1

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: sally_adel_2000@yahoo.com

2

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

4

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

5

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

7

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

9

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

10

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

11

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.

12

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;

13

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

14

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

15

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.

17

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:

18

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

20

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

22

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

23

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

24

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

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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