Just Accepted by Drug Development and Industrial Pharmacy

Formulation and Development of ophthalmic In situ gel

for the treatment ocular inflammation and infection us-
ing application of Quality by Design concept
Nirav Patel, Vaishali Thakkar, Viral Metalia, Lalji Baldaniya, Tejal Gandhi, Mukesh
Gohel

Doi: 10.3109/03639045.2015.1137306

Abstract
Context: The conventional liquid ophthalmic delivery systems exhibit short precor-
neal residence time and the relative impermeability to the cornea which leads to poor
ocular bioavailability. Objective: The aim of this study was to apply quality by design
(QbD) for development of Dexamethasone sodium phosphate (DSP) and Tobramy-
cin sulphate (TS) loaded thermoresponsive ophthalmic In Situ gel containing polox-
amer 407 and Hydroxyl propyl methyl cellulose (HPMC) K4M for prolonging the pre-
corneal residence time, ocular bioavability and decreases the frequency of
administration of dosage form. The material attributes and the critical quality attributes (CQA) of the in-situ gel were identified.
Central composite design (CCD) was adopted to optimize the formulation. Materials and methods: The ophthalmic In Situ
forming gels were prepared by cold method. Materials attributes were the amount of poloxamer 407 and HPMC and CQA
identified were Gel strength, mucoadhesive index, gelation temperature and % of drug release of both drug. Results and
discussion: Optimized batch (F*) containing 16.75% poloxamer 407 and 0.54% HPMC K4M were exhibited all results in
acceptable limits. Compared with the marketed formulation, optimized in-situ gel showed delayed Tmax, improved Cmax
and AUC in rabbit aqueous humor, suggesting the sustained drug release and better corneal penetration and absorption.
Conclusion: According to the study, it could be concluded that DSP and TS would be successfully formulated as in situ gelling
mucoadhesive system for the treatment of steroid responsive eye infections with the properties of sustained drug release,
prolonged ocular retention and improved corneal penetration.

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Formulation and Development of ophthalmic In situ gel for the treatment ocular inflammation
and infection using application of Quality by Design concept

Nirav Patel1, *, Vaishali Thakkar2, Viral Metalia2, Lalji Baldaniya2, Tejal Gandhi2, Mukesh Gohel2
1
Dept. of Pharmaceutical Sciences, Saurashtra University, Rajkot 360 005, Gujarat, India
2
Anand Pharmacy College, Anand, 388 001, Gujarat, India

Keywords
Dexamethasone Sodium Phosphate, Tobramycin Sulphate, Central composite design, Poloxamer
407, Hydroxy propyl methyl cellulose (HPMC) K4M, Eye irritation study, Pharmacokinetic

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study

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*
Corresponding Author:
Nirav Patel
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Dept. of Pharmaceutical Sciences,
Saurashtra University, Rajkot 360 005,
Gujarat, India
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(M): +91-9898303419
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E-mail: [email protected]
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Abstract
Context: The conventional liquid ophthalmic delivery systems exhibit short precorneal residence
time and the relative impermeability to the cornea which leads to poor ocular bioavailability.
Objective: The aim of this study was to apply quality by design (QbD) for development of
Dexamethasone sodium phosphate (DSP) and Tobramycin sulphate (TS) loaded
thermoresponsive ophthalmic In Situ gel containing poloxamer 407 and Hydroxyl propyl methyl
cellulose (HPMC) K4M for prolonging the pre-corneal residence time, ocular bioavability and
decreases the frequency of administration of dosage form. The material attributes and the critical
quality attributes (CQA) of the in-situ gel were identified. Central composite design (CCD) was
adopted to optimize the formulation. Materials and methods: The ophthalmic In Situ forming

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gels were prepared by cold method. Materials attributes were the amount of poloxamer 407 and

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HPMC and CQA identified were Gel strength, mucoadhesive index, gelation temperature and %
of drug release of both drug. Results and discussion: Optimized batch (F*) containing 16.75%
poloxamer 407 and 0.54 % HPMC K4M were exhibited all results in acceptable limits.
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Compared with the marketed formulation, optimized in-situ gel showed delayed Tmax, improved
Cmax and AUC in rabbit aqueous humor, suggesting the sustained drug release and better corneal
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penetration and absorption. Conclusion: According to the study, it could be concluded that DSP
and TS would be successfully formulated as in situ gelling mucoadhesive system for the
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treatment of steroid responsive eye infections with the properties of sustained drug release,
prolonged ocular retention and improved corneal penetration.
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Introduction
Eye is one of the most challenging organs for drug delivery because it’s unique anatomy.
Ocular barriers are essential and exclusive to ocular anatomy and physiology making it a
challenging task for drug delivery scientists as they restrict drug absorption into the deep tissues.
The Conventional eye drops of more than 90% of marketed formulations are available due to
1-4
simple instillation into the eye with accuracy of doses . Conversely, this eye drops has certain
disadvantages like rapid pre-corneal elimination by protective mechanisms of the eye such as
blinking reflex, lacrimal fluid dilution and nasolacrimal duct drainage 1. Currently, a major
progress in development of ophthalmic formulations has been occurred by the ophthalmic gel
technology in the development of droppable gels called an “In Situ Gel” which consists of

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certain polymers which undergoes sol–gel phase transition by an induction of environment
conditions like pH 5, 6, specific ions 3 and temperature 7, 8. In situ hydro gel formulations 9 applied

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as solutions or suspensions that undergo gelation after instillation. These systems are more
acceptable for the patients, since they are administered into the eye as a solution and undergo an
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immediate gelation whilst in contact with the eye. Studies have shown that the pre-corneal
residence time of some In Situ gelling systems are for several hours. Various polymeric
10
combinations , have been successfully used for formulation and desired release profile. It has
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been suggested that a good ophthalmic thermo responsive In Situ gel should have sol–gel
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transition temperature higher than room temperature and form gel at pre-corneal temperature
(350C) to avoid keeping in a fridge before administration which may sometime lead to eye
irritation due to cold eye drops 7, 11, 12.
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Dexamethasone Sodium Phosphate (DSP) and Tobramycin Sulphate (TS) are used in
treatment of post-operative ocular inflammation and prevention of infection after cataract
surgery. Tobramycin is an antibacterial which can help to treat certain types of eye
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infections. Dexamethasone reduces inflammation and can help to relieve the symptoms of
inflammatory eye problems. TOBRADEX® (Tobramycin and Dexamethasone ophthalmic
suspension- FDA approved), is a sterile, multiple dose antibiotic and steroid combination for
topical ophthalmic use. The disadvantages of current marketed formulations are burning,
stinging, irritation, itching to the eyes due to dispersed particles 13.
One of well-known polymer types possessing thermoresponsive behaviour is Pluronics,
so called Poloxamers. They are a triblock copolymer poly (ethylene oxide)-b-poly (propylene
oxide) - b-poly (ethylene oxide) (PEO–PPO–PEO) showing amphiphilic behavior due to
hydrophilic ethylene oxide domains and hydrophobic propylene oxide domains. The gelation
mechanism of Pluronics could be explained by the changes in micellar structure as a function of
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concentration and temperature . Pluronics have been widely used as an ocular drug delivery
system because they could prolong drug release and present satisfactory inertia for eye tissue 8.
However, a major disadvantage of Pluronics is their low mucoadhesive activity; therefore, some
Pluronic-based ophthalmic formulations have been improved by adding polymers providing
mucoadhesive property such as Hydroxypropyl methyl cellulose low viscosity grade (HPMC
K4M), Carbopol 4, sodium hyaluronate 7.
QbD approach encompasses designing and developing formulations, in which

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manufacturing processes ensure pre-defined product specifications. The important part of this

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approach is to understand how process and formulation parameters affect the product quality and
14-15
subsequent optimization parameters with respect to final specifications . QbD uses
multivariate experiments to understand product and process and to establish a design space
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through design of experiments (DOE). DOE is an organized method to determine the relationship
between the inputs and outputs of a process 16.
In the current research, the Dexamethasone Sodium Phosphate (DSP) and Tobramycin
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Sulphate (TS) ophthalmic in situ gel using Poloxamer 407 in the combination with HPMC K4M
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polymers with different ratios were prepared to increase pre-corneal residence time and
decreases the frequency of administration of dosage form. The goal of this research work is to
prepare in-situ ophthalmic gel of high therapeutic efficacy with lacking the undesirable effects of
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the market product. The formulation was optimized in terms of Gelation Temperatures, Gelation
strength, Mucoadhesive Index, DSP and TS Release in 9 hrs by two-factor and five-level Central
Composite Design. The best formulation was studied for In-Vivo skin irritation and elimination
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pharmacokinetic performance in Albino rabbits and compared with marketed formulation.

Materials and methods

Materials
Dexamethasone Sodium Phosphate (DSP) and Tobramycin Sulphate (TS) were obtained as the
gift sample from MARCK Bioscience Ltd, Kheda, India. Poloxamer 188 and 407 were obtained
as gift sample from BASF Pvt. Ltd., Mumbai, India. Hydroxyl propyl methyl cellulose (HPMC
K4M) and Benzalkonium chloride (BKC) were commercially purchased from MEPRO
pharmaceuticals and Sigma-Aldrich respectively. All other reagents were of analytical grade and
commercially available.

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Experimental methods

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Identification of Material Attributes of Excipients Used for the Preparation of In-situ gel
Preliminary trials for the selection of polymers amongst ion, pH and thermo sensitive polymer
like Poloxamer 188, Poloxamer 407, Hydroxy propyl methyl cellulose (HPMC K4M), Gelrite
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(gellan gum), Chitosan, Carbopol 934, Polyox were done on the basis of literature survey.
Polymers were screened out on the basis of viscosity and their rheological behaviour. In addition,
Poloxamers were screened out on the basis of sol-gel transition temperature (Gelation
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Temperature). Solutions of Poloxamer 188, Poloxamer 407, Gelrite, Chitosan, Carbopol 934,
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Polyox, Hydroxy Propyl Methyl Cellulose (HPMCK4M) were prepared using 100 ml sterile
water for injection (WFI). Briefly, Poloxamer 188 & 407 in required amount weighed and was
mixed thoroughly at 40C in phosphate buffer (pH. 7.4) and volume was made up with deionised
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double distilled water. Dispersion was stored over 24 h, until clear solution formed. Viscosity of
above prepared solutions were measured using Brookfield Viscometer at shear rate 10, 20, 30,
50, 60, 75, 100, 120, 150 and 200 rpm by using LVDV Spindle No. 61, 62, 63 and 64 at
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temperature maintained below 200C. The flow behaviour was detected from the graphs of
Viscosity vs. Shear rate. Gelation temperature of prepared Poloxamer mixture was measured by
heating the 10 ml formulation in a 15 ml borosilicate glass test tube. Into each test tube, a
magnetic bar was placed. A thermometer with an accuracy rating of 0.10C was immersed in the
sample solution. The solution was heated at the rate of 10C/1–2 min with constant stirring of 5
rpm. The temperature at which the magnetic bar completely stopped moving because of gelation
was regarded as the GT. Each sample was measured at least in triplicate. These data were used to
optimize concentrations as well as polymers for the preparation of ophthalmic in situ gel.

Drug Polymer Compatibility Studies 17

Fourier Transform Infrared Spectroscopy (FTIR)
The Infra-Red spectra of DSP, TS, Physical mixture of DSP and TS, Physical mixture of drug
and polymers were obtained on Fourier Transform Infrared Spectrophotometer (Perkin Elmer –
spectrum Bx, USA) in order to detect the existence of interaction between drug and polymer.
The sample was dispersed in KBr to prepare 10 % of mixture and ground in mortar-pestle before
being compressed into pellets. This pellet was placed in light path and spectrum was recorded at

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a resolution of 2 cm-1 over a frequency range of 4000 to 400 cm-1. The background spectrum of

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KBr was used as blank for determination.

Differential Scanning Calorimetric (DSC)

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Differential scanning calorimeter (DSC) spectra were obtained using Perkin Elmer instruments,
(Perkin Elmer DSC-7, Norway, USA.) to study the thermal behaviour of DSP, TS and mixture of
drug and polymers. The instrument comprised of calorimeter (DSC-60), flow controller (FCL-
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60), Thermal analyser (TA-60) and operating software (TA-60). The samples (2-4 mg) were
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heated in hermetically sealed flat-bottomed aluminium pans under nitrogen flow (20ml/min) at a
scanning rate of 100c/min from 250C to 3400C. Empty aluminum pans were used as the
reference standard.
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Preparation of in-situ gel with the Selected Variables Using Central composite design 18, 19
Design of Experiment: A two-factor, three-level central composite design used to optimize the
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Poloxamer 407 and HPMC K4M concentration and rheological property of DSP and TS In
Situ gel. The design consisted of ten experimental points that included fractional 6 factorial
points, two star points and two replicates at the centre point. Central composite statistical
screening design was used to optimize and evaluate the effects of the material attributes on the
CQAs of ophthalmic In-Situ gel. A design used is suitable for exploring quadratic response
surfaces and constructing second-order polynomial models with Design Expert (Version
7.0.0, Stat-Ease Inc., Minneapolis, MN, USA).
The linear & quadratic models for predicting the optimal point were expressed using the
following equation:
Linear model:
Y = β0 + β1 X1+ β2 X2+ β12 X1 X2…..…………………..................................... (1)
Quadratic model:
Y=β0 + β1 X1+ β2X2+ β12 X1 X2+ β3 X12+ β4 X22+ β5 X1X22+ β6 X12 X2 ................(2)
Where, Y is the measured response associated with each factor level combination, β0 is intercept,
β1 to β6 are regression coefficients computed from the observed experimental values of Y, and
X1, X2, and X3 are the coded levels of independent variables. The terms X1X2 and X2i (i=1, 2, or
3) represent the interaction and quadratic terms, respectively. The validity of the model was

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determined by comparing the experimental and predicted values. Statistical validity of the

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polynomials was established on the basis of ANOVA provision in the design expert software.
Subsequently, the feasibility and grid searches were performed to locate the composition of
optimum formulations. Also 3- D response surface graphs were constructed using the design
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expert software. Materials attributes and CQAs (process output) selected with the results are
shown in Table 1. Materials attributes studied were Poloxamer 407 conc. (%) (X1) and HPMC
K4M conc. (%) (X2). The The CQAs were gel strength (Y1), Gelation Temperature (Y2),
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mucoadhesion index (Y3) and cumulative percentage drug release for DSP (Y4) and TS (Y5) after
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9 h. The optimum formulation of this study based on the constrains for CQAs given in Table 1.

Formulation & Development of In Situ Gel 20, 21

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The compositions of prepared In-situ gel containing various gelling agents were shown in Table
2. In situ gel was prepared by dispersing required amount of Poloxamer 407 in 50 ml cold
ultrapure water (UPW). Solution was kept in a refrigerator for at least 24 hr. to ensure complete
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dissolution of polymer and clear solution obtained. The required quantity of HPMC K4M was
weighed in separate beaker containing 40 ml of UPW. This mixture was heated to 70oC and then
placed on magnetic stirrer until clear solution obtained. Above two polymer solutions were
allowed to cool at room temperature and mixed properly. DSP (0.1%), TS (0.3%) was added
separately in deionized double distilled water and mixed with polymer solution at room
temperature. Volume was made up to 100 ml with UPW and the pH of all formulations was kept
between 6.5 - 7.4 using 0.5 M NaOH. The sodium chloride (0.2%) was added to make up
osmolality in range of 290 to 310 mOsm kg-1. Disodium EDTA (0.05%) and Benzalkonium
chloride (0.001%) were also added in all the formulations. Formulations were sterilized using
autoclave at temperature 1210C and pressure 15 PSI for 15 min 22.

Evaluation of In Situ Gel

Clarity, pH, viscosity and osmolality
Clarity is one of the most important characteristic features of ophthalmic preparations. All
developed formulations were evaluated for visual inspection of each container under a good light
with viewed against reflection into the eyes and also against a black and white background, with
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the contents set in motion with a swirling action . pH of prepared in situ gel was be measured

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with pH meter. The dynamic viscosity of the prepared in situ gel was measured using a

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viscometer (BROOKFIELD DV-II+ Pro EXTRA, Programmable Rheometer, BROOKFIELD
ENGINEERING LABORATORIES, INC., USA) at a shear rate 10-200. The developed
formulation was poured into programmable viscometer containing spindle no.62 and the angular
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velocity (shear rate) was increased gradually from 10 to 200 rpm. The hierarchy of the angular
velocity was reversed. Viscosity of in situ gel was measured before and after gelation. Gelation
was induced in formulation (50 mL) by adding simulated tear fluid (STF) at the ratio of 40:7
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with temp. 370C, as the conventional commercial eye dropper delivers an average drop volume
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about 40 µl while available tear fluid is 7 µl. The measurements were performed in triplicate and
the mean viscosity of the all batch was calculated 23. The osmolality of sterilized In Situ gel was
determined with a vapour pressure osmometer 24.
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Development of First order derivative Spectrophotometric method for the simultaneous estimation
of DSP and TS and Validation of Analytical Method
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Spectrophotometric method was developed for simultaneous determination of DSP and TS. In the
first order derivative Spectrophotometric method, the first derivative amplitudes at 247 nm and 239
nm were selected for assay of DSP and TS, respectively. Standard solutions of both DSP and TS in
the range of 5–35 μg/mL were separately prepared by appropriate dilutions of their respective
working standard solutions in water and withdrawn 1 ml solution and follow derivatization
procedure then they were scanned in the range of 200–400 nm. For derivatization, 0.5 ml
phenylisocyanate (PIC) and 0.5 ml Tri ethyl amine (TEA) solutions were added in 1 ml of standard
solutions 25. They were heated for 10 min at 700C. Absorbance was measured at wavelength 247 and
239 nm for DSP and TS respectively 26, 27. Absorbance spectra were converted in 1st derivative and
zero cross over point (ZCP) for DSP and TS was measured. Absorbance was measured for DSP at
ZCP of Tobramycin and for TS at ZCP of Dexamethasone. The validity of the method for linearity,
specificity, accuracy, repeatability and precision according to recommendations were tested.
Methods validation has been performed as per the International Conference on Harmonization (ICH)
guidelines ICH, 2005 and USP requirements.

Gelation temperature
Two glass tubes of 10 ml capacity, one containing 1 g of the formulations and the other

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containing 1 ml of water, were transferred to a water bath. A thermometer was placed in the

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water filled glass tube. The temperature of the water bath was increased and the temperature at
which the in situ gel solution stopped flowing upon inverting the tube was recorded (T1). Then
the temperature was decreased and the temperature at which the gel started flowing again was
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recorded (T2). The average value of T1 and T2 was the critical gelation temperature (Average 0C).
Meanwhile, the formulation was diluted by artificial isotonic tears (40:7, v: v), and the gelation
temperature was detected 22.
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Gel strength and Refractive index

The gel strength was determined using texture analyzer. (TA-XT2 Texture analyzer, RK Puram,
New Delhi) The method by which the properties of polymeric system may be conveniently
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determined is texture profile analysis. To mimic the situation where, In Situ gel system, upon
ocular instillation, was diluted with the available tear fluid and gelled in a thermostat at 37 °C.
The in situ gelling system was mixed with STF in proportion 40:7 and transferred to a 100 ml
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graduated cylinder. The experiment was done by placing the gels in standard beaker below the
probe. In this an analytical probe is then immersed into the sample. The Texture Analyzer was
set to the ‘gelling strength test’ mode. An aluminum probe of 7.6 cm diameter was used for all
the samples. The study was carried out at room temperature. The force required to penetrate the
gel was measured as gel strength. Refractive index of the formulations was determined by
refractometer (Abbe's refractometer, DR-A1, ATAGO India Instruments, Mumbai). Refractive
index was measured form the scale. The value should be in acceptable range (Refractive index of
tear fluid is 1.340-1.360) which is essential for clean vision 28.

Mucoadhesion index (MI)

Mucin Dispersion (MUC) 29
Dried mucin was hydrated with phosphate buffer (pH 7.4) by stirring for 12 h at room
temperature yielding a dispersion of 20 % (w/w). Fifteen grams of this dispersion were mixed for
20 min with 5 g of prepared sol before measurement. The final concentration of mucin was 15 %
(w/w). Viscosity of prepared sol/mucin system (ɳt) and that of 15 % (w/w) MUC (ɳm) were
measured at 32oC at shear rates of 25, 50, 100 and 150 S-1. The viscosity of the prepared sol

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/phosphate buffer (pH 7.4) (ɳp) was determined in same way. Viscometric measurement

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performed for 1 min after exactly 3 min of application of shear stress.
The viscosity component due to bioadhesion (ɳb) was obtained by following equation,
ƞb= ƞt – ƞm - ƞp…………………………………………………………….... (3)
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The mucoadhesion index M [Pa] was calculated using the shear rate D[S ] and the viscosity
component ƞb [mPas] according to the following equation,
M= ƞb* D………………………………………………………………….... (4)
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In vitro drug release (Diffusion study) 30

The Franz diffusion cell was used for studying the in vitro release of the in situ gel. A cellulose
acetate membrane (Dialysis membrane with 25 mm diameter.0.5 μm pore size) was adapted to
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the terminal portion of the cylindrical donor compartment. Three ml in situ gel containing drug,
sufficient for establishing sink conditions for the assay was placed into the donor compartment.
The receptor compartment contained 15 ml of Phosphate buffer solution of pH 7.4 was
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maintained at 37 °C under mild agitation using a magnetic stirrer. At specific time intervals,
aliquots of 1 mL was withdrawn and immediately restored with the same volume of fresh
phosphate buffer. The amount of drug released was assessed by measuring the absorbance at 247
and 239 nm for DSP and TS respectively using derivatizing UV spectroscopy.

Kinetics of drug release 31

In order to analyse the drug release mechanism, the goodness-of-fit method for in vitro release
data was applied and different kinetic models like zero-order, first order, Higuchi, and
Korsmeyer-Peppas were applied to ascertain the kinetic modelling of drug release.

Sterility testing 32
The sterility testing of optimized formulation was performed for the aerobic, anaerobic
bacteria & fungi by using alternative thioglycolate medium and soybean casein digest
medium. The positive control (growth promotion), negative control (sterility) test was carried
out. Bacillus subtilis, Bacterio desvulgatus and candida albicans were used as a test organism in
the case of aerobic bacteria, anaerobic bacteria fungi test respectively. Incubation was carried in

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all cases and growth was checked. All the samples were inoculated separately in to Alternative

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Thioglycolate Media (ATGM) and Soya bean Casein Digest Agar Media (SBCD) media and
incubated at 350C and 20-250C, respectively for 7 days. Similarly control samples were prepared
(without In Situ gel) separately in to ATGM and SBCD media and incubated at 350C and 20-
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250C, respectively for 7 days. A control evaluation was also carried out.
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Preservative Efficacy Test (PET) 33

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Preservative efficacy test were performed as per USP method. Culture of bacteria (Escherichia
coli (ATCC 4352), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC
6538)) and fungi (Candida albicans (ATCC 10231), Aspergillus Niger (ATCC 16404)) were
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grown in Sabouraud glucose agar medium and Soya bean casein digest agar medium for
respective bacteria and fungi. Both culture of organism were diluted aseptically with sterile WFI
to obtain 10-6 CFU/ml as per USP. All the cultures were transferred into 5 test tubes containing
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10ml prepared eye drops and 0.1% of prepared cultures in each. Initial counts were noted. These
solutions were poured into petri plate containing Soya bean casein digest agar medium for
bacterial cultures and Sabouraud glucose agar medium for fungi. They were incubated at
32.5±2.50C and 22.5 ± 2.5 ºC for bacteria and fungi respectively. The numbers of colony of
microorganism at 7th, 14thand 28th day were recorded. The criteria for preservative effectiveness
for their acceptable range were also checked.
In Vivo Studies 34
New Zealand Albino rabbits weighing 3.0-3.5 kg were used for in vivo studies. They were
treated as prescribed in the “Guide for the Care and Use of Laboratory Animals” (NIH
Publication No. 92-93, revised 1985). Prior to the experiments, the animals were housed in
standard cages in a light-controlled room at 19±10C and 50±5% relative humidity, with no
restriction of food or water. During the experiments the rabbits were placed in restraining boxes,
where they could move their heads and eyes freely. All the experiments were carried out under
veterinary supervision, few rabbits were utilized for pharmacokinetic study and few were used to
check ocular irritancy. The preclinical experimental protocol (Protocol no. 1222, Anand
Pharmacy College, Anand had been approved on 24th November 2012) was approved by

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institutional animal ethics committee as per the guidance of CPCSEA, Ministry of social justice

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and empowerment, Government of India (Ethical committee Registration number is
277/CPCSEA). EP
Ocular Irritation study
The in vivo eye irritation test of the optimized F* In Situ gel was performed in a group of eight
New Zealand albino rabbits. Twenty microliters of the representative formulation was instilled
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into the lower conjunctival sac of the rabbit’s right eye, while the left was kept as a control
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without manipulation. The test eye was observed at each of the following time intervals: 0, 5, 10,
30 min, 1, 6, 12, 24, 48 and 72 h for the changes of cornea, iris, conjunctiva and chemosis
compared to the control. The degree of eye irritation was also scored following the modified
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Draize test 35.

HPLC method for estimation of DSP and TS in tear fluid of rabbit 36-38
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A liquid chromatographic method for the determination of the Dexamethasone Sodium

Phosphate (DSP) and Tobramycin sulphate (TS) in their synthetic binary mixture was
established. Both drugs were pre column derivatized with phenylisocyanate (PIC) and
triethylamine for 10 min at 70 ºC and chromatographed on an enable C18G 250 x 4.6 mm
column. The separation was achieved on simple isocratic method. The mobile phase contains a
mixture of Tris buffer: acetonitrile PH adjusted with Sulphuric (pH 6.5) in the ratio of 70: 30 v/v.
1ml triethylamine was added. The flow rate for analysis was 1.0 mL/min and the injection
volume was 10μL. The detector wavelength was set to 240 nm for both title drugs because at this
wavelength the sensitivity was higher than in any other more characteristics wavelengths.
Double distillation deionized water is used as diluents. The retention times were 2.7 and 7.8
minutes for DSP and TS respectively. The described method was validated with respect to
system suitability, specificity, linearity, precision, accuracy and robustness.

Pharmacokinetic study 34
Kinetic studies were performed on four rabbits, each weighing 2–3 kg. Two of them were
received marketed conventional eye drops and remaining two had received optimized
formulation. One drop of each optimized batch or marketed eye drops (40µL) was instilled into

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the lower conjunctival sac by a micro pipette. Tear fluid samples were collected by washing eyes

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with 0.5 ml simulated tear fluid at 15, 30, 60, 90, 120, 180 min from the lower marginal tear strip
using disposable glass capillaries. After further dilution, samples were analyzed by HPLC
method. Concentration of DSP and TS in tear fluid was determined and further calculations were
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done based on non-compartmental kinetics technique. The areas under the concentration-time
curves (AUCs) were calculated by the trapezoidal method. The other pharmacokinetic
parameters were calculated using a non-compartmental pharmacokinetic model and Microsoft
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Excel software.
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Accelerated Stability study 39

To assess the drug and formulation stability, Accelerated stability studies were done according to
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ICH guidelines. Optimized formulation was packed in 5 mL white LDPE plastic dropper bottles
and kept in a stability chamber at specified temperature and humidity (250C ± 20C/ 60% ±5%
RH) for six month. The chemical stability of the formulation was assessed by the estimation of
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the percentage drug remaining in the formulation and physical stability was evaluated by
monitoring any change in pH, drug assay, preservative assay, viscosity and appearance.
3. Result and Discussion

Identification of the Material Attributes and Potential CQAs required for Development of
ophthalmic In-Situ gel
According to QbD, pharmaceutical development includes identifying potential CQAs of the drug
product, determining material attributes of excipients, selecting an appropriate manufacturing
40
process (CPPs) and defining a control strategy . Preliminary trials were carried out for the
selection of polymers, concentration and combination ratio. The role of Polymers used in
ophthalmic formulations is to increase the residence time on the ocular surface and thereby to
increase permeability and bioavailability. In particular, an ophthalmic thermoresponsive in situ

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gel, possesses liquid characteristic at low temperature and forms gel when comes into contact

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with a certain temperature defined as a sol–gel transition temperature. It has been suggested that
a good ophthalmic thermoresponsive in situ gel should have sol–gel transition temperature
higher than room temperature and form gel at precorneal temperature (350C) to avoid keeping in
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a fridge before administration which may lead to eye irritation from use of cold eye drops 41. One
of well-known polymer types possessing thermoresponsive behavior is Pluronics, so called
Poloxamers. In present study, initially two types of temperature sensitive polymers, Poloxamer
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188 and Poloxamer 407 were used for in situ gelation purpose. Concentrations of above
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polymers were selected based on literature survey. Poloxamer 407 gives a colourless and
transparent gel but requires somewhat higher concentration of about 15 to 30 % (m/V) to exhibit
sol-gel phase transition at 37°C when used alone. So, an attempt was made to decrease the conc.
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of P407 in formulation by combining other poloxamer, P188, and developed a series of

combinations. Their final Screening was based on Gelation Temperature (GT) and Viscosity
study of prepared formulations. The GT of preliminary batches was ranging from 30 to 37 °C but
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viscosity was very high in sol form due to the combinations of P407 and P188, which may
hinders the vision of the eye and generate difficulty during instillation, hence the combination of
two poloxamers approach was omitted in further trials and P407 was selected for further batches,
as the gels of poloxamer 407 are particularly suitable for ophthalmic formulation due to the
reverse thermosensitive gelation, low toxicity, mucomimetic properties, optical clarity and
controlled drug delivery, and have been used successfully in clinics for a variety of ocular
42
diseases . However, a major disadvantage of Pluronics is their low mucoadhesive activity,
therefore, some Pluronic-based ophthalmic formulations have been improved by adding
polymers providing mucoadhesive property such as Carbopol, sodium hyaluronate. Over the past
years, many researchers have addressed, Hydroxypropyl methyl cellulose (HPMC) could be used
safely for ophthalmic formulations. HPMC K4M was selected as the best choice of polymer in
the current study due to its mucoadhesive property and helps to increase gel strength, reliability,
and the appropriateness of its physicochemical structure. As concentration of HPMC K4M is
increased, viscosity is also increased. Appropriate concentrations of HPMC K4M were taken to
formulate less viscous formulation. From results of preliminary trials, P407 was selected as in
situ gelling polymer and HPMC K4M was selected as rate controlling and muacoadhesive
polymer. Further optimization was carried out via Central Composite experimental design. The

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potential CQAs for development of ophthalmic In-situ gel were identified to gelation temp.,

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gelation strength, mucoadhesive index, and % Cumulative release for both drugs after 9 hrs.

Drug Polymer Compatibility Studies

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Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectra of DSP, TS, Physical mixture of drugs and with polymers are shown in Figure 1.
An FTIR spectrum of Pure DSP and TS shows principal peaks at wave numbers 1300, 1713,
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1453, 1138 cm-1 and 3340.53, 2980, 1334, 1127 cm-1 respectively. The FTIR spectra of the
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physical mixtures of drug and with polymers had all the characteristic peak and band values of
pure DSP and TS that all the functional groups of both drugs are well preserved (Figure 1). This
study clearly indicate absence of any chemical interaction between the both drug (DSP and TS)
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and the polymers (Poloxamer 188, Poloxamer 407 and HPMC K4M) in physical mixture and in
final formulation and thus confirming that the drug is compatible with all the polymers used in
the present investigation.
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Differential Scanning Calorimetric (DSC)

DSC thermograms of pure DSP, TS, and drug-Polymer Physical Mixture are shown in Figure 2.
The DSC thermogram of pure DSP and TS exhibited a single sharp endothermic peak at
262.920C and 3320C respectively corresponding to their melting point. The endothermic peak of
both drugs does not show any shift in the physical mixture with polymers. The presence of
polymer affects the peak shape and intensity of the drugs which leads to lowering of purity.
Hence, the some minor changes observed in peak values of drugs do not indicate of any potential
incompatibility.

Characterization of In Situ gel

Clarity, pH, viscosity and osmolality
The values of the results of all parameters with ± SD are expressed in Table 3. Clarity of all
formulations was found satisfactory. The formulations were transparent. The pH was within
acceptable range (6.8 to 7.4) and would not cause any irritation upon administration of the
formulation into the eye. The main prerequisite of gelling system is viscosity and gelling
capacity (speed and extent of gelation). Moreover, to facilitate sustained release of drug to the

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ocular tissue, the In Situ formed gel should preserve its integrity without dissolving or eroding

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for a prolonged period of time. The successful use of In Situ gels for ocular delivery is not only
dependent on its properties after administration. It is also important that they are easy to
administer, by dropping, into the eye. It should exhibit low viscosity for reproducible dose
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administration. It is seen from Figure 3A and 3B, that all formulations in sol form exhibit
Newtonian flow at 250C and 370C so they can be easily administered via dropper. All the
selected formulations were shear thinning, exhibiting pseudo plastic behaviour. All the
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formulations were liquid at room temperature and underwent rapid gelation upon raising the pH
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to 7.4. Poloxamer containing formulations were mixed with STF in 40:7 dilution shows drastic
difference in viscosity confirming phase transitions from sol to gel. This was due to temperature
change in the STF and Poloxamer is temperature sensitive polymer. In this condition,
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formulations possess Non-Newtonian flow, i.e., by increasing shear stress, shear thinning of
formulation was observed. This increasing shear rate mimic ocular shear rates associated with
normal blinking which is extremely wide, ranging from 0.03-28500 S-1. To mimic physiological
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condition, formulations were mixed with STF in a ratio of 40:7. This scenario resulted into
slightly diluted gel with a relatively low Temperature. Viscosity of prepared formulations was
found in range of 10 to 150 cps and exhibited Non-Newtonian flow (Figure 3C). Pseudoplastic
properties of formulation under physiological conditions are in favours of sustaining the drainage
of drugs from the conjunctival sac of the eye without the unwanted tendency for undergoing
shear thinning. The osmolality of lachrymal fluid is between 280 and 293 mOsm/kg on waking.
As a result of evaporation when the eyes are open, osmolality may vary between 231 and 446
mOsm/kg 43. Depending on the drop size, solutions with an osmolality lower than 100 mOsm/kg
or higher than 640 mOsm/kg are irritant; however, the original osmolality is restored 1 or 2 min
after instillation of the nonisotonic solution depending on the drop size 43. The osmolality of all
the formulations are in the range (200 to 480 mOsm/L), which indicates the formulations are
compatible with lachrymal fluid and non-irritant.

Development of First order derivative Spectrophotometric method for the simultaneous

estimation of DSP and TS
First order derivative spectrophotometric method was successfully developed for simultaneous
determination of DSP and TS. Chemical derivatization of DSP and TS were done with

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Phenylisocyanate (PIC) and triethylamine (TEA). The λmax for DSP and TS were found to be

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247 nm and 239 respectively. Thereafter, individual first order derivative spectra were recorded
for both drugs and zero crossing points were determined (Figure 4). First order derivative
spectrum for DSP and TS showed zero crossing points at 247 and 242 nm respectively, was
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selected for determination of DSP and TS in the mixture since it showed adequate absorbance at
this wavelength. All the batches shows % assay in the standard limit as per USP (Table 3).
Assay of BKC was also estimated by U.V. spectrophotometric method and it is also within the
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acceptable range (Table 3). All the results of validation parameters like accuracy, precision,
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recovery of marketed formulations are shown in Table 4, they were in the range and method was
validated with high degree of assurance.
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Gelation temperature
The critical gel temperature of different thermoresponsive sol–gel systems is shown in Table 5.
The mixture solution containing P407 (15.17%) and HPMC K4M (0.4%) showed higher critical
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gel temperature 30.10 0C and 37.30 0C before and after dilution with tear fluid respectively. The
ideal characteristics of thermo responsive system is it should be free flowing solution at room
temperature and forms gel after instilled into eyes. Poloxamers or Pluronics are triblock
copolymers of ethylene oxide (EO) and propylene oxide (PO). The amphiphilic properties are
depend on their PEO/PPO weight ratio. In aqueous solutions, P407 molecules self-assemble into
micelles at the critical micellization temperature due to PPO blocks dehydration. In situ gel
forming systems developed by mixing P407 and different polymeric additives such as cellulose
derivatives have been investigated previously 44. In this work, hydroxyl propyl methyl cellulose
(HPMC K4M) was used as an additive polymer to lower the needed concentration of P407, due
to its higher biocompatibility and better gel capacity.

Gel strength (GS) and Refractive index

Gel strength of ophthalmic in situ gel is a measurement of ability to develop and retain a gel
form. All formulations exhibited good gel strength (Table 3). Gel strength is important because
strong gels will support a much higher pressure than weak gels before they are washed out of the
site of administration so, the higher gel strength is good for the maintenance of overall integrity
of the gel but it should not higher than 150 dyne/cm2, otherwise it may hinder the release of the

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drug form the formulation and produce the irritation into the eyes. It has been observed from the

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results that as the concentrations of P407 and HPMC K4M increases, the gel strength increases.
The batch F8 contains lowest amount of P407, which has very low gel strength 67.33 dyne/cm 2,
while the batch F9 contains highest amount of P407, which has higher gel strength 164.5
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dyne/cm2. The same effect has been observed for the HPMC K4M also. Refractive index is the
net value of the components of in situ gel and indicates the isotropic nature of the formulation. It
is recommended that eye drops should have refractive index values not higher than 1.47. Also,
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Refractive index measurements detect possible impairment of vision or discomfort to the patient
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after administration of formulation 45. The refractive index of all batches was found to be in the
range of 1.351 to 1.358 which is within the recommended values.
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Mucoadhesive Index (MI)

The mucoadhesive index is an important physicochemical parameter for In Situ forming
ophthalmic gels because it prevents the formulation from rapid drainage and hence lengthens its
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precorneal residence time. Results of the determination of mucoadhesive forces of all the
prepared formulae at different shear rate are presented in Figure 5. From results it can be seen
that the MI depends both the type and concentration of polymer. A combination of Poloxamer
407 and HPMC K4M leads to a higher MI. Indeed, there was a synergistic influence of the
concomitant presence of poloxamer and HPMC K4M in the gels. This result is obviously of
interest for practical applications, because it is likely to ensure a prolonged adhesion of the
hydrogel at the mucosal surface, following its opthalmic administration. This was due to wetting
and swelling of HPMC K4M, permits intimate contact with eye tissue, interpretation of
mucoadhesive HPMC K4M chains with mucin molecules leading to entanglement and formation
of weak chemical bond between entangled chains. Due to stronger mucoadhesive force, it can
prevent the gelled solution coming out of eyes.

In-Vitro Drug Release

The drug release profiles of DSP and TS from the prepared formulations are shown in Figure 6A
and 6B. The drug release study from In Situ gel containing HPMC K4M clearly indicates that the
release of the drug was influenced by the concentration of the HPMC K4M. The viscosity of
HPMC K4M was an important factor and affects the release behavior of the drug from In situ

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gel. Formulation containing 0.6% w/v HPMC K4M shows incomplete drug release over 9 hrs.

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because of very highly viscous solution and may form H-bond with drug molecule which reduce
diffusion capacity. Formulations without HPMC K4M fail to sustain the drug release up to 9 hr.
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Kinetics of drug release
To study the drug release mechanism of DSP and TS from In Situ gel, the release data were fitted
to the kinetics models like Zero order, Higuchi and Korsmeyer Peppas. Korsmeyer-Peppas
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model describes the release mechanism of drug from matrix. Linearity was observed with zero
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order plots because the correlation coefficient (R2) was ranged from 0.939 to 0.99 for both drugs.
Release profile of log fraction released versus log time was plotted and its slope value is ‘n’
value. Korsmeyer-Peppas equation was applied and n values were obtained in the range of 0.44
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to 0.78, which indicate non-fickian diffusion. It has been shown that in the case of hydrophilic
polymers, swelling and erosion of the polymer occur simultaneously, and both of them
contribute to the overall drug-release rate 46.
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Optimization of CQAs using Experimental Design and Data Analysis

For the response surface methodology involving CCD, a total of 10 experiments were performed
for three factors at three levels each. The experiment runs with material attributes and the
observed CQAs for the 10 formulations are shown in Table 1. A suitable polynomial equation
involving the individual main effects and interaction factors was selected based on the estimation
of several statistical parameters, such as the multiple correlation coefficient (r2), adjusted
multiple correlation coefficient (adjusted r2) and the predicted residual sum of squares (PRESS),
provided by the Design-Expert software® (8.0.7.1) Trial Version. The approximations of
response values (Gelation Temperature, Gel strength, Mucoadhesion Index and Drug releases at
9hr) based on the quadratic model and linear model were most suitable because its PRESS was
lowest. PRESS is a measure of the fit of the models to the points in the design. The smaller
PRESS value is the indication of best model fits to the given data points. The values of the
coefficients X1, X2 and X3 exhibit the effect of these variables on the response. Mathematical
relationships, generated using MLRA (Multiple linear regression analysis), gives an insight into
the effect of the different independent variables. The polynomial equations comprise the
coefficients for intercept, first-order main effects, interaction term, and higher order effects. A

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positive sign of coefficient indicate a synergistic effect while negative term indicates an

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antagonistic effect upon the response. The significant coefficients (p<0.05) are represented in
bold face letters in Table 6. For the estimation of significance of model, ANOVA was
determined as per the provision of design expert using 5 % significance level. A model is
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considered as significant if p <0.05.

Response surface analysis

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Three-dimensional response surface plots are useful to study the interaction effects of the factors
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on the responses. The relationship between the dependent and independent variables was
elucidated by constructing response surface plots.
For Dependent variable R1 (GT) if, X1 from −1 to +1 level increased and keeping X2 at lower
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level GT increase from 28-35oC . If keeping X1 constant and X2 level increased from -1 to +1 GT
will decrease up to 28oC. It was due to nature of poloxamer 407 which is temperature sensitive
and also X2 have negative effect on GT. As outlined in Table 1, the acceptable criteria are
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maximizing 34-370C. At low level of X2, and low to high level of X1, these criteria not satisfied
(Figure 7A). At middle to high level of X2, satisfactory GT was observed at all levels of X2. It is
concluded that by appropriately choosing the levels of X1 and X2, the first constraint (32-34 oC)
can be satisfied.
For Dependent variable R2 (GS) For the R2 response, the interaction between factors X1 and X2
can be elucidated by using response surface plot as illustrated in Figure 7B. By X1X2 interaction
effect, X1 increased from -1 to +1, the GS increase from 40 to 140. At higher level and lower
level of X2, criteria were not satisfied. At middle to high level of X1, satisfactory GS was
observed at all levels of X1. It is concluded that by appropriately choosing the levels of X1, the
second constraint (≤150 dyne/cm2) can be satisfied.
For Dependent variable R3 (MI) if, X1 from −1 to +1 level increased and keeping X2 at lower
level MI increase from 2000-6000 Cps. If keeping X1 constant and X2 level increased from -1 to
+1, MI is increase up to 10,000 Cps. It may due to nature of HPMC K4M which forms hydrogen
bond with mucin. As outlined in Table 1, the acceptable criteria are less than 15,000 Cps. At low
level of X2, and low to high level of X1, these criteria are not satisfied (Figure 7C). At middle to
high level of X2, satisfactory MI was observed at all levels of X2. It is concluded that by
appropriately choosing the levels of X1 and X2, the third constraint (≤ 15,000 Cps MI) can be

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satisfied.

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For Dependent variable R4 and R5 (CDR at9h) For the R4 and R5 response, the interaction
between factors X1 and X2 can be elucidated by using response surface plot as illustrated in
Figure 7D and 7E. If, X1 level increased from -1 to +1 drug release was retarded to 88%w/w in
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X1X2 interaction. At middle level of X1, the drug release to meet criteria and at lower level it
fails. At higher levels of X2 in X1X2 response drug release retarded; this may be due to highly
viscous solution and formed H-bond with drug molecule which reduced diffusion capacity. A
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higher level of both X1X2 response the drug release drastically reduced this may be due to higher
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gelation capacity of poloxamer 407 fail to release drug. Lower levels of X1 and X2 failed to
sustain the drug release because of formed gel gets erode and do not maintain its integrity. At
middle level of X2, satisfactory drug release was observed at all levels of X1. It is concluded that
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by appropriately choosing the levels of X2, the second constraint (CDR at 9hr.) can be satisfied.

Optimization of formula and Validation of Response Surface Methodology

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After generating the polynomial equations relating the dependent and independent variables, the
in situ formulations were optimized for the responses R1, R2, R3, R4 and R5. The optimum values
of the variables were obtained by graphical and numerical analyses using the Design-Expert
software which are based on criterion of desirability. The optimized formula (F*) was achieved
with 16.75 % of Poloxamer 407 and 0.54 % of HPMC K4M. Therefore, to verify the evolved
models, the optimum formulation was prepared according the above values of the factors and
subjected to the analysis of responses. It was demonstrating that the observed value of a new
batch was quite closer to predicted value.

Sterility Testing
From the study of sterility test of optimized batch F* in alternative thioglycolate media (TGM)
and Soya bean casein digest media (SCM), it has been observed that turbidity in media was
absent after 21 days of incubation period. This indicates the absence of any microbial
contamination.

Preservative Efficacy Testing (PET)

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All ophalmic preparation required optimum concentration of preservative to inhibit microbial

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growth. PET was done to evaluate efficacy of preservative. Fungi candida albicans and
Aspargilus niger show inhibition in growth after 7th, 14th and 28th day from initial count (Table
7). The microbial count (Escherichia coli, Pseudomonase aeruginosa, Stephaylococcuse aureuse)
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also decrease as per USP, one log reduction after 7th days, 3 log reductions after 14th days and no
growth in population as compare to 14th day after 28th days. In case of fungi, as per USP, there
should be no growth for PET. Same inhibition was found in the F* batch formulation which
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follows standard limit and pass the all criteria.

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In Vivo Studies
Ocular Irritation Test 47
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The result of eye irritation test of optimized batch F* studied in rabbits following modified
Draize test was shown in Table 8. It indicated that F* did not irritate rabbits’ eyes as seen in the
total score of an eye irritation assessment equalling to 0. Therefore, F* could be accepted as safe
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for ophthalmic use.

HPLC method for estimation of DSP and TS

The HPLC analytical method for quantitative estimation of DSP and TS in tear fluid had been
developed and validated. Chromatogram of both the drug is shown in Figure 8. Retention time
for DSP and TS were found 2.7 and 7.8 min respectively. All the results of validation parameters
are shown in Table 9, they were in the range and method was validated with high degree of
assurance.

Pharmacokinetic study
The HPLC method for the quantitative estimation of DSP and TS in tear fluid has been
developed and validated. Based on results of CCD, optimized formula F* was screen for in vivo
study in New Zealand albino rabbits of either sex. The F* was instilled to rabbit eye and
concentration of DSP and TS in tear (Ctf) was estimated by HPLC method. Results of the
pharmacokinetic parameters and comparison of two formulation (F* and marketed eye drops
(MF) (Toba DM)) on rabbits are illustrated in Figure 9A and 9B. Various pharmacokinetic

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parameters of two formulations are listed in Table 10. It is clearly seen from the data that rate of

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excretion of drug from F* is decreased than marketed eye drops. This is due to effects of
polymer on Mean resident time (MRT) and RTmax. These bioadhesive polymers being
hydrophilic in nature would retain/stabilize tear fluid on eye surface thereby slowing down
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drainage and also formed gel maintained its integrity for longer time and sustaining the drug
release. Ultimately this situation prolongs drug precorneal resident time reflected via RT max.
From Figure 10A and 10B, the average peak excretion rate (Cmax) of the F* is 3.23 ± 1.23 µg
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h/ml for DSP and 4.44 ± 1.23 µg h/ml for TS, which is almost 10 fold higher than that of MF
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0.34± 1.55 and 0.47± 1.55 µg h/ml of respective drug. Tmax of In Situ gel was significantly
delayed as compared to MF it indicating the better sustained release characteristic. The
comparison of constant of absorption rates (Ka) and elimination rates (Ke), suggested that DSP
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and TS from the commercial eye drop was absorbed into the aqueous humor and then eliminated
from the aqueous humor more quickly than that of F*. Higher AUC value of F* indicates that
lower release rate and prolong residence time of the dosage form in the eye. All kinetics data
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were analyzed with ANOVA: Single factor. Fcal > F crit and P value was 0.0025 which is less than
0.005. Hence, we can conclude that the results are significant and there is significant difference
between optimized formulation and marketed preparation.
Accelerated Stability Studies
Accelerated Stability Study was carried out with optimized batch F* for six month. The storage
condition of the dosage form is in refrigerator. Table 11 shows the results of various
physicochemical properties and assay of both drugs over period of 30 days. From T-Test study, it
has been observed that there are no any significant changes between different time intervals
during accelerated stability study period.

Conclusion

In the present research work, QbD was applied for pharmaceutical development of
Dexamethasone Sodium Phosphate (DSP) and Tobramycin Sulphate (TS) loaded ophthalmic In
Situ gel containing Poloxamer 407 as in situ gelling polymer in the combination with HPMC
K4M which acts as a bioadhesive polymers to increase precorneal residence time and decrease
frequency of administration. A Central Composite design (CCD) for 2 factors at 3 levels each

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was employed systemically to optimize formulation. Polynomial mathematical models were

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generated for various response variables using multiple linear regression analysis and those are
found to be statistically significant (p<0.050). Each batch shows Newtonian flow behaviour in
sol form, but when they comes in contact with simulated tear fluid (STF), they formed
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viscoelastic gel and shows Non-Newtonian behaviour. Batches containing combination of above
polymers gives drug release up to 9 hr. followed by non-fickian drug diffusion. Batches
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containing HPMC K4M show excellent Mucoadhesion index. As per Quality Based design CCD,
it is concluded that batch F* [0.1 % of DSP, 0.3 % of TS, 16.75 % of Poloxamer 407 and 0.54 %
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HPMC K4M] was optimized. The results of the animal experiments clearly demonstrated that the
optimized batch was safe, therapeutically efficacious and suitable for the eye instillation. When
F* was compared with marked formulation (MF) for elimination pharmacokinetics, AUC of F*
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was found to be 10 fold greater and Tmax of optimized formulation was significantly delayed as
compared to MF. This is evident of prolong retention and decreased frequency of dosage
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administration. Stability studies revealed that there was no decomposition in physicochemical

properties of In-situ gel and assay of DSP, TS and BKC stored at 25 ºC and 60 % RH after six
months. Therefore, the optimized formulation of thermoresponsive DSP/TS ophthalmic In Situ
gel developed in this study had potential for using as an alternative to conventional DSP/TS eye
drops to increase patient compliance.

Conflict of interest
The authors do not have any declarations of interest.
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systems: Recent advances. Prog Retin Eye Res 1998; 17: 33-58.
29. Oechsner M, Keipert S. Polyacrylic acid/polyvinylpyrrolidone bipolymeric systems. I.

D
Rheological and mucoadhesive properties of formulations potentially useful for the

TE
treatment of dry-eye-syndrome. Eur J Pharm Biopharm 1999; 47: 113-18.
30. Fang J, Yu S, Wu P, Huang Y, Tsai Y. In vitro skin permeation of estradiol from various
proniosome formulations. Int J Pharm 2001; 215: 91-99.
EP
31. Mayol L, Quaglia F, Borzacchiello A, Ambrosio Lrotonda M. A novel
poloxamers/hyaluronic acid in situ forming hydrogel for drug delivery, Rheological,
mucoadhesive and in vitro release properties. Eur J Pharm Biopharm 2008; 70: 199-06.
C

32. Gupta H, Jain S, Mathur R, Mishra P, Mishra A, Velpandian T. 2007. Sustained Ocular Drug
AC

Delivery from a Temperature and pH Triggered Novel In Situ Gel System. Drug Delivery
2007; 14: 507-15.
33. Zignani M, Tabatabay C, Gurny R. Topical semi-solid drug delivery, kinetics and tolerance
ST

of ophthalmic hydrogels. Adv Drug Deliver Rev 1995; 16: 51-60.

34. Dicolo G, Zambito Y, Zaino C, Sansa M. Selected polysaccharides at comparison for their
mucoadhesiveness and effect on precorneal residence of different drugs in the rabbit model.
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Drug Dev Ind Pharm 2009; 35: 941-49.

35. Bozdag S, Dillen K, Vandervoort J, Ludwig A. The effect of freeze-drying with different
cryoprotectants and gamma-irradiation sterilization on the characteristics of ciprofloxacin
HCl-loaded poly D,L-lactide-glycolide nanoparticles. J Pharm Pharmacol 2005; 57: 699-
708.
36. Fang JY, Chen JP. Characterization & Evaluation of Silk protein hydrogels for drug delivery.
Chem. Pharm. Bull. 2006: 44: 373-77.
37. Wagenlehner F, Sorgel F. Pharmacokinetics of Ciprofloxacin XR (1000mg) versus
Levofloxacin (500mg) in Plasma and Urine of Male and Female Healthy Volunteers
Receiving a Single Oral Dose. Int. J. of Antimicrobial Agents. 2006; 27: 7-14.
38. Russ H, McCleary D, Katimy R, Montana JL, Miller RB, Krishnamoorthy R, Davis CW, J.
Development and Validation of a Stability-Indicating HPLC Method for the Determination
of Tobramycin and Its Related Substances in an Ophthalmic Suspension. J. Liq. Chrom. &
Rel. Technol. 1998; 21(14): 2165-81.
39. Baranowski P, Karolewicz B, Gajda M, Pluta J. Ophthalmic Drug Dosage Forms,
Characterisation and Research Methods. The Scientific World J 2014; 1-14.
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and control. Pharm Res 2008; 25 (4):781–91.

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41. Wei G, Xu H, Ding PT, Li SM, Zheng JM. Thermosetting gels with modulated gelation
temperature for ophthalmic use: the rheological and gamma scintigraphic studies. J Control
Rel 2002; 83 (1): 65–74.
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42. Amelie B, Elias F, Jean LG, Francis P, Patrick C. Characterization of a new ocular delivery
system based on a dispersion of liposomes in a thermosensitive gel. Int. J. Pharm. 1998;
162: 119–127.
C

43. Terry J. Human Tear Osmotic Pressure. Archives of Ophthalmology 1978: 96: 120-32.
AC

44. Ruel-Gariepy E, Leroux JC. In situ-forming hydrogels—review of temperature-sensitive

systems. Eur. J. Pharm. Biopharm. 2004; 58: 409–426.
45. Fialho S, Silva-Cunha A. New vehicle based on a microemulsion for topical ocular
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administration of dexamethasone. Clin Exp Ophthalmol 2004; 32: 626-32.

46. Patel N, Chotai N, Patel J, Soni T, Desai J, Patel R. Comparison of in vitro dissolution
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Biopharm 2008; 70: 260-269.
Figure captions
Figure 1. FTIR Spectra of (A) DSP (B) TS (C) Physical mixture of DSP and TS (D) Physical mixture
of drug and polymers.
Figure 2. DSC spectra of (A) DSP (B) TS (C) Physical mixture of drug and polymers
Figure 3. Rheograms of viscosity vs. shear rate of formulations at (A) 250C (In Sol form), (B)
37oC (In Gel form) and (C) diluted gel (40:7) form
Figure 4. First Derivative UV Spectra of DSP and TS
Figure 5. Mucoadhesive index at various shear rates for all batches
Figure 6. In Vitro Release of (A) DSP and (B) TS from formulations
Figure 7. Response surface plot showing the relationship between various levels of polymer on

D
(A) GT, (B) GS, (C) MI, (D) DSP release at 9hr and (E) TS release at 9hr

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Figure 8. The representative HPLC chromatogram of DSP and TS in tear fluid
Figure 9. Profiles of (A) DSP and (B) TS elimination from tear fluid of rabbits following
instillation of optimized and marketed formulation.
EP
Figure 10. Average Log Excretion Rate (Log (dU/dt)) Versus Mid-Point of Time Plots for (A)
DSP and (B) TS after Instillation of Optimized and Marketed Formulation in Two Rabbits
C
AC
ST
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 D
TE
EP
C
AC
ST
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Figure 1. FTIR Spectra of (A) DSP (B) TS (C) Physical mixture of DSP and TS (D) Physical mixture
of drug and polymers.
 D
TE
EP
C
AC
ST

Figure 2. DSC spectra of (A) DSP (B) TS (C) Physical mixture of drug and polymers
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 60 Viscosity at 250C F1
F2
50 F3
F4
40

Viscosity cps
F5
30 F6
F7
20
F8
10 F9
F10
0
0 50 100 150 200
Shear Rate RPM

D
(A)

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Viscosity at 37 0C F1
500
EP
F2
450
F3
400
F4
C
350
F5
300
Viscosity cps

AC

F6
250
F7
200
F8
150
ST

100 F9

50 F10
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0
0 50 100 150 200
Shear Rate RPM

(B)
 Viscosity after Tear fluid dilution
500 F8
450 F1
400 F9
350 F5
Viscosity (cps)

300 F4
250 F7
200 F6
150 F2
100 F10
50 F3
0
0 50 100 150 200

D
Shear stress (RPM)

(C)

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Figure 3. Rheograms of viscosity vs. shear rate of formulations at (A) 250C (In Sol form), (B)
37oC (In Gel form) and (C) diluted gel (40:7) form
EP
C
AC
ST
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Figure 4. First Derivative UV Spectra of DSP and TS

 12000

10000
Mucoadhesion Index

8000 25
50
6000 100
150
4000

2000

D
F1 F2 F3 F4 F5BatchF6 F7 F8 F9 F10

TE
Figure 5. Mucoadhesive index at various shear rates for all batches

In Vitro Release Profile of DSP

EP
F1
100 F2
F3
%Cumulative Drug Release

C
80 F4
F5
AC

60 F6
F7
40
F8
F9
20
F10
ST

0
0 1 2 3 4 TIME5 (Hr) 6 7 8 9 10
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(A)
 110
In Vitro Release Profile of TS
F1
100
F2
90
F3
% Cumulative drug Release

80
F4
70
F5
60
F6
50
F7
40
F8
30
F9
20
F10
10

D
0
0 1 2 3 4 TIME5(hrs) 6 7 8 9 10

TE
(B)
Figure 6. In Vitro Release of (A) DSP and (B) TS from formulations
EP
C
AC
ST
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(A) (B)
 D
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(C) (D)
EP
C
AC
ST
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(E)
Figure 7. Response surface plot showing the relationship between various levels of polymer on
(A) GT, (B) GS, (C) MI, (D) DSP release at 9hr and (E) TS release at 9hr
Figure 8. The representative HPLC chromatogram of DSP and TS in tear fluid

D
TE
0.9
0.8 DSP Elimination
EP
0.7 MF*
Conc. (µg/ml)

0.6 F*
0.5
C

0.4
0.3
AC

0.2
0.1
0
0 100 200 300 400 500
ST

Time (min)

(A)
1.5
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TS Elimination
F*
Conc. (µg/ml)

1 MF

0.5

0
0 100 200 Time (min) 300 400 500

(B)
Figure 9. Profiles of (A) DSP and (B) TS elimination from tear fluid of rabbits following
instillation of optimized and marketed formulation.

3.5 DSP Log dU/dt

3 F*

2.5 MF*
log(dU/dt)

2
1.5
1

D
0.5
0

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0 50 100 150 200 250 300 350 400 450
Time (min)

(A)
EP
2 TS Log dU/dt F*
1.5 MF
C
log dU/dt

1
AC

0.5

0
0 50 100 150 200 250 300 350 400 450
ST

Time (min)

(B)
Figure 10. Average Log Excretion Rate (Log (dU/dt)) Versus Mid-Point of Time Plots for (A)
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DSP and (B) TS after Instillation of Optimized and Marketed Formulation in Two Rabbits
Table 1. Material attributes and CQAs of ophthalmic In-situ gel in Central-composite Design
with the results obtained and their constrains
Material attributes and CQAs of In-situ gel in Central-composite Design

Variables Level of variables

Independent (Material attributes) -1.414 -1 0 1 1.414

X1 = Conc. of Poloxomer 407(%) 15.17 16 18 20 20.82

X2 = Conc. of HPMCK4M (%) 0.034 0.2 0.4 0.6 1.165

Dependent (CQAs) Criteria for selection

Y1=Gelation Temperatures 34-370C

Y2=Gelation strength less than 150 dyne/cm2

D
Y3=Mucoadhesive Index less than 15000 cps

TE
Y4=DSP Release in 9 hrs. >95%

Y5=TS Release in 9 hrs. >95%

No. Coded Factor (Actual value) Response

EP
X1(Conc. of X2(Conc. of Y1 Y2( Y3( Y4 Y5
Poloxamer 407 (%)) HPMC K4M GS) MI)
Batc (%)) (GT) 0C %CD %CD
h dyne Cps R at9h R at9h
C

No. /cm2
AC

F1 -1 (16) -1 (0.2) 39.66 58.5 1770 96.49 99.77

±0.0 ±0.02 ±1.2
2

F2 0 (18) 0 (0.4) 34.33 114. 2265 98.64 99.94

33 ±2.2 ±2.2 ±0.20
ST

F3 0 (18) -1.414 (0.034) 35.33 69 2040 98.41 101.5

±1.5 ±2.32 8±1.6
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F4 1 (20) 1 (0.6) 31.66 149 9300 92.23 96.86

±2.3 ±1.2 ±1.90
2

F5 0 (18) 1.414 (1.165) 33.33 156. 3330 96.14 99.66

13 ±1.2 ±1.2 ±2.10
3

F6 0 (18) 0 (0.4) 34.33 114. 2265 98.64 99.94

3 ±1.2 ±2.21 ±1.30
1
F7 1(20) -1 (0.2) 32.67 119. 3780 91.1± 93.59
66 ±2.2 1.65 ±2.24
1

F8 -1.414 (15.17) 0 (0.4) 39.33 67.3 2030 98.56 99.17

3 ±1.6 ±2.54 ±1.36
5

F9 1.414 (20.82) 0 (0.4) 32.33 164. 5004 91.5± 94.24

5 ±2.5 2.36 ±2.32
4

F10 -1 (16) 1 (0.6) 36.33 100. 4140 96.92 99.38

83 ±2.3 ±2.36 ±2.6
6

D
TE
EP
C
AC
ST
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Table 2. Formulation compositions of thermo responsive Dexamethasone Sodium Phosphate and
Tobramycin Sulphate ophthalmic in situ gels

Ingredients Content of ingredients in each formulation (%, w/w)

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Dexamethasone
Sodium Phosphate
(DSP)
0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3
Tobramycin
Sulphate (TS)

D
Poloxamer 407 16 18 18 20 18 18 20 15.17 20.82 16

HPMC K4M 0.2 0.4 0.034 0.6 1.165 0.4 0.2 0.4 0.4 0.6

TE
0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Sodium chloride
0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
Disodium EDTA
EP
0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001
Benzalkonium
chloride
C

100 100 100 100 100 100 100 100 100 100
Ultrapure water
AC

(UPW) Q.S. to

Sodium Chloride was added to keep the pH of all formulations in the range of 6.5 - 7.4.
ST
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Table 3. Physiochemical evaluation of prepared ophthalmic In-situ gels
Batch No Gelation Time Gel Strength (dyne/cm2) Assay Assay Assay
(Sec) (BKC) (DSP) (TS)
F1 No Gelation* 58.50±0.57 97.23±0.21 98.35±0.79 98.41±1.3
F2 No Gelation* 114.33±0.94 98.44±0.14 100±0.58 98.4±0.75
F3 30 ± 0.55 69.00 ±0.4 97.38±0.11 98.73±0.43 98.92±1.1
F4 25 ± 0.79 149.00 ±0.57 99.33±0.17 97.84±0.79 99.6±0.79
F5 25 ± 0.38 156.13 ±0.57 97.63±0.26 99.5±0.43 99.6±0.11
F6 20 ± 0.43 114.33±0.47 97.42±0.13 98.6±0.38 99.1±1.86

D
F7 19 ± 0.38 119.67±0.81 100.1±0.11 98.6±0.38 96.6±1.11

TE
F8 17 ± 0.57 67.33± 0.5 100.1±0.10 99.3±0.21 99.7±0.08
F9 15 ± 0.58 164.50± 0.76
EP 102.05±0.2 98.1±0.38 99.5 ±0.55
F10 14 ± 0.46 132.45± 0.82 99.02±0.4 99.2±0.12 98.9 ±0.25

* No Gelation within one min, All Value Expressed as Mean ± SD (n=3)

C
AC
ST
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Table 4. Validation Results of 1st order derivative method for the simultaneous estimation of
DSP and TS by UV Spectroscopy method
Parameters 1st order derivative UV spectroscopy

Drugs DSP TS

Wavelength(nm) 247nm 239nm

Linearity range 5- 25µg/ml 5-25 µg/ml

Regression equation y=0.0383x+0.04 y=0.0144x+0.0006

Correlation coefficient(r) 0.9929 0.9992

S.D.of Slope 0.0021 0.0004

D
S.D. of Intercept 0.0002 0.0051

TE
Precision(S.D.)
(a)Intraday(n=6) 0.0021 0.0051
EP
(b)Interday(n=6) 0.0011 0.0053

Specificity 0.5383 0.2144

C
% RSD 0.5657 0.3683

Limit of Detection and (LOD) 0.21 1.21

AC

Limit of Quantification (LOQ) 0.65 0.367

Recovery studies
Marketed Drug Label claim Found Recovery ±SDa %RSDa
(%)
ST

Formulation (mg/ml) (mg/ml)

Trix DM DSP 100 98.50 98.49% 0.687 0.697

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TS 300 297.90 99.30% 0.233 0.234

Toba DM DSP 100 99.44 99.44% 0.149 0.150
TS 300 298.60 99.37% 0.134 0.135

a
denote n=6 average of three determination
Table 5. The critical gelation temperature of all formulations before and after dilution with tear
fluid
Batch No Before dilution After dilution with artificial tear fluid
Average (0C) Average (0C)

F1 26.67 ± 1.25 37.32 ± 1.10

F2 21.87 ± 0.60 33.32 ± 1.80

F3 22.74 ± 1.05 34.12 ± 2.05

F4 17.87 ± 1.45 28.97 ± 1.05

D
F5 20.92 ± 0.85 31.22 ± 1.25

TE
F6 21.10 ± 1.10 33.40 ± 1.50

F7 18.10 ± 1.15 29.60 ± 0.80

EP
F8 30.10 ± 1.30 37.30 ± 0.80

F9 16.02 ± 0.90 28.12 ± 1.20

C

F10 25.22 ± 1.55 37.50 ± 0.65

AC
ST
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Table 6. Analysis of variance for CQAs from central composite design
Source D.F. Sum Mean F Value p value
Square Square
Gelation Temperature (R1)
X1 1 168.572 168.57 110.41 0.0005

X2 1 5.226 5.226 3.423 0.137

X1X2 1 0.562 0.562 0.368 0.576

X12 1 15.540 15.540 10.178 0.033

X22 1 0.143 0.143 0.093 0.774

D
Gel Strength ((R2)

TE
X1 1 7610.55 7610.55 56.823 0.002

X2 1 4747.85 EP 4747.85 35.449 0.004

X1X2 1 42.25 42.25 0.315 0.604

X12 1 4.744 4.74 0.035 0.859

X22 1 33.17 33.17 0.247 0.644

C

Mucoadhesive Index (R3)

AC

X1 1 4422338 4422338 1.63 0.32

X2 1 832050 832050 0.30 0.63

X1X2 1 2480628 2480628 0.91 0.43

ST

X12 1 4921900.07 4921900.07 1.81 0.31

X22 1 1766380.74 1766380.74 0.65 0.50

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X12 X2 1 4599035 4599035 1.69 0.32

X22 X1 1 1098257.49 1098257.49 0.40 0.58

DSP Release at9hr(R4)

X1 1 50.322 50.322 10.055 0.015

X2 1 0.340 0.340 0.068 0.801

TS Release at9hr(R5)
 X1 1 15.713 15.713 9.609 0.036

X2 1 8.585 8.585 5.250 0.083

X1X2 1 16.486 16.483 10.080 0.033

X12 1 10.183 10.183 6.227 0.067

X22 1 0.152 0.152 0.093 0.775

Table 7. Microbial count of fungi and bacteria for PET

D
Culture Batch Initial 7th day 14th day 28th day

TE
Candida Albicans F* 12 x 106 91 x 105 43 x 104 54 x 103
Aspargilus Niger F* 10 x 106 37 x 105 45 x 104 16 x 103
45 x 105 20 x 104
Escherichia Coli F*
EP 503 335
Pseudomonas Aeruginosa F* 17 x 105 15 x 104 301 228
Pseudomonas Aeruginosa F* 20 x 105 12 x 104 235 225
C
AC
ST
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Table 8. Score obtained from eye irritation assessment of optimized F* in rabbits
Lesion Score Score obtained
for from
each assessment
lesion

A Conjuctival edema (chemosis)

No swelling 0 0

Any swelling 1

Prominent swelling along with partial lid eversion 2

D
Swelling with half-closed lids 3

Swelling with totally closed lids 4

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B Redness in conjunctiva

Absent 0 0
EP
Abnormal conjunctival injections 1

More diffuse and deeper hyperaemia, separate vessels can not 2

C
be seen easily
AC

Diffuse and dense hyperaemia 3

C Secretion

Absent 0 0
ST

Any abnormal secretion 1

Secretion leading to wet eye lashes closer to lids 2

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Secretion leading to wet lids and whole periorbital area 3

D Corneal opacity

Absent 0 0

Scattered or diffused areas-detail of the iris discernible 1

Easy discernable, transparent areas, detail of the iris slightly 2

darkened

Opalescent areas, no details of the iris discernible, size of the 3

 pupil barely discernible

Opaque cornea, iris not discernible

E Iris involvement

Absent 0 0

Pronounced deep folds, congestion, deep swelling, 1

circumcorneal injection, the iris still reacts to light

No response, haemorrhage, marked destruction 2

Total 0
Score

D
TE
EP
C
AC
ST
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Table 9. Validation parameters of HPLC method for estimation of DSP and TS
System suitability test TOBRA DEXA
Retention time (min) (mean ± 7.97 ± 0.007 2.68 ± 0.0167
S.D.,
n = 6)
Repeatability of peak area; 0.0291 0.0178
RSD% = (S.D./mean) × 100
Resolution (Rs) ----- 16.311
Tailing factor (asymmetric factor) 1.310 1.230
USP plate count 2889.03 5778.01
Linearity
2
Analyte Concentration r Slope Intercept
range
DEXA 2 to 12 μg/mL 0.994 10019.1 9950.65
TOBRA 6 to 36μg/mL 0.993 61023 7492.41

D
Accuracy
Analyte Recovery Actual conc. Found %Recovery SD %RSD

TE
level conc.
50% 6 6.72 112.13 2.516 0.022
DEXA 100% 8 7.98 99.87 2.886 0.028
150% 10 9.43 94.37 4.932 0.052
EP
50% 18 18.74 104.12 1.154 0.01
TOBRA 100% 24 23.99 99.99 1.527 0.015
150% 36 27.90 93.02 5.033 0.054
Recovery
C

Formulation Drug Actual conc. Found %Recovery SD %RSD

AC

conc.
TRIX DM Dexa 12 11.98 99.84 1.527 0.015
Tobra 36 35.99 99.99 1.154 0.015

TOBA Dexa 12 12.93 107.81 1.527 0.057

DEX
ST

Precision
Day Analyte 1 2 3 4 5 6 Mean %RSD
Intra-day DEXA 112468 112486 112532 112468 112476 112475 112486.0 0.021
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precision TOBRA 291527 291532 291527 291526 291532 291525 291531.7 0.003
Inter-day DEXA 112468 112586 112552 112468 112586 112568 112539.0 0.048
precision TOBRA 291527 291537 291552 291534 291587 291527 291544.0 0.028
Table 10. Comparative pharmacokinetic parameters of DSP and TS in Optimized and marketed
formulation
Pharmacokinetic parameters DSP TS

F* MF F* MF

AUC0-10 (µg h/ml ) 3.886 ± 25.36 0.371 ± 22.27 8.23± 25.36 0.96 ± 22.27
AUMC0-∞ (μg h/ml) 860.69 0.74 1728.79 1.11
Cmax (µg/ml) 3.23 ± 1.23 0.34 ± 1.55 4.44 ± 1.23 0.47± 1.55
Tmax(hr) 4.00 ± 2.13 0.30 ± 1.97 3.30 ± 1.63 0.20 ± 1.31
Ka (1/h) 0.10 ± 0.21 0.60 ± 0.32 0.21 ± 0.12 0.73 ± 0.09

D
ke (1/h) 0.004 ± 0.005 0.014 ± 0.007 0.006 ± 0.021 ±
0.002 0.014

TE
t1/2 (Ka) (h) 6.95 ± 0.9 EP 1.81 ± 0.4 7.25 ± 0.2 1.79 ± 0.35

each value indicates mean /S.D. for data from two rabbits.

Table 11. Change in Various physicochemical parameters after stability study

C

Parameters Initial 3 month 6 month

AC

(0 Day)
Gelation Temperature 35.76 ± 1.25 36.12 ± 0.75 35.65 ± 1.15
gel strength 112.33± 0.57 109.33 ± 0.78 108.33 ± 0.85
ST

pH 7.3 ± 0.75 7.2 ± 1.01 7.2 ± 0.55

Assay of TS 100.53 ± 0.65 98.89 ± 0.50 96.42 ± 0.51
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Assay of DSP 100.09 ± 1.05 98.02 ± 0.84 97.02 ± 0.45

*Value Expressed as Mean ± SD (n=3)