Journal of Drug Delivery Science and Technology 46 (2018) 138–147

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Journal of Drug Delivery Science and Technology

journal homepage: www.elsevier.com/locate/jddst

Design, optimization and pharmacodynamic comparison of dorzolamide T

hydrochloride soluble ocular drug insert prepared by using 32 factorial
design
Sayali S. Patil, Archana Bade, Amol Tagalpallewar∗
Department of Pharmaceutics, Sinhgad Technical Education Society's, Sinhgad Institute of Pharmacy, Narhe, Pune, 411041, Maharashtra, India

A R T I C LE I N FO A B S T R A C T

Keywords: The bioavailability of therapeutic agents from eye drops is usually limited due to corneal barrier functions and
Dorzolamide hydrochloride effective eye protective mechanisms. The current study aimed to enhance ocular bioavailability of dorzolamide
32 factorial design hydrochloride, a potent antiglaucoma drug by preparing novel ophthalmic drug delivery system like soluble
Glaucoma ophthalmic drug inserts (SODI).
Ex vivo permeation
Various batches of dorzolamide hydrochloride Soluble Ocular Drug Insert were prepared using Polyvinyl
Gamma radiation
Alcohol, Poloxamer 407 as polymer with Propylene Glycol as plasticizer by solvent casting method. The drug
Soluble ocular drug insert
inserts were prepared by using 32 factorial designs (version 10). Solvent casting method was used to prepare
drug inserts which were further evaluated for various parameters like drug interaction studies, physicochemical
characteristics, in vitro release studies, transcorneal permeation study by using excised goat cornea. The opti-
mized batch F2 showed 98.50 ± 0.05% drug release at the end of 6 h and has desired physicochemical prop-
erties. The gamma radiation sterilized batch F2 was subjected to sterility test as per Indian Pharmacopeia 2014
and accelerated stability studies as per International Conference on Harmonization stability testing of new drug
substances and products guideline, ex vivo and Pharmacodynamic studies. The comparative pharmacokinetic
result shows that Dorzolamide Hydrochloride SODI is more effective than the marketed formulation. The pro-
mising pharmacokinetic results and possibility of commercial applicability justifies the need of product in
market.

1. Introduction
Ocular drug delivery is one of the most fascinating and challenging
task faced by pharmaceutical researcher. One of the major barriers of
ocular medication is to obtain and maintain a therapeutic level at the
site of action for prolong period of time [1,2]. It is generally agreed that
the intraocular bioavailability of topically applied drugs is extremely
poor ranging from 5 to 10% of total dose administered. The normal
capacity of cul-de-sac is 7–10 μl which can be extended upto 30 μl
without blinking. Due to this reason conventional eye drops are
eliminated from the precorneal area immediately and only 1–10% of
topically applied drug get absorbed. Moreover, due to tear drainage
more than 75% of the administered dose passes via nasolacrymal duct
into the GI tract, leading to systemic side effects [3] Short residence
time, poor bioavailability, poor permeability and rapid precorneal
drainage are the major problems for eye drops treatment [4]. Glaucoma
is the second leading cause of world's blindness. It has been reported
that nearly 50% of glaucoma patient discontinued topical ocular
therapy within six month because of high frequency of instillation.
Glaucoma is complex disease characterized by ocular hypertension with
a progressive visual loss that could result in blindness due to damage
occurred to the optic nerve. Glaucoma development may be observed
due to aging, genetic predisposition, exogenous environmental and
endogenous factors [5]. Vision loss occurs in some glaucoma patients
due to uncontrollable intra ocular pressure. Main goal of glaucoma
therapy is to reduce increased intraocular pressure in order to prevent
deterioration of the optic nerve and loss of vision. The number of drugs
and new chemical entities with potential for treating glaucoma is
steadily increasing [6]. Topical applied carbonic anhydrase inhibitors
(e.g. DZH eye drop solutions) has become one of the most widely used
medications for glaucoma treatment [7]. Although DZH is commonly
used for glaucoma treatment, its conventional eye drops possess poor

Abbreviations: PVA, Polyvinyl alcohol; PG, Propylene glycol; DZH, Dorzolamide hydrochloride; SODI, Soluble ocular drug insert; S. aureus, Staphylococcus aureus; C. albicans, Candida
albicans; CPCSEA, Committee for Purpose of Control and Supervision of Experiments on Animals; IOP, Intra ocular pressure; HPLC, High pressure liquid chromatography
∗
Corresponding author.
E-mail addresses: sayalipatil1305@gmail.com (S.S. Patil), archanabade94@gmail.com (A. Bade), tirupati21@rediffmail.com (A. Tagalpallewar).

https://doi.org/10.1016/j.jddst.2018.05.010
Received 30 June 2017; Received in revised form 4 May 2018; Accepted 6 May 2018
Available online 12 May 2018
1773-2247/ © 2018 Elsevier B.V. All rights reserved.
S.S. Patil et al. Journal of Drug Delivery Science and Technology 46 (2018) 138–147

Table 1
Composition of different batches of DZH SODI.
Name of Ingredients (%) Different batches of dorzolamide hydrochloride SODI

F1 F2 F3 F4 F5 F6 F7 F8 F9

PVA (w/v) 4 4 4 4 4 4 4 4 4
Poloxamer 407 (w/v) 6.5 6.5 6.5 7.5 7.5 7.5 8.5 8.5 8.5
PG (% of dry polymer) 10 15 20 10 15 20 10 15 20
Dorzolamide hydrochloride (mg) 74 74 74 74 74 74 74 74 74
Distilled Water (upto ml) 20 20 20 20 20 20 20 20 20

Table 2 etc. in order to increase ocular residence time of instilled dose and
Experimental design layout of DZH SODI. enhance the ophthalmic bioavailability. These formulations offer some
Run Factor Factor(X2) Response (Y1) Response Response (Y3)
improvement over conventional liquid dosage forms but have draw-
(X1) PG Poloxamer % Cumulative (Y2) Folding backs of ease of administration, lack of patient compliance, commercial
407 Drug Release Thickness Endurance applicability and blurred vision [7]. To overcome these drawbacks one
(mm) of the new class of drug delivery systems has been developed i.e. so-
luble ophthalmic drug insert, which offer many advantages over con-
1 −1 −1 97.19 0.09 222
2 −1 0 98.50 0.10 242 ventional dosage forms, like increased ocular residence, sustained re-
3 −1 +1 93.92 0.11 264 lease, accurate dosing, and reduced dose frequency. Ophthalmic inserts
4 0 −1 96.49 0.10 283 are defined as sterile preparations, with a thin, multilayered, drug‐im-
5 0 0 94.91 0.10 291
pregnated, solid or semisolid consistency devices placed into cul‐de‐sac
6 0 +1 94.98 0.11 314
7 +1 −1 90.78 0.11 322
or conjunctival sac and whose size and shape are especially designed for
8 +1 0 92.87 0.12 333 ophthalmic application [9]. Poloxamer 407 is gelling agent commonly
9 +1 +1 91.07 0.12 342 used in ophthalmic formulations. Poloxamer 407 used in pharmaceu-
tical applications for transdermal, ophthalmic and injectable sustained
drug release systems [10–12]. The objective of this study is to prepare
bioavailability and several adverse effects at high concentration [8]. To and evaluate DZH SODI based on 32 factorial design using different
overcome the limitations of conventional eye drops various novel polymers containing DZH to reduce dose and dosing frequency, in-
ophthalmic drug delivery systems have been developed such as aqueous creased ocular residence, enhanced ocular bioavailability, sustained
gels, liposomes, nanoparticles, soluble ophthalmic drug inserts, den- release of drug, reduced total cost of treatment and increased patient
drimer, nanomicells, implant, contact lenses, nanosuspension, in situ gel compliance. Ex vivo – in vivo characterization for confirmation of sus-

Table 3
Physicochemical evaluation of SODI.
Different Batches Evaluation Parameter

Thickness (mm) Folding Endurance Surface pH Weight Uniformity (mg) Drug Content (%)

F1 0.10 ± 0.012 222.3 ± 2.5 6.5 ± 0.01 2.3 ± 0.05 99.35 ± 0.08%
F2 0.09 ± 0.006 242.0 ± 2.0 6.6 ± 0.06 2.2 ± 0.01 99.91 ± 0.03%
F3 0.11 ± 0.012 264.7 ± 1.5 6.6 ± 0.01 2.3 ± 0.05 99.56 ± 0.03%
F4 0.12 ± 0.015 283.0 ± 2.6 6.5 ± 0.01 2.4 ± 0.01 99.89 ± 0.07%
F5 0.10 ± 0.012 291.0 ± 2.1 6.6 ± 0.06 2.5 ± 0.06 99.57 ± 0.06%
F6 0.12 ± 0.010 314.3 ± 3.2 6.6 ± 0.06 2.4 ± 0.05 99.92 ± 0.04%
F7 0.12 ± 0.012 322.3 ± 2.5 6.7 ± 0.01 2.6 ± 0.06 99.76 ± 0.04%
F8 0.12 ± 0.010 333.0 ± 1.0 6.7 ± 0.05 2.6 ± 0.10 99.39 ± 0.06%
F9 0.12 ± 0.012 342.0 ± 2.0 6.6 ± 0.01 2.8 ± 0.20 99.57 ± 0.06%

Fig. 1. Tensile strength of optimized batch.

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S.S. Patil et al.
Fig. 2. Comparative in vitro release profile of DZH from SODI.
tained drug delivery with improved ocular bioavailability.
2. Materials and methods
2.1. Materials
DZH was obtained from FDC Ltd (FDC Ltd. Mumbai, India) and
Poloxamer 407 from Sigma-Aldrich Chemicals, Bangalore. PVA pur-
chased from (Qualigens Fine Chemicals Pvt. Ltd Mumbai). All other
ingredients and reagents were of research grade.
2.2. Methods
2.2.1. Preparation of ocular insert
Solvent casting method was used to prepare ocular insert. The re-
quired quantity of polymer is dissolved in 20 ml of distilled water and
stirred for 2 h then weighed quantity of DZH was added and stirred for
2 h on magnetic stirrer to prepare uniform dispersion. After complete
mixing, casting solution was poured on petridish and allowed it to dry.
The dried inserts thus obtained were cut into required size, wrapped in
aluminum foil and stored for further evaluations. Composition of dif-
ferent batches is given in Table 1.
2.2.2. Full factorial experimental design
For optimization of DZH SODI, 32 randomized full factorial design
was selected. The design was applied to study the effect of concentra-
tion of poloxamer 407 and PG on formulation. The amount (%) of
plasticizer, propylene glycol (PG) (X1) The amount (%) of polymer,
poloxamer 407 (X2) were selected as independent variables. These two
factors were evaluated at 3 levels as higher, middle and lower levels
with coding +1, 0 and −1 respectively. Levels of X1 10%, 15%, and
20%. The dependent or response variables included thickness (Y1),
folding endurance (Y2), cumulative % drug release (Y3).
2.3. Validation of model
The optimized compositions of DZH SODI required to obtain desired
responses. Any composition with desirability near to 1 can be chosen as
optimized formulation.
2.3.1. Characterization of prepared dorzolamide hydrochloride ocular
insert
2.3.1.1. Thickness measurement. Thickness of SODI was determined
using a caliper (digital vernier caliper) and recorded as the mean of
measurements [13].
2.3.1.2. Folding Endurance. Folding endurance was determined by
repeatedly folding of SODI at the same place till break it or first sign
of break. Number of time the SODI could be folded at the same place
without breaking gives the folding endurance value [13].
2.3.1.3. Surface pH. The DZH SODI was allowed to swell in closed
petridish at room temperature for 30 min in 1 ml of distilled water. The
swollen device was removed and solution placed under digital pH meter
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Journal of Drug Delivery Science and Technology 46 (2018) 138–147
to determine the surface pH [13].
2.3.1.4. Weight uniformity. From each batch (n = 3), SODI were taken
and weighed individually using digital balance. The mean weight of the
insert was recorded [13].
2.3.1.5. Content uniformity. To check the uniformity of the drug in the
SODI, three inserts were taken from each formulation. Each insert was
placed in a glass vial containing 10 ml of artificial tear fluid. The insert
was dissolved by the aid of a magnetic stirrer, the solution was then
filtered. 1 ml from the filtrate was withdrawn and diluted upto 10 ml
distilled water and absorbance was measured by using optimized &
validated HPLC [13].
2.3.1.6. Tensile strength. Tensile strength of the formulation was
checked by Texture Analyzer (CT-3/10,000, Brookfield, USA)
equipped with a load cell. The SODI of 400 mm2 was randomly
selected and was fixed between the two clamps of probe TA-DGA for
a hold time of 60 s. The lower clamp was held stationary and the SODI
was pulled apart by the upper clamp. It was pulled at a speed of
2.0 mm/s to a distance of 6 mm with trigger load 0.05 N. The force of
the SODI at the point when the film broke was recorded [14]. Tensile
strength of the prepared SODI was calculated according to the following
equation [15].
N
Tensile strength =
mm2
Breaking load N
i.e.
Cross sectional area of the sample mm2
2.3.1.7. In vitro drug release study. In vitro drug release from the
different SODI was studied by using franz diffusion cell and dialysis
membrane. The dialysis membrane acted as a corneal epithelium. The
receptor compartment was filled with freshly prepared artificial tear
fluid. 40 mm2 area of ocular film was placed on the dialysis membrane
and opening of the donor compartment was sealed with a glass cover
slip, while the receptor fluid was maintained at 37 ± 0.5 °C with
constant stirring. 1 ml sample was withdrawn from receptor
compartment at various time intervals upto 6 H and was analyzed by
using reversed phase HPLC. Each sample withdrawn was replaced with
equal volume of artificial tear fluid [13].
2.3.1.8. Ex vivo transcorneal permeation study. Whole eye ball of goat
was transported from local butcher shop to the laboratory in cold (4 °C)
normal saline within 1 h of slaughtering the animal. The cornea was
carefully excised along with 2–4 mm of surrounding scleral tissue and
was washed with cold normal saline till the washing was free from
proteins. Isolated cornea was mounted by sandwiching surrounding
scleral tissue between clamped donor and receptor compartments of all
glass modified franz diffusion cell in such way that its epithelial surface
faced the donor compartment. The receptor compartment was filled
with freshly prepared artificial tear fluid. 40 mm2 area of ocular film
was placed on the cornea and opening of the donor compartment was
sealed with a glass cover slip, while the receptor fluid was maintained
at 37 ± 0.5 °C with constant stirring, using magnetic stirrer bead. 1 ml
sample was withdrawn from receptor compartment at various time
intervals upto 6 h and analyzed by using reversed phase HPLC. Each
sample withdrawn was replaced with equal volume of artificial tear
fluid [15].
2.3.1.9. Drug release kinetics. Drug release mechanisms and kinetics are
the two important characteristics of a drug delivery system in
describing drug dissolution profile. To describe the kinetics of the
drug release from optimized SODI, mathematical models such as zero-
order, first order, Higuchi, Hixson-Crowell and Korsmeyer-Peppas were
used. The criteria for selecting the most appropriate model were chosen
S.S. Patil et al. Journal of Drug Delivery Science and Technology 46 (2018) 138–147

Fig. 3. a) 3D Surface plot b) Contour plot for % Drug Release.

on the basis of the goodness or fit test.
2.3.1.10. Sterilization of ocular insert. The selected ocular inserts were
sterilized by gamma radiation using the Cobalt-60 source located at
ISOMED (Baba Atomic Research Center, Mumbai). SODI were packaged
in amber glass vials and exposed to a total dose of 2.5 Mrad.
2.3.1.12Culture media. Alternate thioglycolate medium and soyabean
casein digest medium was used as a culture medium for bacteria (S.
aureus) and fungi (C. albicans) respectively. Culture media were
prepared according to I.P.2014 and 20 ml was taken in boiling test
tube, properly plugged with cotton and sterilized by autoclaving at
121 °C, 15 lb/inch gauge pressure for 20 min.

2.3.1.13Inoculation and incubation. Formulation was aseptically added

2.3.1.11Sterility testing as per I.P. 2014. It is carried out to detect
in test tube containing respective media and simultaneously positive
presence of viable forms of microorganisms in the sterilized SODI.
and negative control was prepared for each media. The inoculated
The test is based on the principle that if the nutrient media is provided
culture media for bacteria and fungi were incubated at 30 °C - 35 °C and
to the sterilized ocular insert and favourable condition for growth of
20 °C - 25 °C respectively in incubator for 14 days.
microorganism is maintained if the microorganisms grow, the presence
is indicated by the turbidity in medium [16]. The test for sterility on the
sterilized ocular insert was carried out by direct inoculation method. 2.3.1.14Pharmacodynamic study. The CPCSEA protocol was obtained

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Fig. 4. a) 3D Surface plot b) Contour plot for Thickness.
from animal ethical committee (SIOP/IAEC/2016/08). Six adult male
albino rabbits of weight ranging from 1.5 to 2.0 kg were used in this
study. During experiments the test animals were kept in individual
cages with free access to food and water. Isotonic xylocaine solution
(2% v/v) instilled into rabbits eyes to anaesthetize the cornea.
Ophthalmic insert formulation applied into the right eye of three
rabbits, while the left eye was used as a control. Marketed
formulation was applied into the right eye of remaining three rabbits,
while left eye was used as a control. After administration of the test
formulation, the IOP of both eyes was measured every 1 h for 6 h using
a Schiotz tonometer (Riester, Germany). The percentage decrease in
IOP was determined according to the following equation
% Decrease in IOP = IOP control eye – IOP dosed eye / IOP control
eye × 100
2.3.1.15Accelerated stability studies as per ICH guidelines. Stability
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Journal of Drug Delivery Science and Technology 46 (2018) 138–147
studies for the optimized batch F2 was carried out to determine the
effect of presence of formulation additives on the stability of the drug
and also to determine the physical stability of the formulation under
accelerated storage conditions. The optimized batch F2 was subjected
to long term intermediate, accelerated stability studies for six months at
40 ± 2 °C/75 ± 5% RH. Samples were withdrawn at the end of 30, 60,
90 & 180 days and evaluated for physical appearance, and drug content
& in vitro drug release.
3. Results
3.1. Optimization and experimental design of ocular insert
Various batches of PVA and poloxamer 407 ocular insert were
prepared. 32 factorial design was estimated by using design expert
software (version 10). The independent variables were poloxamer 407
and PG concentrations. The independent variable and their levels are
shown in Table 2. The % cumulative drug release, thickness and folding
S.S. Patil et al. Journal of Drug Delivery Science and Technology 46 (2018) 138–147

Fig. 5. a) 3D Surface plot b) Countour plot for Folding Endurance.

endurance of the prepared inserts were taken as response parameters as

Table 4
SODI formulation and their overall desirability. the dependent variable.

Batch Variable Response Desirability

X1 X2 Y1 Y2 Y3 3.2. Thickness measurement

1 64.4 22 97.40 0.09 222 0.985 Inserts thickness was determined using a caliper (digital vernier
2 84.6 22 97.84 0.10 243 0.984 caliper) and recorded as the mean of measurements. The results are
3 43.2 22 96.21 0.10 256 0.997
4 42.4 22 96.54 0.11 266 0.978
given in Table 3. It indicates, prepared SODI were uniform in thickness
5 85.1 22 94.23 0.11 286 0.995 which is found to be directly related to concentration of polymer [15].

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Table 5 3.8. In vitro drug release study

Optimized formula suggested by software.
Batch PG Poloxamer 407 Predicted value VVVVVVVVVVdfsVValuevalue The in vitro permeation studies of DZH SODI were carried out
through the dialysis membrane clamped between donor and receptor
% Drug Thickness Folding compartment of franz diffusion cell. The result indicates that as the
release endurance concentration of poloxamer 407 is increases the release of drug is de-
1. 26.55 33.40 98.4 0.09 222 creases. Poloxamer 407 acts as a release retardant and gives cushioning
2. 26.01 45.01 98.3 0.09 243 effect. The results are shown in Fig. 2 indicating that the in vitro drug
3. 25.36 43.54 97.5 0.09 245 release from SODI (F1 to F9) was sustained for 6 H. Batch F2 shows
4. 24.28 59.21 97.3 0.10 252 drug release in sustained manner and it was more specific than other
5. 26.12 65.01 97.1 0.10 256
batches.
6. 22.87 52.11 96.2 0.10 266
7. 23.54 45.20 96.9 0.11 281
8. 22.15 42.18 95.7 0.11 302 3.9. Selection of optimized formulation
9. 21.80 39.57 94.5 0.12 323

3.3. Folding endurance
The folding endurance of SODI were recorded which reflects the
flexibility of films. This test ensures that the prepared SODI were sui-
table to produce continuous film without breaking or tearing. All the
patches showed good folding endurance (more than 200 was con-
sidered to be good value) [17]. The respective results are given in
Table 3.
3.4. Surface pH
The developed formulation should be non irritant after adminis-
tration. The surface pH of the prepared SODI was found to be in range
of 6.5 ± 0.1 to 6.7 ± 0.1 indicating safe in chronic treatment of eye
infection as shown in Table 3. The pH of therapeutic substances applied
to eye can vary from 3.5 to 8.5 [15].
From the in vitro drug release it was observed that the batch F2
shows appearance (good and clear), thickness (0.10), folding endurance
(242), and cumulative % drug release (98.50%) respectively.
3.10. Fitting data into model
All the responses observed from F1 to F9 were fitted to various
models using design-expert software.
3.10.1. Response surface plots analysis and contour plots
3.10.1.1. Effect of formulation variable on drug diffusion. The model F-
value of 86.02 implies the model is significant. There is only a 4.63%
chance that an F-value this large could occur due to noise. Values of
“Prob > F” less than 0.0500 indicate model terms are significant. In
this case X2 is a significant model term.
The model for response Y1 (% cumulative drug release) is as follows:
Y1 (% Drug Release) = +95.92–0.98X1-1.52 X2-0.25X1.X2+6.66X12-
0.76X22 (1)

Above equation (3) indicates that (X1) concentration of PG and (X2)

3.5. Weight uniformity
The weights of the DZH SODI were found to be in the range of
2.2 ± 0.1 to 2.8 ± 0.2 mg given in Table 3. The uniformity of the
weights of the SODI indicates good distribution of the drug, in polymer
and plasticizer [15].
3.6. Content uniformity
Drug content of PVA films were determined and it was found to be
in between 97.92 and 98.76% given in Table 3. The results indicated
that drug was uniformly dispersed.
concentration of poloxamer 407. Both have significant effect on % drug
release. Effect of (X1) and (X2) can be further explained by response
surface plots and contour plots (Fig. 3).
3.10.1.2. Effect of formulation variable on thickness. The model F-value
of 17.40 implies the model is significant. There is only a 2.00% chance
that an F-value this large could occur due to noise. Values of
“Prob > F” less than 0.0500 indicate model terms are significant. In
this case X2, X12 are significant model term.
The model for response Y2 (thickness) is as follows:
Y2(Thickness) = +0.11 + 6.66 X1 +1.0X2-5.0 X1. X2 -7.81X12-4.29
X2 2 (2)

Above equation (2) indicates that (X1) concentration of PG and (X2)

3.7. Tensile strength
Tensile strength of DZH SODI was found to be 24.33 kg/mm2. The
tensile strength value more than 7 kg/mm2 indicates good tensile
strength. The values showed that the mechanical strength of patches
was enough to bear stress during transport and administration of pat-
ches [17] (see Fig. 1).
concentration of poloxamer 407. Both have significant effect on thick-
ness. Effect of (X1) and (X2) can be further explained by response sur-
face plots and contour plots (Fig. 4).
3.10.1.3. Effect of formulation variable on folding endurance. The model
F-value of 320.58 implies the model is significant. There is only a 0.03%
chance that an F-value this large could occur due to noise. Values of

Table 6
Validation of model.
Variables Predicted value Experimental value % Prediction error

X1 X2 Y1 Y2 Y3 Y1 Y2 Y3 Y1 Y2 Y3

26.5 33.45 98.41 0.10 222 98.11 0.11 226 −0.30 1.09 1.70
25.3 43.54 97.50 0.12 245 98.25 0.10 243 0.76 −1.70 −0.82
24.2 59.21 97.30 0.11 252 96.32 0.12 257 −1.01 1.80 1.90

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Fig. 6. Ex vivo transcorneal cumulative % drug release from SODI.

Fig. 9. Intra ocular pressure after administration of the developed SODI.

Fig. 7. First order kinetic from optimized SODI.

Table 7
Sterility testing for batch F2.
Sr.No. Medium Days Fig. 10. Percentage decrease in IOP after administration of developed SODI and
marketed product.
1. Alternative Thioglycolate 1 2 3 4 5 10 14
Control – – – – – – –
Sterilized film – – – – – – – significant model terms (not counting those required to support
Unsterilized film – – – – – + + hierarchy), model reduction may improve your model. The equation
Positive – – + + + + + in terms of coded factors can be used to make predictions about the
2. Soyabin Casein Digest
response for given levels of each factor. By default, the high levels of the
Control – – – – – – –
Sterilized films – – – – – – – factors are coded as +1 and the low levels of the factors are coded as
Unsterilized films – – – – – + + −1. The coded equation is useful for identifying the relative impact of
Positive – – + + + + + the factors by comparing the factor coefficients.
The model for response Y1 (folding endurance) is as follows:

Y3(FE) = +294.25 + 15.56X1+44.71X2-5.67X1.X2+2.76X12-8.38X22

(3)

Above equation (1) indicates that (X1) concentration of PG and (X2)

concentration of poloxamer 407, both have significant effect on folding
endurance. Effect of (X1) and (X2) can be further explained by response
surface plots and contour plots (Fig. 5).

3.11. Validation of model

The optimized compositions of ocular insert required to obtain de-

Fig. 8. (A) indicates Positive, (B) indicates unsterilized SODI, (C) indicates
sterilized SODI, and (D) indicates as control.
“Prob > F” less than 0.0500 indicate model terms are significant. In
this case X1, X2, X12 are significant model terms. Values greater than
0.1000 indicate the model terms are not significant. If there are many in
sired responses are shown in Table 3. Any composition with desirability
near to 1 can be chosen as optimized formulation (Table 4).
Software suggested all possible combination of independent vari-
able other than those used for optimization and their effect on depen-
dent variable as shown in Table 5. Out of all suggestions, any combi-
nations of independent variable i.e. concentration of PVA and PG can be
selected randomly and used for validation of suggested model. Ex-
periments were carried out by using batch 1, 2, 3. Data obtained by

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Fig. 11. Rabbit eye after measurement IOP for 6 h.

presence of fungi. Optimized SODI were used to perform sterility testing

Table 8 and results are given in Table 7 and Fig. 8. In Table 7 (−) sign indicates
Accelerated stability studies. no growth of microorganisms and (+) sign indicate the growth of mi-
Time in month Temperature/%RH Parameters croorganisms. Optimized batch F2 of SODI indicates that the sterilized
SODI were free from microorganism.
Appearance % Drug Content

1 40 ± 2 °C/75 ± 5 No change 99.05 3.13.3. Pharmacodynamic study

2 40 ± 2 °C/75 ± 5 No change 99.74 The IOP of normotensive rabbits decreased by 20% ± 0.2 after
3 40 ± 2 °C/75 ± 5 No change 99.49 administration of single dose per day of developed SODI where as three
6 40 ± 2 °C/75 ± 5 No change 99.32
doses of conventional ophthalmic eye drop per day showed only 10% of
decrease as shown in Fig. 9. The difference in IOP with reference to
time indicating developed SODI lead to faster onset of drug action as
experiment was used for % prediction error calculation. Results ob-
compared to the marketed product given in Fig. 10. It is also concluded
tained were showed in Table 5.
that developed formulation remain sustained and effective up to 6 H
Percent prediction error was calculated by using formula as given
while marketed formulation showed effect that is for 3–4 H. After
below
checking IOP for 6H there was no signs of irritation as like redness,
= Experimental Value –Predicted Value / Experimental Value × 100 swelling and haemorrhage given in Fig. 11. It indicated that prepared
SODI is safe [18].

As % prediction error was found to be less than 4% in all cases,

model suggested by software was found to be valid (see Table 6). 3.14. Accelerated stability studies

Stability study was carried out on optimized SODI formulation for

3.12. Ex vivo transcorneal permeability study
Goat cornea was used for ex vivo transcorneal permeability study of
DZH SODI. Cumulative percent drug of DZH SODI (batch F2) is shown
in Fig. 6.
3.13. Drug release kinetics
The drug release data obtained from in vitro release experiments
(optimized formulation) was subjected to various kinetics equations to
evaluate the drug release kinetics. The prepared batch F2 shows first
order kinetic release (Fig. 7) i. e the release of drug is dependent on
concentrations.
3.13.1. Sterilization of ocular insert
There was no change observed in sterilized SODI when exposed to
2.5 Mrad radiation. Indicating the method & dose is suitable for ster-
ilization. These results proved the efficiency of gamma radiation as a
method of sterilization at the given dose 2.5 Mrad [19].
three months. It was found that formulation remained stable at various
conditions of temperature and relative humidity used as per ICH
guidelines. The results obtained are shown in Table 8. The results
showed that there was no change in physical appearance of SODI. Drug
content showed no marked change after six months. These results
concluded that SODI were chemically and physically stable at different
temperature and humidity conditions for three months.
4. Conclusion
The developed Dorzolamide Hydrochloride SODI shows sustained
release of drug for upto 6 h. Hence, the studies indicated that the de-
veloped formulation may prove to be a possible alternative to con-
ventional ophthalmic eye drop in terms of ease of administration with
added benefits of sustained effect, safety, bioavailability and ther-
apeutic activity.
Acknowledgements

3.13.2. Sterility testing as per IP 2014
In this study the presence of microorganisms is checked after ster-
ilization. SODI were sterilized by gamma radiations. For this study al-
ternative thioglycolate medium (ATGM) was used to analyze presence
of bacteria and soybean casein digest medium (SBCD) was used for
Authors are thankful to FDC Mumbai for providing gift sample of
dorzolamide drug. Authors are grateful to Principal Dr. K. G. Bothara
for providing excellent facility for research work. (Sayali S. Patil, Dr.
Amol Tagalpallewar, Archana Bade declare that they have no conflict of
interest).

146
S.S. Patil et al.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.jddst.2018.05.010.
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