Patel JK, Patel RP, Amin AF, Patel MM.

Formulation and Evaluation of

Mucoadhesive Glipizide Microspheres. AAPS PharmSciTech. 2005; 06(01): E49-
E55. DOI:  10.1208/pt060110

Formulation and Evaluation of Mucoadhesive Glipizide Microspheres

Jayvadan K. Patel,1 Rakesh P. Patel,1 Avani F Amin,2 and Madhabhai M. Patel1
1
Shree S.K. Patel College of Pharmaceutical Education and Research, Ganpat
Vidyanagar, Mehsana-Gozaria Highway, Kherva-382711, India
2
Nirma Institute of Pharmacy, Nirma University of Science and Technology,
Sarkhej-Gandhinagar Highway, Ahmedabad-380061, India

Correspondence to:
Jayvadan K. Patel
Tel: 0091-2762-286082
Fax: 0091-2762-286082
Email: [email protected]

Received: July 16, 2004; Accepted: November 12, 2004; Published: September 20,
2005

Abstract

The purpose of this research was to formulate and systematically evaluate in vitro
and in vivo performances of mucoadhesive microspheres of glipizide. Glipizide
microspheres containing chitosan were prepared by simple emulsification phase
separation technique using glutaraldehyde as a cross-linking agent. Results of
preliminary trials indicate that volume of cross-linking agent, time for cross-
linking, polymer-to-drug ratio, and speed of rotation affected characteristics of
microspheres. Microspheres were discrete, spherical, and free flowing. The
microspheres exhibited good mucoadhesive property in the in vitro wash-off test
and also showed a high percentage drug entrapment efficiency. A 32 full factorial
design was employed to study the effect of independent variables, polymer-to-drug
ratio (X1), and stirring speed (X2) on dependent variables percentage mucoadhesion,
t80, drug entrapment efficiency, and swelling index. The best batch exhibited a high
drug entrapment efficiency of 75% and a swelling index of 1.42; percentage
mucoadhesion after 1 hour was 78%. The drug release was also sustained for more
than 12 hours. The polymer-to-drug ratio had a more significant effect on the
dependent variables. In vivo testing of the mucoadhesive microspheres to albino
Wistar rats demonstrated significant hypoglycemic effect of glipizide.
Keywords: chitosan, mucoadhesive microspheres, glipizide, factorial design, in
vivo study

Introduction

Microsphere carrier systems made from the naturally occurring biodegradable

polymers have attracted considerable attention for several years in sustained drug
delivery. Recently, dosage forms that can precisely control the release rates and
target drugs to a specific body site have made an enormous impact in the
formulation and development of novel drug delivery systems. Microspheres form
an important part of such novel drug delivery systems.1-3 They have varied
applications and are prepared using assorted polymers.4 However, the success of
these microspheres is limited owing to their short residence time at the site of
absorption. It would, therefore, be advantageous to have means for providing an
intimate contact of the drug delivery system with the absorbing membranes.5-8 This
can be achieved by coupling bioadhesion characteristics to microspheres and
developing bioadhesive microspheres. Bioadhesive microspheres have advantages
such as efficient absorption and enhanced bioavailability of drugs owing to a high
surface-to-volume ratio, a much more intimate contact with the mucus layer, and
specific targeting of drugs to the absorption site.9-12 Chitosan (obtained by
deacetylation of chitin) is a cationic polymer that has been proposed for use in
microsphere systems by a number of authors.13-17 Chitosan was selected as a
polymer in the preparation of mucoadhesive microspheres because of its good
mucoadhesive and biodegradable properties.

Glipizide is a second-generation sulfonylurea that can acutely lower the blood

glucose level in humans by stimulating the release of insulin from the pancreas and
is typically prescribed to treat type II diabetes (non-insulin-dependent diabetes
mellitus). Its short biological half-life (3.4 ± 0.7 hours) necessitates that it be
administered in 2 or 3 doses of 2.5 to 10 mg per day.18 Thus, the development of
controlled-release dosage forms would clearly be advantageous. Researchers have
formulated oral controlled-release products of glipizide by various techniques.12,19,20
Moreover, the site of absorption of glipizide is in the stomach. Dosage forms that
are retained in the stomach would increase the absorption, improve drug efficiency,
and decrease dose requirements. Thus, an attempt was made in this investigation to
use chitosan as a mucoadhesive polymer and prepare microspheres. The
microspheres were characterized by in vitro and in vivo tests, and factorial design
was used to optimize the variables.

Materials and Methods

Materials

Glipizide was obtained as gift sample from USV Ltd (Daman, India). Chitosan
(degree of deacetylation of 85%; intrinsic viscosity, 1390 mL/g in 0.30 M acetic
acid/0.2 M sodium acetate solution; and viscometric molecular weight, 4.08 × 105
Da) was obtained as gift sample from Central Institute of Fisheries Technology
(Cochin, India). Dioctyl sodium sulfosuccinate (DOSS) and petroleum ether 80:20
were procured from Willson Lab (Mumbai, India) and S. D. Fine Chemicals Ltd
(Mumbai, India), respectively. Liquid paraffin and glutaraldehyde were purchased
from Loba Chemie Pvt Ltd (Mumbai, India).

Methods

Preparation of Microspheres

Mucoadhesive microspheres of chitosan were prepared by simple emulsification

phase separation technique. Chitosan was used as a polymer and was cross-linked
using glutaraldehyde as per method described by Thanoo et al.13

Chitosan (1.5 g) was dissolved in 150 mL of 1% vol/vol aqueous acetic acid

solution. Five hundred milligrams of drug was dispersed in the polymer solution.
In batches J1 to J15 the polymer-to-drug ratio was kept constant at 3:1. The
resultant mixture was extruded through a syringe (No. 20) in 1 L of liquid paraffin
(heavy and light, 1:1 ratio) containing 0.2% DOSS, and stirring was performed
using a propeller stirrer (Remi, Mumbai, India) at 1000 rpm. After 15 minutes,
glutaraldehyde (25% vol/vol aqueous solution) was added and stirring was
continued. The amount of cross-linking agent and cross-linking time were varied in
batches J1 to J15 from 10 to 60 mL and 1 to 3 hours, respectively, as shown in
Table 1. In factorial design batches B1 to B9, 50 mL of glutaraldehyde was used as
a cross-linking agent and cross-linking time was kept to 1 hour. The polymer-to-
drug ratio and stirring speed were varied in batches B1 to B9 as shown in Table 2.
All other variables were used as mentioned in preliminary trial batches.
Microspheres thus obtained were filtered and washed several times with petroleum
ether (80:20) to remove traces of oil. They were finally washed with water to
remove excess of glutaraldehyde. The microspheres were then dried at room
temperature (at 25°C and 60% relative humidity [RH]) for 24 hours. The effect of
formulation variables on characteristics of the microspheres is summarized in
Tables 1 and 2.

Table 1. Result of Preliminary Trial Batches*

 In Vitro Wash-
Cross- off Test Drug
Volume of linking (% Entrapment
Batch Glutaraldehyde Time Mucoadhesion Efficiency Sphericity of
Code (mL) (h) After 1 h) (%) Microspheres

J1 10 1 85 38 (±0.6) Very irregular

J2 10 2 79 40 (±1.2)
J3 10 3 74 42 (±1.9)
J4 20 1 81 52 (±2.1) Slightly
irregular
J5 20 2 75 57 (±1.3)
J6 20 3 68 59 (±0.8)
J7 40 1 72 58 (±2.4) Spherical free
flowing
J8 40 2 66 60 (±1.1)
J9 40 3 59 62 (±0.8)
J10 50 1 74 68 (±2.1) Spherical free
flowing
J11 50 2 65 70 (±1.1)
J12 50 3 60 74 (±1.8)
J13 60 1 61 67 (±2.1) Spherical free
flowing
J14 60 2 54 70 (±1.4)
J15 60 3 47 69 (±0.5)

*All the batches were prepared at a polymer-to-drug ratio of 3:1.

Table 2. 32 Full Factorial Design Layout*

Variable In Vitro Wash-

Levels in off Test Drug
Coded Form (% Entrapment Particle
Batch Mucoadhesion t80 Efficiency Swelling Size
Code X1 X2 After 1 h) (minutes) (%) Index (µm)
 B1 −1 −1 51 236 52 0.866 60.4
B2 −1 0 45 234 50 0.819 56.7
B3 −1 1 42 220 47 0.808 49.3
B4 0 −1 74 207 70 1.172 67.1
B5 0 0 65 239 68 1.113 64.9
B6 0 1 59 250 64 0.982 61.6
B7 1 −1 78 476 75 1.423 96.0
B8 1 0 71 456 71 1.282 88.2
B9 1 1 65 380 68 1.229 71.8

Translation of Coded Levels in Actual Units

Variables Level Low (−1) Medium (0) High (+1)

Polymer-to-drug ratio (X1) 1:1 3:1 6:1

Stirring speed (X2) rpm 500 1000 1500

*All the batches were prepared using 50 mL of glutaraldehyde and a cross-linking

time of 1 hour.

Assay of Glipizide

Glipizide was estimated by ultraviolet visible (UV/Vis) spectrophotometric method

(Shimadzu UV-1601 UV/Vis double beam spectrophotometer, Kyoto, Japan).
Aqueous solutions of glipizide were prepared in phosphate buffer (pH 7.4) and
absorbance was measured on UV/Vis spectrophotometer at 276 nm.21 The method
was validated for linearity, accuracy, and precision. The method obeys Beer’s Law
in the concentration range of 5 to 50 µg/mL. When a standard drug solution was
analyzed repeatedly (n = 5), the mean error (accuracy) and relative standard
deviation (precision) were found to be 0.8% and 1.3%, respectively.

Drug Entrapment Efficiency

Microspheres (50 mg) were crushed in a glass mortar and pestle, and the powdered
microspheres were suspended in 10 mL of phosphate buffer (pH 7.4). After 24
hours, the solution was filtered and the filtrate was analyzed for the drug content.
The drug entrapment efficiency was calculated using the following formula:
Practical drug content / Theoretical drug content × 100 . The drug entrapment
efficiency for batches J1 to J15 and B1 to B9 are reported in Tables 1 and 2,
respectively.

Particle Size and Swelling Index of Microspheres

The particle size of the microspheres was determined by using optical microscopy
method.22 Approximately 100 microspheres were counted for particle size using a
calibrated optical microscope (Labomed CX RIII, Ambala, India).

For estimating the swelling index, the microspheres (∽100) were suspended in 5
mL of simulated gastric fluid USP (pH 1.2).23 The particle size was monitored by
microscopy technique every 1 hour using an optical microscope (Labomed CX
RIII). The increase in particle size of the microspheres was noted for up to 8 hours,
and the swelling index was calculated as per method described by Ibrahim.24 The
swelling index for microspheres of batches B1 to B9 is reported in Table 2.

In Vitro Wash-off Test for Microspheres

The mucoadhesive properties of the microspheres were evaluated by in vitro wash-

off test as reported by Lehr et al.25 A 1-cm by 1-cm piece of rat stomach mucosa
was tied onto a glass slide (3-inch by 1-inch) using thread. Microspheres were
spread (∽50) onto the wet, rinsed, tissue specimen, and the prepared slide was
hung onto one of the groves of a USP tablet disintegrating test apparatus. The
disintegrating test apparatus was operated such that the tissue specimen was given
regular up and down movements in a beaker containing the simulated gastric fluid
USP (pH 1.2). At the end of 30 minutes, 1 hour, and at hourly intervals up to 10
hours, the number of microspheres still adhering onto the tissue was counted. The
results of in vitro wash-off test of batches B1 to B9 are shown in Tables 1 and 2
respectively.

Drug Release Study

The drug release study was performed using USP XXIV basket apparatus
(Electrolab, TDT-06T, Mumbai, India) at 37°C ± 0.5°C and at 50 rpm using 900
mL of phosphate buffer (pH 7.4) as a dissolution medium (n = 5) as per USP
XXVI dissolution test prescribed for glipizide extended release tablets.21
Microspheres equivalent to 10 mg of glipizide were used for the test. Five
milliliters of sample solution was withdrawn at predetermined time intervals,
filtered through a 0.45 µm membrane filter, diluted suitably, and analyzed
spectrophotometrically. An equal amount of fresh dissolution medium was
replaced immediately after withdrawal of the test sample. Percentage drug
dissolved at different time intervals was calculated using the Lambert-Beer’s
equation ( y = 0.0218 x + 0.0147 , R 2 = 0.9979 ) described above. The t80 was
calculated using the Weibull equation.26 The average values of t80 for batches B1 to
B9 are mentioned in Table 2. The percentage drug release of batch B7 is shown in
Figure 1.

Figure 1. In vitro dissolution of glipizide from mucoadhesive microspheres of

batch B7.

Scanning Electron Microscopy

A scanning electron photomicrograph of drug-loaded chitosan mucoadhesive

microspheres was taken. A small amount of microspheres was spread on glass
stub. Afterwards, the stub containing the sample was placed in the scanning
electron microscope (JSM 5610 LV SEM, JEOL, Datum Ltd, Tokyo, Japan)
chamber. The scanning electron photomicrograph was taken at the acceleration
voltage of 20 kV, chamber pressure of 0. 6 mm Hg, original magnification ×800.
The photomicrograph is depicted in Figure 2.

Figure 2. Scanning electron photomicrograph of glipizide-loaded chitosan

mucoadhesive microspheres (batch B7). The parameters of scanning electron
microscopy were acceleration voltage of 20 kV, chamber pressure of 0.6 mm Hg,
original magnification ×800.

Factorial Design

A statistical model incorporating interactive and polynomial terms was used to

evaluate the responses:

(1
Y = b 0 + b 1 X 1 + b 2 X 2 + b 12 X 1 X 2 + b 11 X 1 2 + b 22 X 2 2 ,
)

where, Y is the dependent variable, b0 is the arithmetic mean response of the 9 runs,
and bi is the estimated coefficient for the factor Xi. The main effects (X1 and X2)
represent the average result of changing one factor at a time from its low to high
value. The interaction terms (X1X2) show how the response changes when 2 factors
are simultaneously changed. The polynomial terms ( X 1 2 and X 2 2 ) are included
to investigate nonlinearity.

In Vivo Study
In vivo evaluation studies for glipizide mucoadhesive microspheres were
performed on normal healthy Wistar rats weighing 250 to 300 g each. The
approval of the Institutional Animal Ethics Committee was obtained before starting
the study. The study was conducted in accordance with standard institutional
guidelines. Two groups of Wistar rats (5 in each group) that were fasted (with
water) at least 12 hours before the experiments were used for the study. Before
drug administration, a blood sample as a control was taken from each rat from
behind the eyeball through the angle of ocular cavity using small capillary tubes.
The blood glucose level for the control and test samples was determined using the
glucose-measuring instrument Medisence (Abbott Laboratories, Bedford, MA).
The instrument was self-calibrated, and the samples were allowed to dry before the
results were read to avoid contamination of the lens. Pure glipizide and
mucoadhesive microspheres of glipizide were administered orally to each group
using stomach intubations. A dose of 800 µg/kg of glipizide was administrated in a
suspension form (freshly prepared) for each rat. Blood samples were collected at
predetermined time at 1-hour intervals up to 24 hours, and the blood glucose level
was performed as per method described earlier. The percentage reduction in blood
glucose level was measured and is depicted in Figure 3.
Figure 3. Percentage reduction in blood glucose levels following oral
administration of glipizide (-●-) and its mucoadhesive microspheres (-▲-) in
Wistar rats.

Results and Discussion

Preliminary Trials

The mucoadhesive microspheres of chitosan were prepared by simple

emulsification phase separation technique. Chitosan was selected as a polymer for
the preparation of mucoadhesive microspheres owing to its biodegradable and
mucoadhesive properties. Different concentrations of acetic acid from 1% vol/vol
to 6% vol/vol were used for preparing the polymer solution, but no significant
effect of concentration of acetic acid was observed on percentage mucoadhesion or
drug entrapment efficiency, therefore 1% vol/vol of acetic acid was used. This
finding could be owing to good solubility of chitosan in acetic acid.

One of the important factors related to microspheres as reported by Lee et al27 is

the viscosity of the polymer solution. Polymer concentrations of 0.5%, 1%, and 2%
wt/vol were selected for preliminary trials. Flake formation was observed when
chitosan concentration was used at a level of 0.5%, whereas maximum sphericity
was observed at the 1% level. The chitosan solution was found to be too viscous to
pass through the syringe when used at the 2% level. Therefore 1% wt/vol of
chitosan in 1% vol/vol acetic acid was found to be the optimum concentration for
the polymer solution. A 1:1 mixture of heavy and light liquid paraffin was found to
be suitable as the dispersion medium. The addition of 0.2% wt/vol of DOSS to the
dispersion medium was found to be essential to minimize aggregation of
microspheres.

Batches J1 to J15 were prepared to study the effect of the volume of cross-linking
agent (glutaraldehyde), time for cross-linking, and stirring speed on the percentage
mucoadhesion, drug entrapment efficiency, and characteristics of the microspheres.

The volume of glutaraldehyde was varied from 10 to 60 mL. Discrete spherical

microspheres were obtained using 40, 50, and 60 mL of glutaraldehyde (batches J7
to J15). Batches J1 to J6 prepared using 10 and 20 mL of glutaraldehyde yielded
irregular microspheres. The higher amount of glutaraldehyde appears to favor the
cross-linking reaction, and hence spherical free-flowing microspheres were
obtained. The microspheres of batches J7 to J15 also showed significant effect on
the percentage mucoadhesion and drug entrapment efficiency (Table 1).
Microspheres of batches J7 to J9 prepared using 40 mL of glutaraldehyde showed
good percentage mucoadhesion, but drug entrapment efficiency was below 63%.
Batches J10 to J12 also showed good mucoadhesion as well as 70% drug
entrapment efficiency. In the microspheres of batches J13 to J15, the drug
entrapment efficiency was above 68%, but mucoadhesion decreased. The decrease
in mucoadhesion could possibly be attributed to the greater amount of cross-
linking agent giving a more rigid cross-linked polymer whose adhesion is
decreased. Thus, we can conclude that 50 mL of glutaraldehyde was the optimum
amount. Increase in the cross-linking time (1 to 3 hours) in batches J1 to J15
inversely affected the percentage mucoadhesion. The cross-linking polymer
probably becomes more rigid and thus mucoadhesiveness decreases. The cross-
linking time did not have a significant effect on the percentage drug entrapment
efficiency.
On the basis of the preliminary trials a 32 full factorial design was employed to
study the effect of independent variables (ie, polymer-to-drug ratio [X1] and the
stirring speed [X2]) on dependent variables percentage mucoadhesion, t80, drug
entrapment efficiency, and swelling index. The results depicted in Table 2 clearly
indicate that all the dependent variables are strongly dependent on the selected
independent variables as they show a wide variation among the 9 batches (B1 to
B9). The fitted equations (full models) relating the responses (ie, percentage
mucoadhesion, t80, drug entrapment efficiency, and swelling index) to the
transformed factor are shown in Table 3. The polynomial equations can be used to
draw conclusions after considering the magnitude of coefficient and the
mathematical sign it carries (ie, positive or negative). The high values of
correlation coefficient (Table 3) for the dependent variables indicate a good fit.
The equations may be used to obtain estimates of the response since small error of
variance was noticed in the replicates.

Table 3. Summary of Results of Regression Analysis

Coefficient b0 b1 b2 b11 b22 b12 R2

% Mucoadhesion 65.22 12.67 −6.17 −1 −7.33 1.17 0.9956

t80 241.66 103.73 −11.61 −20.0656 101.62 −14.84 0.9621
Drug entrapment efficiency 68.17 11.0 −2.97 −0.475 −7.0 −0.30 0.9981
Swelling index 1.08 0.24 −0.07 −0.03 −0.02 0.01 0.9907

Factorial Equation for Percentage Mucoadhesion and Swelling Index

The in vitro wash-off test for percentage mucoadhesion after 1 hour varied from 51
to 78 and showed good correlation coefficient (0.9956). Results of the equation
indicate that the effect of X1 (polymer-to-drug ratio) is more significant than X2
(stirring speed). Moreover, stirring speed had a negative effect on the percentage
mucoadhesion (ie, as the stirring speed increased, the percentage mucoadhesion
decreased). This finding may be attributed to the change in particle size that affects
mucoadhesion. Similar results were obtained for swelling index. The amount of
polymer directly affected the solvent transfer rate; thus, as the polymer
concentration increased the swelling index also increased. The swelling index
varied from 0.866 to 1.423 and showed good correlation coefficient (0.9907).
Thus, we can conclude that the amount of polymer and stirring speed directly
affects the percentage mucoadhesion and swelling index.
Factorial Equation for Drug Entrapment Efficiency and t80

The drug entrapment efficiency and t80 are important variables for assessing the
drug loading capacity of microspheres and their drug release profiles, thus
suggesting the amount of drug availability at site. These parameters are dependent
on the process of preparation, physicochemical properties of drug, and formulation
variables. The drug entrapment efficiency varied from 47% to 76% and showed
good correlation coefficient (0.9981). Results of the equation indicate that the
effect of X1 (polymer-to-drug ratio) is more significant than X2 (stirring speed).
Moreover, stirring speed had a negative effect on drug entrapment efficiency (ie, as
the stirring speed increased, the particle size decreased, and thus drug entrapment
efficiency decreased).

The mucoadhesive microspheres of all the batches of the factorial design were
spherical and free flowing. They ranged in particle size from 48 to 96 µm. Results
depicted in Table 3 indicate that the percentage drug released in vitro is highly
dependent on the polymer-to-drug ratio and the stirring speed. The stirring speed
has a negative effect on t80 because as the particle size increases the drug released
decreases. Higher levels of polymer-to-drug ratio favor the cross-linking reaction
and thus higher t80 is obtained. Batch B7 exhibited a high t80 of 476 minutes and
seems to be a promising candidate for achieving drug release up to 12 hours. The
drug release profile of batch B7 is shown in Figure 1. The figure reveals that drug
release rate was slowed after 4 hours. The study focus was the preparation of
mucoadhesive microspheres, thus the microspheres of batch B7 were also
evaluated in simulated gastric fluid USP (pH 1.2). The results indicated that no
significant difference was observed between dissolution profiles at pH 7.4 and pH
1.2 as the f2 value was 65.2. The particle size of microspheres of batch B7 was 96
µm. The photomicrograph of batch B7 is depicted in Figure 2, which indicates
good sphericity of the microspheres. The dissolution data of batch B7 were further
analyzed to ascertain the mechanism of drug release.28 The release profile fitted
best to the Weibull equation ( F = 9 . 05 ) . The value of the correlation coefficient
was found to be 0.987. The values of slope and intercept were found to be 1.22 and
−3.06, respectively.

In Vivo Study

In vivo efficiency of the optimized batch B7 was performed in healthy normal

Wistar rats by measuring the hypoglycemic effect produced after oral
administration. The drug was administered at a dose equivalent to 800 µg/kg pure
glipizide, and glipizide mucoadhesive microspheres were used for the study. Pure
glipizide drug was administered in a suspension form at the same dose. When pure
glipizide suspension was administered, a rapid reduction in blood glucose levels
was observed and maximum reduction of 48% was observed within 2 hours after
oral administration. Blood glucose levels were recovered rapidly to the normal
level within 8 hours (Figure 3). In the case of glipizide mucoadhesive
microspheres, the reduction in blood glucose levels was slow and reached
maximum reduction within 4 hours after oral administration. This reduction in
blood glucose levels was sustained over longer periods of time (12 hours). Kahn
and Shechter29 have suggested that a 25% reduction in blood glucose levels is
considered a significant hypoglycemic effect. Significant hypoglycemic effect
(25%) was maintained only from 0.5 to 5 hours after oral administration of
glipizide, whereas in the case of mucoadhesive glipizide microspheres, significant
hypoglycemic effect (25%) was maintained for a period of 2 to 12 hours. The
sustained hypoglycemic effect observed over a longer period of time in the case of
mucoadhesive microspheres is due to the slow release and absorption of glipizide
over longer periods of time. Glipizide sustained release formulation is significantly
more effective than the immediate release formulation of glipizide in reducing
fasting plasma glucose levels and side effects.30 Formulation of glipizide as
mucoadhesive sustained release dosage form could also exhibit a decrease in side
effects.

Conclusion

The results of a 32 full factorial design revealed that the polymer-to-drug ratio and
stirring speed significantly affected the dependent variables percentage
mucoadhesion, t80 drug entrapment efficiency, and swelling index. The
microspheres of the best batch exhibited a high percentage mucoadhesion of 78%
after 1 hour, 75% drug entrapment efficiency, and swelling index of 1.42. The t80
of 476 minutes indicates that the mucoadhesive microspheres of glipizide could
sustain the release of the drug for more than 12 hours. The in vivo study
demonstrated significant hypoglycemic activity of the mucoadhesive microspheres
of glipizide.

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