F#13 and F#14 respectively.
The reduction in the swelling ratio (Q) of SPHC than SPH indicates that
the overall crosslinking density is increased. Since Ac-Di-Sol is not expected to
participate as a chemical crosslinking agent, it is thought to participate as a
physical crosslinking agent. It is highly likely that the polymer chains can
physically entangle through Ac-Di-Sol particles. When Ac-Di-Sol was mixed
with the monomer solution, it swelled so that monomers and crosslinker (Bis)
were absorbed into its network. During the synthesis of SPHC, the absorbed
34
monomers and crosslinker, along with those that were not absorbed, all
participated in the polymerization, leading to the formation of an
interpenetrating polymer network (IPN). IPN formation was limited to the Ac-
Di-Sol particles and thus the localized IPNs in Ac-Di-Sol particles provided
additional crosslinking. The decrease in swelling ratios of Ac-Di-Sol
incorporated SPH was partially due to the increase in physical crosslinking (34)
.
3.7.2. Determination of Apparent Density:
Solvent displacement method was used to determine the volume of the
sample of SPHC.
The apparent densities for superporous hydrogel composites formulae
were 0.34, 0.75 and 0.49 g/cm3 for F#12, F#13 and F#14 respectively. It is clear
that the apparent density of the SPHC is higher in comparison to the SPH
polymer. This may be due to the incorporation of the cellulosic fibers within the
polymer structure (18)
.
All apparent densities are less than density of the gastric fluid (≈1.004)
(50) and caused floating of the prepared formulae. So their gastric retention may
be due to floating together with large volume after swelling.
As observed in SPH evaluation results, also in the SPHC increasing the
swelling ratio was accompanied by decrease in the apparent density. This was in
35
accordance with Dorkoosha et al. in evaluation of their synthesized superporous
hydrogels and superporous hydrogel composites (18)
.
3.7.3. Incorporation of Chromium Picolinate into Superporous Hydrogel Composites:
Chromium picolinate was incorporated in the SPHC formulae by adding
it just before addition of NaHCO3, so as effervescences occurred after the
addition of NaHCO3 promoted efficient mixing of the drug with the rest of the
components. Composition of F#15, F#16 and F#17 (medicated SPHC) are
shown in table (II).
The swelling ratio (Q) for chromium picolinate SPHC were 18.56, 14.04
and 15.63 while apparent densities were 0.5, 0.78 and 0.56 g/cm3 for F#15,
F#16 and F#17 respectively.
3.7.4. Estimation of Drug loading:
The chromium picolinate content was 99.14, 128.08 and 120.27 g per one
mg of SPH for F#15, F#16 and F#17 respectively.
As previously mentioned that SPHC prepared from AM (F#15) possessed
the highest swelling ratio, so it is suggested that it possessed the lowest drug
content because it contained more open interconnected capillary channels which
cause escape of the drug during acidification.
3.7.5. In-Vitro Chromium Picolinate Release Study from SPHC Formulae:
36
Figure (1B) shows the release profile of chromium picolinate from
different formulae of superporous hydrogel composites using different
monomers in pH 1.2 at 37 °C. F#15, F#16 and F#17 showed a flush of 15.1,
20.94 and 13.11 % respectively during the first 5 minutes. The initial burst
effect may result from rapid dissolution of drug crystals at or near the surface of
the outer matrix of the SPHC. After 12 hours of the release, the SPHC formulae
released 95.32, 95.12 and 95.54 % for F#15, F#16 and F#17 respectively.
F#17 showed the least burst effect and the highest percentage released
after 12 hours.
Practically using SPAK/AM as a monomer mixture facilitates the
preparation of more homogenous polymers.
The release data were kinetically treated, where the computed
determination coefficient (r2) was taken as a criterion for estimation of the order
of chromium picolinate release from its different superporous hydrogel
composite formulae in pH 1.2 followed by mathematical, statistical and kinetic
constants computation. The release of chromium picolinate from F#15, F#16
and F#17 followed first order release (data not shown).
The value of n for the prepared F#16 was lower than 0.43 and this could
be due to high flush at early times and a marked retardation of the transport for
37
longer times, leading to lower n values (39)
. Some SPHC had 0.43 < n values < 1,
thus followed anomalous transport.
For more confirmation of the release order mechanism, the following
equation was applied: Mt/M∞ = K1t1/2
+ K2 t
Kinetic analysis of the release data (data not shown) reveals that chromium
picolinate was released from SPHC formulae by (case I transport = Fickian
diffusion) mechanism.
The respective values for half-life for the prepared SPHC formulae were
2.9, 3.2 and 2.94 hours for F#15, F#16 and F#17 respectively.
It was concluded that the three SPHC formulae retarded the drug release
as their values of half-life are longer than plain drug (0.22 hour) and possessed
high extent of swelling which favored their use as gastric retention formulae.
Their visual mechanical strength was higher than those of SPH due to
physical entanglement of Ac-Di-Sol fibers.
3.7.6. Morphological Analysis of Superporous Hydrogel Composites:
The morphology of porous structure of the selected plain formula (F#14)
was examined with scanning electron microscope (SEM).
Figure (3) shows the inner structure of superporous hydrogel composite
under SEM. The pictures showed that the pores were connected to each other to
38
form extensive capillary channels. Polymer layer formed around hollow Ac-Di-Sol
particles and Ac-Di-Sol fibers were interlocked with SPH matrix to form an
integral unit.
3.8. In-vitro Evaluation of the Synthesized Radio-opaque Chromium
Picolinate Superporous Hydrogel Composites® (RO-CP-SPHC)
Labeling of chromium picolinate dosage forms is an essential step for
their in-vivo study in animals. Introduction of barium sulphate as a radio-
opaque material during synthesis of our dosage forms might have changed their
physical characteristics. Therefore in-vitro evaluation of the radio-opaque
dosage forms is essential before subsequent in-vivo evaluation.
3.8.1. Swelling Studies:
The swelling ratios (Q) for CP-SPHC (F#17) and RO-CP-SPHC were
15.63 and 10.35 respectively.
It is obvious that the swelling ratio decreased after addition of BaSO4 and
squeezing of the RO-CP-SPHC to fit in 000 gelatin capsule. Squeezing caused
partial closure of the capillary channels (41)
, also BaSO4 powder may interrupt
water passage in the open capillary channels. These may be the reasons for
decreased swelling ratio of RO-CP-SPHC.
3.8.2. Determination of Apparent Density:
39
Solvent displacement method was used to determine the volume of the
sample of SPHC.
The apparent densities for superporous hydrogel composites formulae
were 0.56 and 0.834 g/cm3 for CP-SPHC (F#17) and RO-CP-SPHC
respectively. It was clear that the apparent density of the RO-CP-SPHC is
higher in comparison to the CP-SPHC. This may be due to the squeezing which
blocked some of the capillary channels together with the addition of heavy
powder of BaSO4(41)
.
It was observed that the decrease in swelling ratio was accompanied by
increase in apparent density. This was in accordance with Jun Chen et al. (41)
who studied the gastric retention properties of superporous hydrogel
composites.
All apparent densities are less than density of the gastric fluid (≈1.004) (50)
and caused floating of the prepared formulae.
3.8.3. Estimation of Drug loading:
The chromium picolinate content was 128.08 and 412.17 g per one mg
of SPHC for CP-SPHC (F#17) and RO-CP-SPHC respectively.
As previously mentioned, CP-SPHC (F#17) possessed higher swelling
ratio than RO-CP-SPHC, so it is suggested that it possessed lower drug content
40
because it contained more open interconnected capillary channels which cause
escape of the drug during acidification.
3.8.4. In-Vitro Chromium Picolinate Release Study from RO-CP- SPHC:
Figure (4) shows the release profile of chromium picolinate from
different formulae of superporous hydrogel composites in pH 1.2 at 37 °C
compared to the market formula. Market Formula (capsules) and RO-CP-SPHC
showed a flush of 31.93and 12.07 % respectively during the first 10 minutes.
The initial burst effect of the drug from SPHC may result from rapid dissolution
of drug crystals at or near the surface of the outer matrix of the SPHC.
The market formula released 100% of the drug after 2 hours while after
12 hours of the release, the RO-CP-SPHC released 98.01.
The release of chromium picolinate from the market formula together
with RO-CP-SPHC followed first order release (data not shown). The main
transport mechanism of RO-CP-SPHC was fickian diffusion since n value was
0.81 (anomalous) and k1/k2 ratio > 1 confirming fickian transport.
The values for half-life were 0.15and 2.21 hours for market formula and
RO-CP-SPHC respectively.
It was concluded that the SPHC formulae retarded the drug release as
their values of half-life are longer than market formula (0.15 hour) and
41
possessed high extent of swelling which favored their use as gastric retention
formulae.
3.8.5. Morphological Analysis of Radio-opaque Chromium Picolinate
Superporous Hydrogel Composite:
The morphology of porous structure of the radio-opaque formula was
examined with scanning electron microscope (SEM).
Figure (5) shows the inner structure of superporous hydrogel composite
under SEM. The picture showed that the pores are connected to each other to form
capillary channels. Polymer layer formed around hollow Ac-Di-Sol particles and
Ac-Di-Sol fibers were interlocked with SPH matrix to form an integral unit. Excess
drug, together with BaSO4 powder was precipitated on the surface of SPHC matrix
as shown in figure (5 a). Some pores are partially blocked due to squeezing as
shown in figure (5 b).
It is clear that the effect of BaSO4 on the physical properties of SPHC did
not counteract its gastric retention properties so; RO-CP-SPHC can be further
used in assessing the in-vivo properties of SPHC.
3.8.6. Radiographic Examination of the Selected Radio-opaque Chromium
Picolinate Superporous Hydrogel Composite:
A series of X-ray images showing the gastric retention property of RO-
CP-SPHC were taken. Figure (6c) shows the SPHC 15 minutes after dosing. The
42
gel appeared swelled in the stomach after the dissolution of the hard gelatin
capsule which is a prerequisite to ensure success of such systems, as otherwise
they could be evacuated from the stomach by the normal gastric emptying
processes, including the interdigestive migrating myoelectric complex (41)
. After
2 hours, figure (6d) reveals that the gel remained in the stomach and not
swapped by the housekeeper wave. This result proved that the gel had the
sufficient mechanical strength to remain intact and conserve its geometrical
shape against the gastric motility. By monitoring the gel, it appeared intact and
retained in the stomach till 12 hours with the same geometry as shown in figures
(6c-6g). At 24 hours, figure (6h) shows that the gel somehow deformed but still
retained in the stomach. These results were in accordance with Jun Chen et al.
(41) who studied the gastric retention properties of superporous hydrogel
composites.
Promising results were obtained from gastric retention experiments in
dogs. It was clear that the dogs after a few hours entered into fasted condition
RO-CP-SPHC was able to achieve one of our goals which were to retain in the
stomach for sustained period of time in an attempt to release the drug gradually
to the transporters in the intestine.
3.8.7. Bioavailability and Pharmacokinetic Studies of the Selected
Chromium Picolinate Gastroretentive Dosage Forms:
43
Bioavailability is usually defined in terms of the systemic levels of the drug.
Therefore, the blood level of the drug has been measured in order to relate
formulation effects to bioavailability.
Chromium picolinate is absorbed through the intestinal wall by active
carrier transport (32)
. Saturation of these carriers might limit the overall
absorption of the drug. The in-vivo drug release from the gastroretentive dosage
forms tended to be the rate-limiting step in the cascade of events prior to arrival
in the systemic circulation, and chromium picolinate concentrations that were
available for absorption following its release from the dosage forms were below
the saturation limit of the transporters. These results were in accordance with
Eytan A. Klausner et al.(42)
in their evaluation of the gastroretentivity of levodopa
in dogs.
Chromium picolinate pharmacokinetic parameters (Cmax., Tmax., MRT,
AUC (0-24), AUMC (0-24) and % bioavailability) following single oral
administration of one capsule of different chromium picolinate formulae were
calculated using Winnonlin 1.1 software and compiled in table (III) and
illustrated in figure (7).
Table (III) compiles the mean bioavailability and pharmacokinetic
parameters of chromium picolinate following administration of single oral dose
(200 g) of RO-CP-SPHC capsule to six healthy dogs in comparison to market
capsule
44
Thus the mean Tmax, MRT, AUC(0-24) and AUMC(0-24) following the
administration of the gastroretentive formulae in comparison to market capsules
can be arranged as follows : RO-CP-SPHC > market capsule.
The mean Cmax., following the administration of the gastroretentive formulae
in comparison to market capsules can be arranged as follows : market capsule >
RO-CP-SPHC
Cmax was lower for the gastroretentive dosage forms than the immediate
market capsules as expected. This was in accordance with Iman S. A.(55)
PhD
thesis which dealt with the in-vitro and in-vivo testing of gastric retention device.
The multiple peaks observed in the serum-concentration time curves of
gastroretentive dosage forms indicated that the drug arrived at different times to
the active transporters in the small intestine(55,56)
. This explained the longer tmax,
MRT and the higher AUC and AUMC for the GRDF over the market capsules
where the drug was released all at once and reached the active transporters at
high concentration causing saturation of these transporters with certain amount
of the drug and the rest was eliminated without being absorbed.
Finally, the mean percentage relative bioavailability following the
administration of the gastroretentive formula in comparison to market capsules is
298.8±15.9%.
3.8.8. Statistical comparison of pharmacokinetic parameters:
The One-Way Analysis of Variance (ANOVA) was performed to determine
the significance of difference between the tested systems followed by Least
45
Significant Difference (LSD) multiple comparison tests were used to assess the
statistical significance of difference between the results following extravascular
mode of administration using Social Package for Statistical Studies (SPSS). A P
value of less than 0.05 was considered significant. Data were presented as mean
± S.D.
It was found out that there was a significant difference between the prepared
RO-CP-SPHC formula and market capsules with respect to the MRT, Tmax, Cmax
AUMC(0-24) and AUC(0-24). This verifies that the improvement recorded in the
pharmacokinetic parameters for the new gastric retention dosage form
formulation is significant.
4- Conclusion
Based on the in-vitro evaluations, the pharmacokinetics properties and
radiographic examinations, it was concluded that chromium picolinate
superporous hydrogel composites capsules would optimize the therapy of this
drug owing to the extension of the absorption phase in comparison to non-
gastrotetentive dosage form (market formula). This enabled the desired
therapeutic concentration to be achieved in a controlled and sustained manner
providing continuous supply of the drug to its absorption site in the small
intestine, and yielding a sustained and prolonged chromium picolinate input to
the systemic circulation. Thus these controlled release gastroretentive dosage
46
form could be good candidate for novel drug delivery device to improve the
bioavailability of narrow absorption window drugs.
5. Declaration of interest
The authors report no declaration of interest
47
6. References
1. Verma RK, Garg S, Current Status of Drug Delivery Technologies and
Future Directions. PharmTechnol, 25 (2): 1–14, 2001.
2.Yao Y, Zhan X, Zhang J, Zou X, Wang Z, Xiong Y, Chen J, Chen G, A
specific drug targeting system based on polyhydroxyalkanoate granule binding
protein PhaP fused with targeted cell ligands,Biomaterials 2008, 29: 4823–4830
3. Yokoyama M, Drug targeting with nano-sized carrier systems. J Artif
Organs, 8: 77–84, 2005.
4. Streubel A, Siepmann J, Bodmeier R, Drug delivery to the upper small
intestine window using gastroretentive technologies. Curr Opin in Pharmacol, 6:
501–508, 2006.
5. Kagan L, Hoffman A, Selection of drug candidates for gastroretentive dosage
forms: Pharmacokinetics following continuous intragastric mode of
administration in a rat model. Eur J Pharm Biopharm, 69: 238–246, 2008.
6. Hoffman A, Stepensky D, Lavy E, Eyal S, Klausner E, Friedman M,
Pharmacokinetic and pharmacodynamic aspects of gastroretentive dosage
forms. Int J Pharm, 277: 141–153, 2004.
7. Liu Y, Zhang J, Gao Y, Zhu J, Preparation and evaluation of glyceryl
monooleate-coated hollow-bioadhesive microspheres for gastroretentive drug
delivery. Int J Pharm, 413:103 –109, 2011.
48
8. Punda S, Joshi A , Vasu K, Nivsarkar M, Shishoo C, Gastroretentive delivery
of rifampicin: In vitro mucoadhesion and in vivo gamma scintigraphy. Int J
Pharm, 411:106–112, 2011.
9. Lohan A, Chaudhary GP, Mucoadhesive microspheres: A novel approach to
increase gastroretention. Chron Young Sci, 3:121-128, 2012.
10. Darandale SS, Vavia PR, Design of a gastroretentive mucoadhesive dosage
form of furosemide for controlled release. Acta Pharm Sinic B, 2(5):509–517,
2012.
11.Abdul Ahad H , Sreeramulu J , Narasimha R D , Guru P P, Ramyasree P,
Fabrication and In vitro Evaluation of High density Gastro retentive
Microspheres of Famotidine with Synthetic and Natural Polymers. Ind J Pharm
Edu Res, 46(1):45-51, 2012.
12. Goole J, Vanderbist F, Amighi K, Development and evaluation of new
multiple-unit levodopa sustained-release floating dosage forms. Int J Pharm,
334: 35–41, 2007.
13.Sauzet C, Claeys-Bruno M, Nicolas M, Kister J, Piccerelle P, Prinderre P,
An innovative floating gastro retentive dosage system: Formulation and in vitro
evaluation. Int J Pharm, 378: 23–29, 2009.
14. Amrutkar PP, Chaudhari PD, Patil SB, Design and in vitro evaluation of
multiparticulate floating drug delivery system of zolpidem tartarate. Colloid
Surface B, 89: 182–187, 2012.
49
15. Chen Y, Ho H, Lee T, Sheu M, Physical characterizations and sustained
release profiling of gastroretentive drug delivery systems with improved
floating and swelling capabilities. Int J Pharm, 441 :162– 169, 2013.
16. Klausner EA, Lavy E, Friedman M, Hoffman A, Expandable gastroretentive
dosage forms. J Control Release, 90: 143–162, 2003.
17. Kumar A, Pandey M, Koshy M K, Saraf SA, Synthesis of fast swelling
superporous hydrogel: effect of concentration of crosslinker and acdisol on
swelling ratio and mechanical strength. Int J Drug Deliv, 2:135-140, 2010.
18.Dorkoosh FA, Brussee J, Verhoef JC, Borchard G, Rafiee-Tehrani M,
Junginger HE, Preparation and NMR characterization of superporous hydrogels
(SPH) and SPH composites. Polymer, 41: 8213–8220, 2000.
19.Spiller KL, Laurencin SJ, Charlton D, Maher SA, Lowman AM,
Superporous hydrogels for cartilage repair: Evaluation of the morphological and
mechanical properties. Acta Biomaterialia, 4: 17–25, 2008.
20.Kuang J, Yuk KY, Huh KM, Polysaccharide-based superporous hydrogels
with fast swelling and superabsorbent properties. Carbohyd Polym, 83: 284–
290, 2011.
21. Omidian H, Park K, Swelling agents and devices in oral drug delivery. J.
Drug Del. Sci. Tech, 18 (2): 83-93, 2008.
22. Ahmed IS, Ayres JW, Bioavailability of riboflavin from a gastric retention
formulation. Int J Pharm, 300: 146–154, 2007.
50
23. Dorkoosh FA, Verhoef JC, Ambagts MHC, Rafiee-Tehrani M, Borchard G,
Junginger HE, Peroral delivery systems based on superporous hydrogel
polymers: release characteristics for the peptide drugs buserelin, octreotide and
insulin. Eur J Pharm Sci, 15: 433–439, 2002a.
24. Dorkoosh FA, Verhoef JC, Borchard G, Rafiee-Tehrani M, Verheijden
JHM, Junginger HE, Intestinal absorption of human insulin in pigs using
delivery systems based on superporous hydrogel polymers. Int J Pharm, 247:
47-55, 2002b.
25.Yin L, Ding J, Fei L, He M, Cui F, Tang C, Yin C, Beneficial properties for
insulin absorption using superporous hydrogel containing interpenetrating
polymer network as oral delivery vehicles. Int J Pharm, 350: 220–229, 2008.
26. Gümüşderelioğlu M, rce D, TDemirtaş T, Superporous
polyacrylate/chitosan IPN hydrogels for protein delivery. J Mater Sci Med, 22
(11): 2467-2475, 2011.
27. Gupta N V, Shivakumar HG, Preparation and characterization of
superporous hydrogels as gastroretentive drug delivery system for rosiglitazone
maleate. DARU, 18 (3): 200-210, 2010.
28. Mahmoud E A, Bendas ER, Mohamed MI, Effect of Formulation
Parameters on the Preparation of Superporous Hydrogel Self-Nanoemulsifying
Drug Delivery System (SNEDDS) of Carvedilol. AAPS Pharm SciTech, 11(1):
221-225, 2010.
51
29. Chavda H, Patel Ch, Preparation and In vitro evaluation of a stomach
specific drug delivery system based on super porous hydrogel composite. Indian
J of Pharm Sci, 73 (1): 30-37, 2011.
30. Porter, D.J., Raymond, L.W. and Anastasio, G.D., Chromium: friend or
foe?, Arch. Fam. Med., 8(5), 386-390, 1999.
31. Cefalu ,W.T., Oral chromium picolinate improves carbohydrate and lipid
metabolism and enhances skeletal muscle Glut-4 translocation in obese,
hyperinsulinemic (JCR-LA corpulent) rats, J. Nutr., 132(6), 1107-1114, 2002.
32. King, J.C. and Keen, C.L. In: Modern Nutrition in Health and Disease. 9th
Edition. Baltimore, MD, (eds): Williams & Wilkins, 223-239, 1999.
33. Chen, J., Park, H. and Park, K., Synthesis of superporous hydrogels:
Hydrogels with fast swelling and superabsorbent properties, J. Biomed. Mater.
Res., 44 (1), 53-62 , 1999.
34. Chen, J. and Park, K., Synthesis and characterization of superporous
hydrogel Composites, J. Control. Rel. 1;65(1-2), 73-82, 2000.
35. Qiu, Y. and Park, K., Superporous IPN Hydrogels Having Enhanced
Mechanical Properties, AAPS PharmSciTech, 4(4), 406-412, 2003.
36. Simonelli, A.P.; Mehta, S.C. and Higuchi, W.I., Dissolution rates of high
energy polyvinylpyrrolidone (PVP)-sulfathiazole coprecipitates J. Pharm. Sci.,
58(5), 538-549, 1969.
52
37. Higuchi, T., J., Mechanism of sustained-action medication, theoretical
analysis of rate of release of solid drugs dispersed in solid matrices, Pharm. Sci.,
52, 1145-9, 1963.
38. Scwartz, J.B., Simonelli, A.P., Higuchi, W.I. and Higuchi, T., Drug release
from wax matrices. I. Analysis of data with first-order kinetics and with the
diffusion-controlled model, J. Pharm.Sci., 57(2), 274-277, 1968.
39. Ritger, P.L. and Peppas, N.A., A simple equation for description of solute
release. I. Fickian and non-Fickian release from non-swellable devices in the
form of slabs, spheres, cylinders or discs, J. Control. Rel., 5, 23-36, 1987.
40. Yu, L., Zhang, H., Cheng, S.X., Zhuo, R.X. and Li, H., Study on the drug
release property of cholesteryl end-functionalized poly (-caprolactone)
microspheres. Part B: Applied Biomaterials, J. Biomed. Mat. Res., 77(1), 39-46,
2006.
41. Chen, J., Blevins, W.E., Park, H. and Park, K., Gastric retention properties
of superporous hydrogel composites; J. Control. Rel., 64, 39-51, 2000.
42. Klausner, E. A., Eyal, S., Lavy, E., Friedman, M. and Hoffman, A., Novel
levodopa gastroretentive dosage form: in vivo evaluation in dogs,
J.Control.Rel., 88, 117-126, 2003.
43. Christensen, J.M., Hoist, E., Bonde, J.P. and Knudsen, L., Determination of
chromium in blood and serum: evaluation of quality control procedures and
estimation of reference values in Danish subjects, The Science of the Total
Environment, 132 (1), 11-25, 1993.
53
44. Davis, P.D., Kraus, L.A., Thompson, A.G. and Olerich, M., Percutaneous
absorption of salicylic acid after repeated (14-day) in vivo administration to
normal, acnegenic or aged human skin, J. Pharm. Sci., 86(8), 896-9, 1997.
45. Marzouk, A.H.M., Formulation, pharmacokinetic and bioavailability studies of
certain drugs in TDDS, Ph.D. Thesis, Faculty of Pharmacy, Cairo University, 1999.
46. Park, H. and Park, K., Hydrogel foams: A new type of fast swelling
hydrogels. In: The 20th Annual Meeting of the Society for Biomaterials,
[Abstract #158], 1994.
47. Park, H., and Park, K., Honey, I blew up the hydrogels! Proc. Int. Symp.
Control. Rel. Bioact. Mater., 21, 21, 1994.
48. Van Phan, D. and Trokhan, P.D., Superabsorbent polymer foam. U.S. Patent
No. 5,506,035, 1996.
49. Kellenberger, S.R., Shih-Schroeder, W.H. and Wisneski, A.J., Absorbent
structure, U.S. Patent No. 5,149,335, 1992.
50. El-Gibaly, I., Development and in vitro evaluation of novel floating chitosan
microcapsules for oral use: comparison with non-floating chitosan
microspheres, Int. J. Pharm., 249, 7-21, 2002.
51. Kim, H. and Fassihi, R., A new ternary polymeric matrix system for
controlled drug delivery of highly soluble drugs; I. Diltiazem hydrochloride,
Pharm. Res., 14, 1415-21, 1997.
52. Kanig, J.L. and Rudnic, E.M., The mechanisms of disintegrant action,
Pharm. Tech., April, 50-63, 1984.
54
53. Shangraw, R., Mitrevej, A. and Shah, M., A new era of tablet disintegrants,
Pharm. Tech., October, 49-57, 1980.
54. Shangraw, R.F., Wallace, J.W. and Bowers, F.M., Morphology and
functionality in tablet excipients for direct compression: part ii, Pharm. Techn.,
October, 44-60, 1981.
55. Saad, I.M., In Vitro and in vivo testing of a gastric retention device,
development and evaluation of a new colonic delivery system, Ph.D. Thesis,
Faculty of Pharmacy, Oregon State University, 2002.
56. kawashima, Y., Niwa, T., Takeuchi, H., and Ito, Y., Preparation of multiple
unit hollow microspheres with acrylic resin containing tranilast and their drug
release characteristics (in vitro) and floating behavior (in vivo), J. Control. Rel.,
16(3), 279-289, 1991.
55
Table (I): Synthesis of Superporous Hydrogel
Form
ula
nu
mb
er
Monomer
Type
Monomer
(l)
Crosslinker
(2.5 % Bis)
(l)
Water
(l)
Foam Stabilizer
(10 % PF 127)
(l)
Acid
(l)
Initiator
(20%APS)
(l)
Initiation
catalyst
(20%
TEMED)
(l)
Drug
(gm)
Foaming
Agent
(NaHCO3)
(mg)
Drying
Condition
a b
F#1 AM 1000 (50% AM) 200 460 100 45
(AA) 40 40 ------- 90 √ √
F#2 AA
(Na salt) 1000 (pH 6) 200 460 100
25
(AA) 40 40 ------- 90 √ √
F#3 ATMS 1000
(30% ATMS) 40 ----------- 50
30
(AA) 20 20 ------- 90 √ √
F#4 HEMA♣
700 100 ----------- 100 --------- 50 50 ------- 80 √ √
F#5 SPAK 1000
(30% SPAK) 40 ----------- 50
30
(AA) 20 20 ------- 90 √ √
F#6 AM +
SPAK
440 (50% AM )+
300(50% SPAK) 250 ----------- 50
10
(AA) 30 20 ------- 100 √ √
F#7 AM + AA 300 (50% AM) +
200(50% AA) 100 330 30 --------- 20 20 ------- 120 √ √
F#8 NIPAM +
AM
1000
(25% NIPAM) +
200 (20% AM)
400 ----------- 100 50 (6N
HCL) 50 50 ------- 60 √ √
F#9 AM 1000 (50% AM) 200 460 100 45
(AA) 40 40 0.1 90 ----- √
F#10 SPAK 1000
(30% SPAK) 40 ----------- 50
30
(AA) 20 20 0.1 90 ----- √
F#11 AM +
SPAK
440 (50% AM )+
300(50% SPAK) 250 ----------- 50
10
(AA) 30 20 0.1 100 ----- √
♣= Adjust temperature to 60 oC during synthesis
56
Table (II): Synthesis of Superporous Hydrogel Composites
Form
ula
nu
mb
er
Monomer
Type
Monomer
(l)
Crosslinker
(2.5 % Bis)
(l)
Water
(l)
Foam
Stabilizer
(10 % PF
127)
(l)
Acid
(l)
Initiator
(20%APS
(l)
Composite
Material
(Ac-Di-Sol®)
(mg)
Initiation
Catalyst
(20%
TEMED)
(l)
Drug
(gm)
Foaming
Agent
(NaHCO3)
(mg)
F#12 AM 1000
(50% AM) 200 460 100
45
(AA) 40 270 40 ------ 100
F#13 SPAK 1000
(30% SPAK) 40 ---------- 50
30
(AA) 20 270 20 ------ 100
F#14 AM +
SPAK
1200 (50%
AM )+ 900
(50% SPAK)
450 ---------- 90 30
(AA) 45 270 45 ------ 100
F#15 AM 1000
(50% AM) 200 460 100
45
(AA) 40 270 40 0.1 100
F#16 SPAK 1000
(30% SPAK) 40 ---------- 50
30
(AA) 20 270 20 0.1 100
F#17 AM +
SPAK
1200 (50%
AM )+ 900
(50% SPAK)
450 ---------- 90 30
(AA) 45 270 45 0.1 100
57
Table (III): Mean Bioavailability and Pharmacokinetic Parameters of Chromium Picolinate Following
Administration of Single Oral Dose (200 g) of RO-CP-SPHC Capsule to Six Healthy Dogs in
Comparison to Market Capsule
Formulae C max. *
(ng/ml)
Tmax. *
(hr)
MRT*
(hr)
AUC(0-24) *
(ng.hr/ml)
AUMC(0-24) *
(ng.hr2/ml)
% Relative
Bioavailability*
RO-CP-SPHC 25.77 ± 2.66 12 ± 0.9 11.09 ± 0.26 401.54 ± 21.39 4448.16 ± 137.63 298.80 ± 15.91
Market Capsule 32.2 ± 2.2 1.33 ± 0.18 7.54 ± 1.05 134.38 ± 17.95 1024.05 ± 274.76 100
*The data are mean values of six healthy dogs ± S.D.
58
List of Figures
Figure (1): Percentage Cumulative Release of Chromium Picolinate from Different Formulae
of Superporous Hydrogels (A) and Superporous Hydrogel Composites (B) using Different
Monomers in pH 1.2 at 37 °C
Figure (2): Scanning Electron Microscope Pictures of Poly (AM-co-SPAK) Superporous
Hydrogel Dried under Condition (a)(F#2a) and Condition (b) (F#2b)
Figure (3): Scanning Electron Microscope Pictures of Plain (F#14) and Medicated Poly (AM-
co-SPAK) (F#17) Superporous Hydrogel Composite
Figure (4): Percentage Cumulative Release of Chromium Picolinate from Radio- opaque
Superporous Hydrogel Composites Compared to Market Formula in pH 1.2 at 37 °C.
Figure (5): Scanning Electron Microscope Pictures of Radio-opaque Chromium Picolinate
Poly (AM-co-SPAK) Superporous Hydrogel Composite at Different Magnification Power
Figure (6): Radiographs for One Dog Showing RO-CP-SPHC in Stomach (a) before dosing, (b)
after eating, (c) 15 minutes, (d) 2hr, (e) 4hr, (f) 8hr, (g) 12 hrs and (h) 24 hr after dosing.
Figure (7): Mean Serum Concentration of Chromium Picolinate Following the Administration of
a Single Oral Dose (200 µg) of Different Chromium Picolinate Capsules to Six Healthy Dogs
59
60
61
62
63
64
65