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Introduction
Scopolamine (SCOP), a naturally occurring antimuscarinic
agent, has been used for the prevention and treatment of
nausea and vomiting associated with motion sickness for
almost 200 years. However, this drug has a low and variable
oral bioavailability (10.7%_48.2%) because of extensive
hepatic first-pass metabolism[1]. The variability in absorption
and poor bioavailability of oral SCOP indicate that this route
is neither reliable nor effective for this drug. In addition, the
SCOP transdermal patch has been developed.
Pharmacokinetic studies showed that peak plasma concentrations
(Cmax) were reached about 12_16 h after
dosing[2,3]. Moreover, SCOP concentrations in plasma also declined more slowly after the
patches were removed than after an iv dose. The reported
delay in the drug reaching the circulation after patch
application, and the potential for prolonged unwanted side
effects such as dry mouth, dizziness, or blurred vision, led
us to look for an alternative route of administration to oral or
transdermal delivery.
About 50 years ago, the nasal absorption of SCOP in
solution was reported by Hyde et
al[4]. They found that SCOP could produce faster responses and greater
therapeutic activity than an equivalent oral dose. Recently,
Scopolamine Hydrobromide 0.2% Nasal Spray was on the market.
However, problems still exist, such as a short duration of
therapeutic effect due to the rapid elimination of the instilled
drug from the nasal cavity by mucociliary beating (a
clearance half-life of 15 min[5,6]), and consequently, a frequent
dosing regimen was needed.
Several approaches have been used in designing nasal
dosage forms with high absorption and lasting drug effects.
For example, hydrogels made of carbopol, methylcellulose,
and hydroxypropyl methylcellulose can increase the drug
contact time with the mucosa, therefore reducing its rapid
clearance and resulting in increased absorption. However,
viscous gels have the disadvantage of being difficult to
administer.
These problems have been overcome by the use of
in situ gels. In situ gels are instilled as low viscosity solutions
into the nasal cavity, and upon contact with the nasal mucosa,
the polymer changes conformation producing a gel. This
type of gel combines the advantages of a solution,
administration convenience, and exact dosing, with the favorable
residence time of a gel. The phase transition can be induced
by a shift in pH, as for cellulose acetate
phthalate[7], a shift in temperature as for the thermo gelling Poloxamer
407[8,9] or by the presence of cations as for gellan
gum[10].
Taken the information into account, in the present study
a nasal in situ gel system for SCOP was developed. Since
nasal mucosa is covered with approximately 0.1 mL mucus in
humans, which consists of sodium, potassium, and calcium
ions, a cation-responsive polymer gellan gum was chosen.
Gellan gum is an anionic deacetylated, exocellular
polysaccharide secreted by Pseudomonas
elodea with a tetrasaccharide repeating unit of
1β-L-rhamnose, 1β-D-glucuronic, acid and 2β-D-glucose. The mechanism of gelation involves
the formation of double-helical junction zones followed by
aggregation of the double-helical segments to form a 3-D
network by complexation with cations and hydrogen
bonding with water[11_13]. Gellan gum has been widely used in
ophthalmic drug delivery[10,14]. Nevertheless, there is little
literature on its potential as a vehicle for nasal application.
The present work aimed to formulate an ion-activated
in situ gel for SCOP with gellan gum and to investigate its
viscosity, in vitro release, nasal ciliotoxicity, the nasal
mucosal residence time, and antimotion sickness capacity.
Materials and methods
Materials SCOP was gifted by the Department of
Pharmaceutics, School of Pharmacy, Fudan University
(Shanghai, China). Gellan gum was purchased from ZhongWei Biochemical Ltd (Shanghai, China). The SCOP
injection (0.3 g/L) was obtained from Shanghai Harvest
(Shanghai, China). Technetium-99m-diethylentriamine
pentaacetic acid (99mTc-DTPA) was prepared in the
Department of Nuclear Pharmacy at Fudan University (China).
The ion compositions of artificial nasal fluid included
150±32 mmol/L Na+, 41±18 mmol/L
K+, and 4±2 mmol/L Ca2+,
prepared according to Lorin et
al[15]. All other reagents were of commercially analytical grade.
Preparation of nasal formulations
A certain amount of gellan gum was added to deionized water and dissolved by
heating to 100 °C with moderate stirring. After cooling to
below 40 °C, SCOP (0.4%, w/v), mannitol (5%,
w/v), and chlorhexidine acetate (0.01%,
w/v) were added and mixed well. Three kinds of SCOP
in situ gels were prepared at the concentrations of gellan gum which were 0.2%, 0.5%, and 1.0%
(w/v), respectively. The pH of all the formulations was
between 4.0 and 6.0.
Viscosity measurement of in situ gel
formulations The viscosity of the gellan gum formulations, either in solution
or in gel made with artificial nasal fluid instead of 5% mannitol,
were determined with a rotational viscometer (NDJ-5S,
Shanghai, China) using a 20 mL aliquot of the sample.
Measurements were performed using suitable spindle number at
6, 12, 30, 60 r/min, and the temperature was maintained at
37 °C. The viscosity was read directly from the viscometer
display. All measurements were made in triplicate.
In vitro release of SCOP
from gels The in vitro release
of SCOP from the gels was measured through a cellulose
acetate dialysis membrane employing Valia-Chien diffusion
cells (2TYTP3A, GongYi Ltd, Shanghai, China) with a
diffusional area of 1.75 cm2 and a receptor compartment volume of
16 mL. The receptor compartment, containing artificial nasal
fluid to allow the establishment of the "sink condition" and
to simulate the physiological condition, was stirred and
thermostated at 37±0.5 °C in an incubator during the
experiment. 2 mL gellan gum preparation loaded with 0.4%
of the drug (n=6) was placed in the donor compartment. At
predetermined time intervals, samples (1 mL) of receiving
solution were withdrawn and replaced with the same volume
of fresh release medium. The amount of SCOP in each sample
was determined by HPLC (LC-10A, Shimadzu Co Ltd, Kyoto,
Japan).
Chromatographic separation was achieved using a Dikma
DiamonsilTM C18 column (Dikma Co Ltd, Beijing, China, 5
μm, 200 mm×4.6 mm) and a precolumn (Nova-Pak, 10
μm, C18 15, 220, Waters) at 40 °C. The mobile phase was a mixture of
0.008 mol/L sodium dodecyl sulphate containing 0.001 mol/L
hydrochloric acid (pH 3.0) and acetonitrile (50:50
v/v) at a flow rate of 1 mL/min. The UV absorbance of the effluent
was monitored (SPD-10A, Shimadzu Co Ltd, Kyoto, Japan)
at a wavelength of 210 nm.
Nasal ciliotoxicity Nasal ciliotoxicity studies were
carried out using an in situ toad palate
model[16]. In brief, the upper palate of the toads (30_40 g, male and female,
Experimental Animal Center of Fudan University, China,
n=6) was exposed and treated with about 0.5 mL
in situ gel (0.5% gellan gum) for 4 h. Then the test formulation was removed by
washing the palate with saline, about 5×3 mm of the palate
was dissected and the mucocilia was examined with a
electron microscope (Nikon Fx-35A, Tokyo, Japan) at
enlargements of 400×. Saline and sodium deoxycholate (one of the
agents with serious nasal ciliotoxicity, 1%
[w/v] solution) were used as the negative and positive controls, respectively.
Scintigraphic studies The nasal mucosal residence time
of the in situ gel was studied using single photon emission
computing tomography (ZLC 3700, Münich, Germany) auto
tuned to detect the 140 KeV radioactivity of
99mTc-DTPA. In situ gel incorporating
99mTc-DTPA at the gellan gum concentration of 0.5% was prepared as described earlier. The
solution (99mTc-DTPA dissolved in phosphate buffer saline) was
used as a control.
Eight male, New Zealand, white rabbits
(2.0±0.2 kg, Experimental Animal Center of Fudan University, China) were
divided into 2 groups. Each group received gel and solution
formulations in a crossover design with at least a 3 d
washout period. The rabbit was positioned 10 cm in front of the
probe and 100 µL of the radio labeled gel or solution, which
were stored in 20 °C for 30 min before use, were instilled into
the nasal surface. Recording started 5 s after administration
and continued for 60 min using a 128×128 pixel matrix. A total
of 78 frames of dynamic images were recorded in a sequence
of 36×20 s followed by 12×40 s then 30×80 s frames.
The images were analyzed by medical system ICONP
workstation (Siemens, Münich, Germany). All of the graphs
were divided into 2 regions of interest. A circular region was
manually drawn around the nose area as region 1, and region
2 represented the rest of the body.
Antimotion sickness efficacy
Animals Adult, male Wistar rats weighing 200_250 g
were used for the study. The animals were deprived of food
24 h prior to the experiment, but were allowed free access to
water until 2 h before the experiment. The animal experiment
was carried out in compliance with the protocol of Animal
Use and Care by Medical Center of Fudan University (China).
Animal handling and drug administration In order to
induce motion sickness in rats, a modified
procedure[17] was used. In brief, the animals were placed individually in
specifically-designed perspex restrainers (20 cm×5 cm) with a
sliding door at one end to adjust the size for adequate
ventilation. With the help of a hook, the restrainers were
hanged to the shaft of the blade of the centrifuge. The
hanging rope was only 20 cm long so as to minimize the
centrifugal effect. The distance at the shaft of the blade was kept
such that the diameter of rotation was fixed at 35 cm. The
blade was run at a speed so as to obtain rotations of 300
rpm(35 g). Each rat was rotated for 15 min.
Sixty rats were used in this experiment and randomly
divided into 6 groups: group 1 was administered intranasally
with 50 µL blank gel (the excipient control); group 2 and
group 3 with 100 µg/kg SCOP aqueous solution
administered subcutaneously and orally, respectively; and groups
4, 5, and 6 were administered SCOP in situ gel (0.5% gellan
gum) at the dose of 25, 50, and 100 µg/kg, respectively.
Fifteen minutes after administration, all the groups were tested
employing the above treatment, except the oral group which
needed 30 min.
After the treatment, the rats were placed on the floor and
their behavior, such as continuous rotation, unilateral
obliquity, and vomiting, were observed for 1 h, the recovery
time was recorded.
Statistical analysis The results were expressed as mean±
SEM. ANOVA was used to test the differences between the
calculated parameters using the SPSS Statistical Package
(Version 10, SPSS Inc, Chicago, IL, USA). Differences were
considered statistically significant when P<0.05.
Results
Viscosity of in situ gel
formulations All gellan gum formulations, either in solution or in gel, showed pseudo
plastic behavior (Figure 1). The viscosity of the test gels
increased with increasing concentrations of gellan gum,
and a large viscosity change was found when gellan gum
underwent sol-gel transition at lower
concentrations (0.2% and 0.5%). Due to a very viscous solution obtained with 1%
gellan gum, a slight viscosity increase was observed after
gel formation.
In vitro release experiments SCOP release from nasal
preparations was moderate under sink condition (Figure 2).
After incubated for 6 h, about 96.1±4.5 %, 80.0±5.4%, and
61.9±4.9% SCOP release from 3 gels was found, respectively.
At fixed drug concentrations, the release rate depended on
gellan gum concentration: the higher the gellan gum
con-cen-tration, the lower the rate of drug release. The
in vitro release data were kinetically analyzed according to zero-order,
first-order, and the diffusion-controlled release mechanism.
The relative high correlation coefficient values obtained from
the analysis of the amount of the drug released versus the
square root of time indicated the release followed the
Higu-chi[18] kinetic model, as shown in Table 1.
Nasal ciliotoxicity A micrograph showed that there was
a great number of cilia at a fast-beating rate on the edge of
the mucosa treated with in situ gel for 4 h (Figure 3), and the
beating lasted for about 12 h after the palate was dissected.
The cilia density was judged normal compared with the saline,
indicating that in situ gel had no obvious effect on cilia
movement.
Scintigraphic studies The mucociliary clearance graphs
of the 99mTc-labeled formulation are shown in Figure 4.
Instilled solution exhibited a rapid initial drainage of
radioactivity due to nasal turnover, followed by a slow decrease in
remaining radioactivity. Ten minutes after administration of
the solution, the radioactivity in region 2 was observed, 60
min later, the radioactivity was found around the whole body.
However, a clear tendency that was less pronounced after
administration of the gellan gum formulation was visualized.
There was about 80% of the gel deposited in the rabbit nasal
cavity 30 min after administration, 60 min later, more than
60% of the gel was in contact with the mucociliary surface as
compared with only about 20% of the applied reference
solution. These results indicated that 0.5% gellan gum could
increase the residence time of the formulation, which was
further demonstrated by the pharmacodynamic.
Antimotion sickness efficacy The present study was
conducted to test the efficacy of in situ gel for motion
sickness in an experimental rat model. In the rats that were
rotated for a period of 15 min after administration of the blank
gel, there were almost complete symptoms of motion
sickness (Table 2). The phenomenon could last 7_8 min, during
which some rats vomited slightly. Fifteen minutes later, the
rats were static and convoluting with the symptom of
nystagmus to alleviate the suffering, which could last about
45_60 min. The rats treated with SCOP aqueous solution (100
µg/kg sc and 100 µg/kg, po) prior to the test also showed the
symptoms of motion sickness after rotation, which could
last 5 min. Twenty minutes later, the rats were static and
convoluting without nystagmus, and these symptoms
disappeared 30 min post rotation. Intranasal SCOP
in situ gel at a dose of 100 µg/kg (group 6) decreased the degree of
motion sickness significantly in the rats compared with the oral
and subcutaneous group (P<0.01). In addition, although
the dose of subcutaneous injection was 2 times that of the
nasal dose, the recovery time was approximately equal for
the rats in groups 2 and 5, suggesting that a reduced nasal
dose might be expected.
Discussion
Motion sickness, also known as travel, car, sea, air, rail,
or space sickness, is induced through whole body
vibrations by stimulation of the vestibular organ. Motion
sickness is a very common disease characterized by various
sym-ptoms, such as pallor, cold sweating, nausea, vomiting,
fatigue, self-rotation, unilateral obliquity, or the slowing of
brain waves[19_20]. SCOP has been used successfully for
motion sickness for almost a century. At present, SCOP in
motion sickness is administered by transdermal patches.
However, the patch must be attached at least 4_6 h before
the onset of action and is therefore unsuitable for acute
therapy. Recently, Scopolamine Hydrobromide 0.2% Nasal
Spray was on the market, which claimed to have a fast onset
of action within 30 min after
administration[20]. However, due to the mucociliary clearance mechanism, solution
formulations, that are not mucoadhesive, are generally
rapidly cleared from the nasal cavity and result in a short duration.
Therefore, to overcome these barriers, a novel in
situ gel system for the nasal delivery of SCOP was developed in the
present study. The results of the animal experiments
revealed that 15 min after intranasal administration,
in situ gel produced obvious antimotion sickness effects on rats at
all the tested doses. Moreover, in situ gel at a dose of
100 µg/kg showed a better therapeutic efficacy when compared
to oral and subcutaneous administration. These results
suggested that SCOP nasal in situ gel is a promising therapeutic
alternative to existing medications for motion sickness.
Further pharmacokinetic investigation about the nasal
absorption of SCOP from gellan gum preparation is in progress in
our laboratory.
In this study, in situ gels at 3 different gellan gum
concentrations were prepared. The preparations behave like a
fluid, but form a rigid gel when exposed to cations. The
viscosity of the test gel increased with higher gellan gum
concentrations. It was proposed that as the concentration
of gellan gum increased, the polymer chains approached
closer, and the number of interactions between the polymer
chains increases which leads to a denser 3-D network
structure[21]. When the concentration of gellan gum achieved 1%,
high viscosity made administration difficult with a
conventional nebulizer.
Because the release rate of a drug directly affected its
absorption process in vivo, SCOP release through the
different gellan gum formulations was examined using the
Valia-Chien diffusion cell method. The results showed that the
release of SCOP from nasal preparations was moderate
without any burst effects. It was most likely that gellan gum
underwent a rapid sol-gel transition when exposed to
artificial nasal fluid as confirmed by the viscosity experiment.
During the hydrogel formation, a portion of SCOP might be
loaded into the hydrogel phase, and thus the drug release
became slow. In addition, the release rate also depended on
the gellan gum concentration. The release from various gellan
gum formulations could be ranked as follows:
0.2%>0.5%>1%. These results indicated that the structure of the gel
became more closely packed and functioned as an
increasingly resistant barrier to drug release as the concentration of
polymer increased. Considering the viscosity and sustained
release capacity, the gel at 0.5% gellan gum concentration
seems to be a preferable formulation for nasal delivery of
SCOP. Therefore, the 0.5% formulation was chosen for
further study.
Nasal mucociliary clearance plays a crucial role in
protecting the respiratory system from damage by inhaled
substances[22]. Therefore, it is vital to examine the influences of
drugs and drug excipients on nasal mucociliary clearance
before clinical application. In the present study, we chose
the toad palate model for the study of ciliotoxicity because
the toad palate is a robust tissue giving reproductive results
and the experimental technique was
easy[16]. It was found that animals treated with the
in situ gel (0.5% gellan gum) showed a mild effect on ciliary beating, suggesting it was
safe for nasal application.
Several methods, both in vitro and in
vivo, have been used to evaluate mucociliary transport
rates[23_26]. Advantages of the gamma scintigraphic technique lie in the ability
to non-invasively monitor the deposition and clearance of
drug formulations, allowing both quantitative and
photographic illustrations of distribution and clearance of the
radiolabeled formulation. Employing this technique to
evaluate the nasal clearance of mucoadhesive preparations
requires a radiotracer which is stable and non-diffusible to
prevent absorption into the vascular compartment.
99mTc tracer is reported as technically easy to perform and more
representative of ciliary function since it investigates a large
surface of the mucosa as a whole and not the fastest flow
rate[26]. Therefore, 99mTc-DTPA was used in this study. As
expected, in situ gel had a longer residence in rabbit nasal
cavity compared with the solution.
Three kinds of SCOP in situ gels were prepared at the
concentrations of gellan gum, which were 0.2%, 0.5%, and
1.0% (w/v), respectively. Its viscosity depended on the
concentration of gellan gum. In vitro release experiments showed
that the release of SCOP from the test gels was moderate.
In situ gel had a longer residence time in the rabbit nasal cavity
compared with the solution, and no nasal ciliotoxicity. The
antimotion sickness experiment confirmed that intranasal
in situ gel produced pronounced antimotion sickness efficacy,
especially at a dose of 100 µg/kg. In conclusion, SCOP nasal
in situ gel is a well-tolerated and promising therapeutic
alternative to existing medications for motion sickness.
Acknowledgments
We wish to express our thanks to professor Jian-hua ZHU
(Department of Nuclear Pharmacy, School of Pharmacy,
Fudan University, Shanghai, China) for his help
in the scintigraphic studies.
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