Thesis Nasal Gel

PREPARATION AND EVALUATION OF IN SITU
OPTHALMIC GEL OF AN ANTI INFECTIVE DRUG FOR

SUSTAINED OCULAR DELIVERY

By
Mr. RAJAS N.J B. Pharm.,
Reg. No.09PU330

Dissertation Submitted to the
Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore
In partial fulfillment of the requirements for the degree of

MASTER OF PHARMACY
IN
PHARMACEUTICS

Under the guidance of
Mrs. K. KAVITHA M. Pharm.,

Department of Pharmaceutics
Bharathi College of Pharmacy
Bharathinagara
2011
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE

DECLARATION BY THE CANDIDATE

I hereby declare that the matter embodied in the dissertation entitled
PREPARATI ON AND EVALUATI ON OF I N SI TU OPTHALMI C GEL OF AN
ANTI I NFECTI VE DRUG FOR SUSTAI NED OCULAR DELI VERY is a
bonafide and genuine research work carried out by me under the
guidance of Mrs. K Kavitha M.Pharm., Department of Pharmaceutics,
Bharathi College of Pharmacy, Bharathinagara. The work embodied in
this thesis is original and has not been submitted the basis for the award
of degree, diploma, associate ship (or) fellowship of any other university
(or) institution.

Date:

Place: Bharathinagara Mr. RAJAS N.J B. Pharm.,

BHARATHI COLLEGE OF PHARMACY
BHARATHINAGARA-571422

CERTIFICATE BY THE GUIDE

This is to certify that the dissertation entitled PREPARATI ON AND
EVALUATI ON OF I N SI TU OPTHALMI C GEL OF AN ANTI INFECTI VE
DRUG FOR SUSTAI NED OCULAR DELI VERY is a bonafide research work
carried out by Mr. Rajas N.J submitted in partial fulfillment for the
award of the degree of Master of Pharmacy in pharmaceutics by the
Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore.

Date: Mrs. K. KAVITHA M Pharm.,
Department of Pharmaceutics,
Place: Bharathinagara Bharathi College of Pharmacy,
Bharathinagara 571422


ENDORSEMENT BY THE HEAD OF THE
DEPARTMENT

award of the degree of Master of Pharmacy in Pharmaceutics by the
Rajiv Gandhi University of Health Sciences, Bangalore, Karnataka. This
work was carried out by him in the library and laboratories of Bharathi
College of Pharmacy, under the guidance of Mrs. K. Kavitha M.Pharm.,
Department of Pharmaceutics, Bharathi College of Pharmacy,
Bharathinagara.

Date: Dr. T. SIVAKUMAR M Pharm., Ph. D.
Professor and HOD,
Place: Bharathinagara Department of Pharmaceutics,
Bharathi College of Pharmacy,
Bharathinagara 571422


ENDORSEMENT BY THE PRINCIPAL / HEAD OF THE
INSTITUTION

award of the degree of Master of Pharmacy in Pharmaceutics by the
Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore. This
work was carried out by him in the library and laboratories of Bharathi
College of Pharmacy, under the guidance of Mrs. K.Kavitha M.Pharm.,
Department of Pharmaceutics, Bharathi College of Pharmacy,
Bharathinagara.

Date: Dr. T. TAMIZH MANI M.Pharm., Ph.D.
Principal,
Place: Bharathinagara Bharathi College of Pharmacy,
Bharathinagara 571422.

COPYRIGHT

DECLARATION BY THE CANDIDATE

I hereby declare that the Rajiv Gandhi University of Health
Sciences, Karnataka shall have the rights to preserve, use and
disseminate this dissertation / thesis in print or electronic format for
academic / research purpose.

Date:
Place: Bharathinagara Mr. RAJAS N.J B.Pharm.,
Rajiv Gandhi University of Health Sciences, Karnataka.

DEDICATED TO MY
BELOVED FAMILY
AND FRIENDS

ACKNOWLEDGEMENT
Gratitude makes sense of our past, brings peace for today and creates a
vision for tomorrow
Many Thanks to Almighty God for it, who began this work in me and
carried it to completion, who has blessesed me with the people whose names I feel
privileged to mention here.
The completion of this dissertation is not only fulfillment of my dreams, but
also the dreams of my Parents who have taken lots of pain for me in completion of
my higher studies.
I consider this as an opportunity to express my gratitude to all the
dignitaries who have been involved directly or indirectly with the successful
completion of this dissertation.
At first, I consider it as a great privilege to express my heartfelt gratitude
and sincere thanks to my esteemed guide Mrs. K.Kavitha, Assistant Professor,
Department of pharmaceutics, Bharathi college of pharmacy, for her valuable
suggestions, encouragement, motivation, guidance and co-operation during my
thesis work.
My deepest appreciation and heartfelt thanks to Dr. T. Sivakumar Head of
Department of Pharmaceutics for his valuable suggestions and help in making my
study a success. I cherish his co-operation throughout my life.
I consider myself to be very fortunate to have Dr. Tamizh Mani Principal
Bharathi college of pharmacy, Bharathinagara, who with his dynamic approach
boosted my morale, which helped me in the completion of this dissertation.

I take this golden opportunity to express my heartful gratitude and respect
Dr.K.P Channabasavaraj, Mr.Ganesh, and Mr.Rupesh Kumar for his constant
guidance and encouragement throughout my project work.
I take this opportunity to express my deep gratitude and heartfelt thanks
Mr. K.M Prasad, Manager Karnataka Antibiotics and Pharmaceuticals Limited,
Bangalore for providing opportunity to do project work in their reputed
organization.
I take this opportunity to express my gratefulness to Ms.Parimala, Manager
Formulation Development and Mr.Thimaraya swamy Karnataka Antibiotics and
Pharmaceuticals Limited, Bangalore for the valuable guidance during the course of
study.
I take this opportunity to express my gratefulness to Mr.Marigoli, Manager
Quality control Department, Ms.Sharmila, Ms.Uma rani, Ms.Sudha KAPL for the
valuable guidance during the course of study.
Thanks is a small word to my Father NS J ayaprakash, mother Gayathri
devi, my brothers Raghavendra, Rakesh, my Atigae Vidya and all family members
for their encouragement and love that served as a source of inspiration, strength at
each and every front of my life to transport my dreams in to reality.
I cordially thank to Mr.Santhosh, Mr.Sumukh for their suggestion, support
and encouragement during throughout my studies.
I specially convey my gratitude to dearest class mates Mr.Mehaboob,
Mr.sandeep, Mr.Pavan, Ms. Ashvini, Mr.Vinay, Mr.Mangesh, Mr.kiran, Mr.Mehul,
Mr.Dipen for their timely help, support and memorable company during my course.

I specially convey my gratitude to my dearest friends Mr.Pavan, Mr.Rahul,
Ms.Nisha, Ms.Bharathi, Mr.Vinod, Mr.Bharthesh, Mr.Almesh, Mr.Naveen,
Mr.Mithun, Mr.Bhadri, Mr.Ranjan, Mr.Sunil, Mr.Raghavendra, Mr.Omkar,
Mr.Ravi, Mr.Ajay, Mr.Shashi, Mr.Chiranjivi, Mr.Gowrish, Mr.Dilshad,
Mr.Phaniraj, Ms.Ramya, Ms.Sowmya, Mr.Srikanth, Mr.Vikshith, Mr.Nandhan,
Mr.Manjunath, Mr.Sridhar and all my friends for their continuous support
encouragement throughout my studies.
I am proud to say it was a fruitful and enjoyable experience to work with
Mr.Lohith, Mr.Ramachandra, Mr.Yusuf, Mr.Guru, Mr.Deepak, Mr.Shivakumar
reddy, Mr.Raju for their continuous support and encouragement throughout my
project in KAPL.
I wish to express my sincere thanks to my seniors Ms.Veena, Mr.Anil,
Mr.punnet, Mr.Shivaraj, Mr.Panner selvam and all my juniors for their help during
my project work.
I take this golden opportunity to express my thanks to Management of
Bharathi College of pharmacy for providing great environment during my studies.
I t is indeed a difficult task to acknowledge the services of all those gentle
people who have extended their valuable suggestion and support directly or
indirectly whose names have been unable to mention as they are like the countless
stars in numerous galaxies.
My Sincere thanks to all.
Date: Rajas N.J B.Pharm
Place: Bharathinagara
LIST OF ABBREVIATIONS

Department Of Pharmaceutics, Bharathi College Of Pharmacy

IP : Indian pharmacopoeia
BP : British pharmacopoeia
CAP : Cellulose acetate phthalate
FTIR : Fourier transform infrared
LEV : Levofloxacin hemihydrate
HPMC : Hydroxy propyl methyl cellulose
r
2
: Regression coefficient
SD : Standard deviation
SRDF : Sustained release dosage form
USP : United states pharmacopoeia
HPC : Hydroxy propyl cellulose
Rpm : Revolutions per minute
UV : Ultraviolet
Hrs : Hours
Avg : Average
G : Gram
mg : Milligram
mm : Milli meter
g : Microgram
pH : Negative logarithm of hydrogen ion concentration
min : Minutes
Conc. : Concentration


Department Of Pharmaceutics, Bharathi College Of Pharmacy

NDDS : Novel Drug Delivery system
Cps : Centipoise
STF : Simulated tear fluid.
F1 : in situ gel prepared with gelrite concentration 0.2%
SD : Standard deviation
ABSTRACT

Department of Pharmaceutics, Bharathi College of Pharmacy

OBJECTIVE
Ocular bioavailability is always poor from conventional ophthalmic drops due
to spillage and nasolachrymal drainage. Ocular in situ gels can increase the drug
residence time thus increasing bioavailability. Purpose of current study is to prepare
sustained release in situ ocular gels of Levofloxacin hemihydrate using gelrite as
gelling polymer, which is used in treatment of various bacterial infections.
METHODS
In situ gels of LEV were prepared by applying principle of ion exchange and
temperature dependant gelation techniques using gelrite as the gelling polymer. In the
present study six different formulations were prepared containing varying
concentrations of gelrite and the formulations were evaluated for physical parameters
like clarity, pH, drug content, gelation, sterility test, rheological studies, in vitro drug
release study and ocular irritancy studies, anti microbial efficiency study and stability
studies.
RESULTS
FTIR spectras revealed that, there was no interaction between LEV and
excipients. The formulated gels were transparent, uniform in consistency and had
spreadability with a pH range of 7.1 to 7.4. Rheological studies revealed that the
formulations were psuedoplastic in nature, drug content of sterile in situ gels was
found to be 92-98%. From the preliminary studies it was observed that as the
concentration of the polymer was increased, the rate of drug release decreased to
produce sustained drug delivery for more than 8 hours with a maximum of 90.2%
ABSTRACT

ABSTRACT


drug release. Release kinetic study showed that the formulation followed first order
diffusion controlled and non fickian release mechanism, the optimized formulations
was having good antibacterial efficacy, results of ocular irritancy studies showed that
formulations were non irritant and stability studies indicated that formulations were
stable at room temperature as well as at 40
0
C.
CONCLUSION
The present study conclusively demonstrates the feasibility of effectively
formulating Levofloxacin in situ gels using gelrite as release retardant to form a
potentially effective sustained release drug delivery system.
Keywords: Gelrite; Rheological; ocular; gels; Bioavailability; Anti microbial; in vitro

CONTENTS


CHAPTER
NO
TITLE PAGE
NO
1 Introduction 1

1.1 Ocular anatomy and physiology 2
1.2 Absorption and bioavailability of the drugs from
the eye
10
1.3 General bacterial infections of eye and their
medications
12
1.4 Ocular drug delivery systems 14
1.5 Methods of preparation of in situ gels 19
2 Aims and objectives 26
2.1 Plan of work 27
3 Review of literature 29

3.1 Drug profile 37
3.1.1 Levofloxacin Hemihydrate 37
3.2 Polymer profiles 40
3.2.1 Gelrite 40
3.2.2 Propylene glycol 41
3.2.3 Benzalkonium chloride 43
4 Materials and Methods 44

4.1.Materials 44
4.2 Methods 45
4.2.1 Preformulation studies 45
4.2.2 Preparation of in situ gel of LEV. 46
4.2.3 Evaluation of prepared in situ gelling system 47
5 Results 54
6 Discussion 79
7 Conclusion 82
8 Summary 83
9 Bibliography 85
10 Annexures 94

CONTENTS

CONTENTS


LIST OF TABLES


TABLE
NO
TITLE PAGE
NO
1.1 Topical antibacterial agents for ophthalmic use. 14
4.1 List of chemicals used with grade and supplier. 44
4.2 List of instruments used. 45
4.3 Formulation of in situ gels of LEV. 46
5.1 Interpretations of IR spectra. 57
5.2 Preliminary evaluation of visual appearance, clarity, pH, and
drug content.
58
5.3 Evaluation of gelling capacity. 59
5.4 Rheological studies of in situ gels before gelation. 60
5.5 Rheological studies of in situ gels after gelation. 61
5.6 Test of sterility. 62
5.7 Standard calibration of LEV. 63
5.8 In vitro release profile of marketed eye drops. 64
5.9 Comparative in vitro release of marketed eye drop and prepared
in situ gels.
65
5.10 Comparative Zero order release kinetics data of in situ gels. 66
5.11 Comparative First order release kinetics data of in situ gels. 67
5.12 Comparative Higuchi release kinetics data of in situ gels. 68
LIST OF TABLES

LIST OF TABLES


5.13 Comparative Peppas release kinetics data of in situ gels. 69
5.14 Regression co-efficient (r2) values of different kinetic models. 72

5.15 Antimicrobial activity of in situ gels. 73
5.16 Stability studies of Formulation F1. 74
5.22 Stability studies of all formulation at room temperature. 77
5.23 Stability studies of all formulations at 40
0
c. 77
LIST OF FIGURES


FIG. NO
TITLE PAGE NO
1.1 Different Parts of human eye. 3
1.2 Routes of ocular absorption of drugs. 10
1.3 Classification of hydrogels. 18
1.4 PEO-PPO-PEO (Poloxamer). 20
1.5 In situ gel formation due to change in temperature. 21
1.6 Structure of Carbomer. 22
1.7 Schematic representation of pH dependent in situ gels. 23
1.8 Structure of deacetylated gellan gum. 24
5.1 IR spectra of LEV 55
5.2 IR spectra of Gelrite. 55
5.3 IR spectra of physical mixture of LEV and Gelrite. 56
5.4 IR spectra of in situ gel of Levofloxacin hemihydrate. 56
5.5 Rheological studies in situ gels before gelation. 60
5.6 Rheological studies in situ gels after gelation. 61
5.7 Calibration curve of Levofloxacin hemihydrate. 63
5.8 In vitro release profile of marketed eye drops. 64
5.9
Comparative in vitro release of marketed eye drop and
prepared in situ gels.
70
5.10 Comparitive zero order release kinetics of in situ gels. 70
5.11 Comparitive first order release kinetics of in situ gels. 71
5.12 Comparative higuchi release kinetics of in situ gels. 71
5.13 Comparative peppas release kineticsof in situ gels. 72
5.14 Stability studies of in situ gels at room temperature. 78
5.15 Stability studies of in situ gels at 40C. 78
LIST OF FIGURES

CHAPTER 1 INTRODUCTION

Department of Pharmaceutics, Bharathi College of Pharmacy 1

Eye is unique and vital organ. It is considered as window of the soul. We can
enjoy and view the whole world only with this organ. There are many eye ailments
which affect this organ and one even can loss the eye sight. Therefore many
ophthalmic drug delivery systems are available. These are classified as conventional
and newer drug delivery systems. Eye drops that are conventional ophthalmic delivery
systems often result in poor bioavailability and therapeutic response, because high
tear fluid turnover and dynamics cause rapid precorneal elimination of the drug. A
high frequency of eye drop instillation is associated with patient non-compliance.
Inclusion of excess drug in the formulation is an attempt to overcome bioavailability
problem, is potentially dangerous if the drug solution drained from the eye is
systemically absorbed from the nasolacrimal duct. Various ophthalmic vehicles such
as inserts, ointments, suspensions and aqueous gels have been developed in order to
lengthen the residence time of instilled dose and enhance the ophthalmic
bioavailability. These ocular drug delivery systems however have not been used
extensively because of some drawbacks such as blurred vision from ointments or low
patient compliance from inserts.
Several in situ gelling system have been developed to prolong the precorneal
residence time of a drug and improve ocular bioavailability. These systems consist of
polymers that exhibit sol to gel phase tansititions due to change in specific physico
chemical parameter (pH, temperature) in their environment, the cul de sac in this case.
Depending on the method employed to cause sol-to-gel phase transition on the eye
surface the following three types of systems are recognized, pH triggered system,
1. INTRODUCTION



temperature dependant system and ion activated system. Using these three methods
above in situ gelling ophthalmic delivery system is developed.
1
Topical anti infectives are commonly used to treat bacterial conjunctivitis and
infection of cornea caused by susceptible strains of bacteria such as S. aureus, S.
epidermidis, S. pneumoniae, Enterobacter cloacae, H. influenzae, P. mirabilis and P.
aeruginosa. They are also indicated for the treatment of bacterial corneal ulcers
caused by susceptible strains of the following bacteria: S. aureus, S. epidermidis, S.
pneumoniae, P. aeruginosa, S. marcescens and Propionibacterium acnes etc.
2

1.1 OCULAR ANATOMY AND PHYSIOLOGY
The human eye is challenging subject for topical administration of the drugs.
The basis of this can be found in the anatomical arrangements of surface tissue and in
permeability of the cornea. The protective operation of eyelids and lacrimal system
are such that there is rapid removal of material instilled into eyes unless the materials
are suitably small in volume and chemically and physiologically compatible with
surface tissues.
3
The eye is referred to as a globe, is actually two spheres, one set in the other, as
shown in Fig. 1.1. The front sphere is the smaller of the two and is bordered anteriorly
by the cornea, whereas the larger posterior sphere is an opaque fibrous shell encased
by the sclera. The combined weight of both spheres has been given as 6.77-7.5 g, with
a volume approximately 6.5 ml. The circumference of the eye is about 75 mm. Along
with the rest of the orbital contents; the eye is located within the boney orbital cavity
of the head.
4



Fig.1.1 Different parts of human eye.

A. Structural support
The orbit
The eyes rest in two boney cavities; i.e, the orbits, located on either side of the
nose. The anterior two-third of the orbit is roughly the shape of a quadrilateral
pyramid, where as the posterior one-third of the orbit narrow to the shape of a
triangular pyramid. The globe occupies approximately 20 % of the cavity, lying
slightly nearer the upper and lateral sides but never in contact with the orbital bones.
4

External ocular tissues
Eyebrows and Eyelids
The eyebrows separate upper eyelid from the forehead and may move as part of
changing facial expressions or in consult with change in the direction of the visual
axis and position of the eyelids. The eyebrows perform a variety of specialized
functions. They have a major influence on nonverbal communication by way of facial


expression. In addition, owing to their position and curvature, the eyebrows help
shield the eyes from bright sunlight coming from directly.
4

The eyelids cover the external part of the eye. These mobile folds protect the
eye from mechanical or chemical injury by sweeping the external surface of the eye at
periodic intervals and when closed as a first line of defense.
4

The eyelids are lubricated and kept fluid-filled by secretion of the lacrimal
glands and specialized cells residing in the bulbar conjunctiva. The anterior chamber
has the shape of a narrow cleft directly over the front of the eyeball, with pocket-like
extensions upward and downward. The pockets are called the superior and inferior
fornices (vaults) and the entire space, the cul-de-sac. The elliptical opening between
the eyelids is called the palpebral fissure.
3

Conjunctiva
The conjunctiva is a vascularized mucus membrane that covers the anterior
surface of the globe with the exception of the cornea. Conjuctival epithelium is
continuous with that of the cornea and with the epidermis of the lids and has a surface
that is about five times of the cornea. Mucus producing goblet cells which are
important for wetting and tear film stability are located in the conjuctiva. In addition
to physical protection of the globe, the conjuctiva has great potential for combating
infection for four reasons.
4

It is highly vascular tissue.
It contains many immune competent cells.
The different types of cells are located within the conjuctiva.
The anatomy and biochemistry of conjuctival cells.



Anatomically the conjunctiva is divided into three areas.
4

a. The palpebral (lid) conjuctiva
b. The conjunctiva fornix
c. The bulbar (globe) conjuctiva
Lacrimal system
Tears
The conjuctival and corneal surfaces are covered and lubricated by a film of
fluid secreted by the conjuctival and lacrimal glands. The secretion of the lacrimal
glands; the tears are delivered through a number of fine ducts into the conjunctival
fornix. The secretion is a clear, watery fluid containing numerous salts, glucose, other
organic compounds, approximately 0.7 % proteins, enzymes and lysozyme.
3

The cul-de-sac normally holds 7-9 l of tears but can retain up to approximately
20-30 l without overflowing if care is taken not to blink. Under baseline conditions,
the normal tear flow rate and tear film thickness are 1 l/min, i.e, approximately
16%/min and 4-9 m, respectively. Additionally, the normal pH of tears is 6.5-7.6.
Since tears are a well buffered system in part, because instilled solutions of lower pH
are quickly returned to physiological conditions.
4

Secretary and Drainage apparatus
The lacrimal system consists of a secretary and collection portion. The secretary
portion of the lacrimal system consists of the main and accessory lacrimal glands.
Secreted tears do not normally flow across the cornea but will be assisted by the wiper
like action of the lids. It is assumed that secreted tears mix relatively rapidly and
thoroughly with tears held in the lower cul de sac.
4

Tears are drained from the lacrimal lake through two small openings, the superior
and inferior canaliculi, which themselves join at the common canaliculus that leads


into the upper part of the nasolacrimal duct, the beginning of which is the lacrimal
sac. The act of blinking exerts a suction force pump action in remaining tears from the
lacrimal lake and emptying them into the nasal cavity.
3

Anterior segment
Anterior chamber

The anterior chamber is bounded in front by the cornea and a small portion of
the sclera. The anterior chamber is deepest centrally and shallowest at the periphery,
holding a volume of aqueous humor that has been shown to be 250 l in the human,
with a turnover rate of 1%/min. naturally, there is some overlap of tissues between the
anterior and posterior segments.
4

Cornea
The cornea or window of the eye is an optically transparent tissue that conveys
images to the back of the eye. The cornea, being an avascular tissue, receives
nutrients and oxygen from the bathing solutions, i.e, the tears and aqueous humor as
well as from the blood vessels that line the junction between the cornea and sclera.
Corneal diameter is about 11.5 mm with a radius of curvature of anterior corneal
surface of 7.8 mm.
4
Limbus
The corneosclerallimbus is a transitional zone 1-2 mm wide between the cornea
proper, sclera and conjuctiva. Externally the limbus is covered by peripheral corneal
epithelium and anterior conjuctival epithelium. Internally the limbus includes aqueous
veins, the canal of schlemm and the trabecular meshwork. The limbal blood supply is
an important source of nutrient and defense mechanism.
4



Trabecular meshwork and Schlemms canal
The trabecular meshwork and canal of schlemm form the conventional pathway
of aqueous humor outflow from the anterior segment of the eye. The trabecular
meshwork is divided into uveal and corneoscleral portions, each with there own
distinct anatomy. Aqueous humor leaves the eye at an angle in the anterior chamber,
where it enters the trabecular meshwork.
4

Posterior chamber
The posterior chamber is somewhat triangular in appearance. The apex of the
triangle is located, where the edge of the iris rests on the lens. Ciliary processes form
the base, the posterior valve is formed by the lens and zonule and the anterior valve is
the posterior surface of the iris. The posterior chamber is filled with about 50 l of
aqueous humor. Aqueous humor is produced by ciliary processes, at the rate of 2.0 -
2.5l/ min, flows into the posterior chamber through the pupil and from there into the
anterior chamber.
4

Iris and Pupil
The pupil is circular opening located near the center of the iris. There are two
sets of smooth muscle in the iris that regulate papillary diameter: the sphincter
(parasympathetically innervated) and the dilator muscle (Sympathetically innervated).
The diameter of the normal pupil varies 2-9 mm. papillary dilation increases the
amount of light entering the eye and enhances the ability of the rods to function. In
contrast, papillary constriction increases depth of the focus of the eye and decreases
optical aberrations from the lens periphery.
4

Ciliary body
The ciliary body is responsible for many functions in the eye. It secretes
aqueous humor that nourishes the lens, provides the muscle power for accommodation


and may secrete the unique zonular fibers. The anterior is known as the pars plicata
and contains ciliary processes where aqueous humor is secreted. The posterior portion
of the ciliary body is known as the pars plana. Ciliary epithelium consists of two
layers an inner non-pigmented layer (NPE) and an outer pigmented layer (PE).
3
Aqueous humor
The aqueous humor is produced both by active and passive secretion from the
ciliary processes. There are two enzymes located in the non-pigmented epithelium of
the ciliary processes that are intimately involved in the active secretion of aqueous
humor. These are the sodium-potassium activated adenosine triphosphate enzyme
(Na
+
-K
+
-ATPase) and carbonic anhydrase.
4

Posterior segment
Lens
The lens is a transparent biconvex structure located behind the iris and in front
of the vitreous. Like all lenses, that of the eye has two surfaces, anterior and posterior
and a border where these surfaces meet i.e, the equator, at its equator the lens has a
diameter of 10 mm. The average radius of curvature of the anterior surface is10 mm,
where as that of the posterior surface is 6 mm.
4

Sclera
The outer coat of the eye is fibrous and serves a protective function. The white
opaque sclera constitutes the posterior five-sixths of the globe, where as the
transparent cornea comprises the anterior one-sixth of the globe. The anterior surface
of the sclera is covered by the episclera and the inner surface is covered by the
laminar fusca.
4



Choroid
The choroid is the vascular coat of the eye and is divided into four layers.
6

Supra choroids
Layer of vessels
Chorio capillaries
Bruchs membrane
Vitreous
The vitreous humor comprises 80% of the internal volume of the eye. Vitreous
humor weighs 4g and occupies a volume of almost 4 ml. The vitreous cavity is
surrounded by the retina and optic nerve posteriorly. Anteriorly, it is bounded by the
ciliary body, zonules, and the posterior surface of the lens. In its normal state, the
vitreous is a clear gel composed almost entirely of water (99%). The vitreous is
impotent as a supporting structure and a metabolic pathway for nutrients for the lens
and retina. However of equal importance is its clarity and ability to transmit light to
the retina.
4

Retina
The retina is the inner most coat of the eye. It is a transparent tissue that lines
the posterior two-thirds of the eyeball. The only firm attachments of the retina are at
its anterior termination, the oraserrata, at the margins of the optic nerve.
4
Photoreceptor cells of the retina consist of rods and cones. The cones are concerned
with visual activity and color discrimination, whereas the rods are concerned with
peripheral vision under decreased illumination. The retina has the highest oxygen
consumption per unit weight of any tissue in the body.
4



Optic nerve
The optic nerve is a bundle of myelinated nerve fibers through which the entire
output of the retina travels. It reveals from the retina to the optic chiasm and is
divided into four portions, intra ocular portion, intra orbital portion, intra canalicular
portion, intra cranial portion.
4
1.2 ABSORPTION AND BIOAVAILABILITY OF THE DRUGS FROM THE
EYE

The drug solution instilled as eye drops into the ocular cavity may disappear
from the precorneal area of the eye by any or a composite of the following routes.
5

shown in Fig.1.2.
1. Nasolacrimal drainage,
2. Tear turnover,
3. Productive corneal absorption,
4. Nonproductive Conjuctival uptake.

Fig.1.2 Routes of ocular absorption of drugs.


Drug administered by instillation must penetrate the eyes and do so primarily
through the cornea. Corneal absorption is much more effective than scleral or
conjuctival absorption, in which removal by blood vessels into the general circulation
occurs.
3

Many ophthalmic drugs are weak bases and are applied to the eye as aqueous
solution of their salts. The free base and the salts will be in an equilibrium that will
depend on the pH and the individual characteristics of the drug molecule. To aid in
maintaining storage stability and solubility, the medication may be acidic at the
moment of instillation but usually, the neutralizing action of the lacrimal fluid will
convert it rapidly to the physiological pH range (pH 7.4) at which there will be
enough free base present to begin penetration of the corneal epithelium. Once inside
the epithelium (lipid rich) the undissociated free base dissociates immediately to a
degree that the dissociated moiety then will tend to penetrate the stroma because it is
water-soluble. At the junction of the stroma (lipid poor) and endothelium (lipid rich),
the same process that took place at the outer surface of the epithelium must occur
again. Finally, the dissociated drug leaves the endothelium for the aqueous humor.
Here it can readily diffuse to the iris and the ciliary body, the site of its
pharmacological action.
3

The topical application of ophthalmically active drugs to the eye is the most
prescribed route of administration for the treatment of various ocular diseases. It is
generally agreed that the intraocular bioavailability of topically applied drugs is
extremely poor.
5
Upon instillation of an ophthalmic solution; most of the instilled
volume is eliminated from the pre corneal area. This loss is mainly due to drainage of
the excess fluid by the nasolacrimal duct or elimination of the solution by tear
turnover, which will results in poor ocular bioavailability.


1.3 GENERAL BACTERIAL INFECTIONS OF EYE AND ITS
MEDICATIONS
Common bacterial infections of eye are listed as below:
7
Conjuctivitis
It is a common superficial eye disorder and may be caused by infection with a
wide range of bacteria, viruses and less frequently fungi. Staphylococci or
Streptococci commonly cause acute bacterial conjunctivitis in adults, and
Haemophilus influenzae and Morexella catarhallis particularly in children. Other
causes of bacterial conjuctivitis include Gonococci and Chlamydia trachomatis.
Uncomplicated bacterial conjunctivitis may be self limiting but empirical treatment
with topical antibacterial is often given.
Blepharitis
It is an infection of the lid margins. It is usually present in a chronic condition
and may require prolonged treatment, typically involving local hygiene to remove
encrustations and topical application of a broad-spectrum antibacterial ointment.
Keratitis
It may be caused by infection of the cornea by bacteria, fungi, viruses or
protozoa usually following trauma to the surface of the eye, including that due to
contact lens wear. Common bacterial pathogens include Staphylococci, Streptococci,
Pseudomonas spp. and Enterobacteriaceae. Bacterial keratitis is potentially sight
threatening and requires prompt aggressive treatment with broad-spectrum
antibacterials. Frequent or continuous topical application of drops or the use of local
drug delivery devices has been used to ensure prolonged elevated drug concentrations.



Endophthalmitis
It is a devastating ocular disease resulting from infection of the ocular cavity,
usually following penetrating trauma or surgery. Depending on the route of infection,
causative organisms commonly include Staphylococci, Streptococci, Haemophilus
influenzae, bacillus cereus and Propionibacterium acnes. Bacterial endophthalmitis
requires immediate aggressive treatment with antibacterials, usually given
intravitreally.
A considerable amount of effort has been made in ophthalmic drug delivery
since the 1970. A number of approaches to the delivery of drugs for ocular treatment
has been investigated and proposed. These range from simple systems such as
aqueous suspensions where the viscosity and hence the residence time, has been
increased by cellulosic polymer to complex system such as penetration enhancers,
external devices (collagen shields, Preformed gels, iontophoresis and pumps),
ion-exchange resins, liposomes, microspheres and micro particles, polymeric films,
inserts, prodrugs, mucoadhesives and metabolism based drug design.
8
The commercially available antibacterial agents for ophthalmic use are as follows:



Table 1.1 Topical antibacterial agents for ophthalmic use.
Generic name

Formulation

Indications

Bacitracin Zinc 500 /g ointment Conjunctivitis Blepharitis
Chloramphenicol 0.5% solution 1% ointment Conjunctivitis Keratitis
Chlortetracyclin 1% ointment Conjunctivitis Blepharitis
Pefloxacin 0.3% solution Conjunctivitis Keratitis
Erythromycin 0.5% ointment Conjunctivitis Blepharitis
Gentamicin sulfate 0.3% solution 0.3% ointment
Conjunctivitis Keratitis
Blepharitis
Norfloxacin 0.3% solution Conjunctivitis
Sulfactamide sodium
10, 15 at solution
10% ointment
Conjunctivitis Keratitis
Blepharitis
Sulfisovarzole 4% solution 4% ointment
Conjunctivitis Blepharitis
Lentitis
Polymixin B
Various solution and various
ointment
Conjunctivitis Blepharitis
Tetracycline 1% solution Conjunctivitis Blepharitis
Tobramycin sulfate 0.3 % solution 0.3% ointment
Conjunctivitis Keratitis,
Blepharitis

1.4 OCULAR DRUG DELIVERY SYSTEMS
9
Solutions and suspensions
Solutions are the pharmaceutical dosage forms most widely used to administer
drugs that must be active on the eye surface or in the eye after passage through the
cornea or the conjunctiva. Solutions also have disadvantages:
The solution stays for a short time on eye surface,
It has poor bioavailability,


The instability of the dissolved drug and the necessity of using
preservatives.
Sprays
Although not commonly used, some practitioners use Mydriatics or
Cycloplegics alone or in combination in the form of eye spray. These sprays are used
in the eye for dilating the pupil or for Cycloplegics examination.
Contact lenses
Contact lenses can absorb water soluble drugs when soaked in drug solutions.
These drug saturated contact lenses are placed in the eye for releasing the drug for a
long period of time. The hydrophilic contact lenses can be used to prolong the ocular
residence time of the drugs.
Artificial tear inserts
A rod shaped pellet of Hydroxy propyl cellulose, without preservative is
commercially available (Lacrisert). This device is designed as a sustained release
artificial tear for the treatment of Dry eye disorder.
Filter paper strips
Sodium fluorescein and bengal dyes are commercially available as drug-
impregnated filter paper strips. These dyes are used diagnostically to disclose corneal
injuries and infections such as herpes simplex and dry eye disorders.
Micro emulsion
Due to their intrinsic properties and specific structures, microemulsions are a
promising dosage form for the natural defense of the eye. Indeed, because they are
prepared by inexpensive processes through auto emulsification or supply of energy
and can be easily sterilized, they are stable and have a high capacity of dissolving the
drugs.


Ocular Inserts
Ocular inserts, one of the new classes of drug delivery systems, which are
gaining worldwide praise, release drugs at a pre-programmed rate for a longer period
by increasing the precorneal residence time.
10
The goal of this delivery system is to
provide a therapeutic amount of drug to the ocular tissues to achieve promptly and
then maintain the desired drug concentration by increasing the contact time between
the preparation and the Conjunctival tissue.
11
To achieve this goal particularly for
chronic diseases such as glaucoma, it would be advantageous and more convenient to
maintain a dosing frequency to once, or at most, twice a week regimen.
12
An
appropriately designed extended release ocular insert can be a major advance in this
direction compared to conventional immediate release dosage forms.
Collagen Shield
Collagen is regarded as one of the most useful biomaterials. The excellent
biocompatibility and safety is due to its biological characteristics, such as
biodegradability and weak antigenicity, these properties made collagen the primary
resource in medical applications. Collasomes show promise among drug delivery
systems to the human eye. They are first fabricated from procine scleral tissue, which
bears a collagen composition similar to that of the human cornea. The shields are
hydrated before they are placed on the eye. Shields are not individually fit for each
patient, as are soft contact lenses and therefore, comfort may be problematic and
expulsion of the shield may occur.
Ocular Iontophoresis
Iontophoresis is the process in which direct current drives ions into cells or
tissues. When iontophoresis is used for drug delivery, the ions of importance are
charged molecule of the drug. Ocular iontophoresis offers a drug delivery system that


is fast, painless and safe; and in most cases; it results in the delivery of a high
concentration of the drug to a specific site. But the role of iontophoresis in clinical
ophthalmology remains to be identified.
Liposomes
Liposomes are phospholipid lipid vesicles for targeting drugs to the specific
sites in the body; provide the controlled and selective drug delivery and improved
bioavailability. Liposomes offer the advantages of being completely biodegradable
and relatively non toxic but are less stable than particulate polymeric drug delivery
systems.
Niosomes
In order to circumvent the limitations of liposomes, such as chemical instability,
oxidative degradation of phospholipids, cost and purity of natural phospholipids,
niosomes have been developed as they are chemically stable compared to liposomes
and can entrap both hydrophilic and hydrophobic drugs. They are non toxic and do
not require special handling techniques.
Mucoadhesive Dosage Forms
The successful development of fewer mucoadhesive dosage forms for ocular
delivery still poses numerable challenges. This approach relies on vehicles containing
polymers, which will attach via noncovalent bonds to conjuctival mucin.
Nanoparticles and Microparticles
Particulate polymeric drug delivery systems include micro and nanoparticles.
The upper size limit for microparticles for ocular delivery is about 5-10 mm, above
this size; a scratching feeling in the eye can result after ocular application. After
optimal drug binding to microspheres or nanoparticles, the drug absorption in the eye
enhanced significantly in comparison to eye drops.


Hydrogels
The most common way to improve drug retention on the corneal surface is
undoubtedly by using polymers to increase solution viscosity.
13
Hydrogels are
polymers endowed with anability to swell in water or aqueous solvents and induce a
liquidgel transition. Currently, two groups of hydrogels are distinguished, namely
preformed and in situ forming gels. Preformed hydrogels can be defined as simple
viscous solutions which do not undergo any modifications after administration. In situ
forming gels are formulations applied as solutions, sols or suspensions that undergo
gelation after instillation due to physicochemical changes inherent to the eye .
14

Fig.1.3 Classification of hydrogels.



In situ gel
Distinguishing from preformed hydrogels, in situ forming gels are formulations,
applied as a solution, which undergoes gelation after instillation due to
physicochemical changes inherent to the biological fluids. In this way, the polymers
which show sol-gel phase transition and thus trigger drug release in response to
external stimuli are the most investigated. In situ hydrogels are providing such
sensor properties and can undergo reversible sol-gel phase transitions upon changes
in the environmental condition. These intelligent or smart polymers play
important role in drug delivery since they may dictate not only where a drug is
delivered, but also when and with which interval it is released.
15

A polymer used to prepare in situ gels should have following charteristics:
16

It should be biocompatible.
It should be capable of adherence to mucus.
It should have pseudo plastic behaviour.
It should have good tolerance and optical clarity.
It should influence the tear behaviour.
The polymer should be capable of decreasing the viscosity with increasing
shear rate there by offering lowered viscosity during blinking and stability of
the tear film during fixation.
1.5 METHODS FOR PREPARATION OF IN SITU GELS
There are many methods have been employed to cause reversible sol-gel phase
transition in cul de sac and some of these methods are change in temperature
18
, pH
19

and electrolyte composition.
20



Thermo reversible in situ gels
These hydrogels are liquid at room temperature (2025
0
C) and undergo gelation
when in contact with body fluids (3537
0
C), due to an increase in temperature.
21

Example: Poloxamers, cellulose derivatives and xyloglucan. The Poloxamers consist
of more than 30 different non ionic surface active agents.
These polymers are ABA-type tri block copolymers (Fig.1.4) composed of
polyethylene oxide (PEO) (A) and polypropylene oxide (PPO) units (B). The
Poloxamer series covers a range of liquids, pastes and solids with molecular weights
and ethylene oxidepropylene oxide weight ratios varying from 1100 to 14,000 and
1:9to8:2.
22

Fig.1.4PEO-PPO-PEO (Poloxamer).
The gelation mechanism of Poloxamer (Fig.1.5) solutions has been
investigated extensively, but is still being debated. Ultrasonic velocity, light-scattering
and small-angle neutron scattering measurements of aqueous Poloxamer solutions
have clearly indicated a micellar mode of association. Micelle formation occurs at the
critical micellization temperature as a result of PPO block dehydration with increasing
temperature, micellization becomes more important and at a definite point, micelles
come into contact and no longer move. In addition, the formation of highly ordered
structures, such as cubic crystalline phase, has been proposed as the driving force for
gel formation, but this hypothesis has been questioned recently.
22
Thus, packing of
micelles and micelle entanglements may be possible mechanisms of Poloxamer
solution gelation with increase of temperature.
23
Furthermore, it has suggested that
intramolecular hydrogen bonds might promote gelation.The Poloxamers are reported


to be well tolerated and non-toxic even though large amounts (25-30%) of polymers
are required to obtained a suitable gel.

Fig.1.5 In situ gel formation due to change in temperature.
Thermo reversible gels can be prepared with naturally occurring polymers. Most
natural polymer aqueous solutions form a gel phase when their temperature is
lowered. Classic examples of natural polymers exhibiting a solgel transition include
gelatin and carrageenan. At elevated temperatures these polymers adopt a random coil
conformation in solution. Upon cooling, a continuous network is formed by partial
helix formation.
24

Cellulose derivatives also cause gelation Eg: Methylcellulose and hydroxy
propyl methyl cellulose (HPMC) are typical examples of such polymers.
Xyloglucan a polysaccharide derived from tamarind seed, forms thermo
responsive gels in water, under certain conditions. Gelation is only possible when the
galactose removal ratio exceeds 35%.
25
The transition temperature is inversely related
to polymer concentration
26
and the galactose removal ratio. For Eg: The solgel
transition of xyloglucan was shown to decrease from 40 to 5
0
C when the galactose
removal ratio increased from 35 to 58%. Xyloglucan is approved for use as a food
additive. However, its relatively low transition temperature (2227
0
C) makes
handling at room temperature problematic.
27



pH sensitive in situ gels
Gelling of the solution is triggered by a change in the pH.Eg: Cellulose acetate
phthalate (CAP) latex and its derivatives such as carbomer (Fig.1.6) are used.
Cellulose acetate derivatives are the only polymer known to have a buffer capacity
that is low enough to gel effectively in the cul-de-sac of the eye.
28
First preliminary
investigations of pH sensitive latexes for ophthalmic administration began in early
1980 and have been extensively studied by Boye.
29

Fig.1.6 Structure of Carbomer.
Cellulose acetate phthalate latex is a polymer with potentially useful properties
for sustained drug delivery to the eye because latex is a free running solution at a pH
of 4.4, which undergoes coagulation when the pH is raised by the tear fluid to pH 7.4.
But the low pH of the preparation can elicit discomfort in some patients.
30
The poly
acrylic acid and its lightly cross-linked commercial forms (Polycarbophil and
Carbopol) exhibit the strongest mucoadhesion,
31
Carbomer (Carbopol) a cross-linked
acrylic acid polymer (PAA) also shows pH induced phase transition as the pH is
raised above its pKa of about 5.5.
32
Different grades of Carbopol are available. The
manufacturer states that Carbopol 934 gel has the lowest cross-linking density, while
Carbopol 981 intermediate and Carbopol 940 have the highest, higher the cross
linking ability more stiff is the gel formed.
All pH-sensitive polymers contain pendant acidic or basic groups that either
accept or release protons in response to changes in environmental pH. The polymers


with a large number of ionizable groups are known as polyelectrolytes. Swelling of
hydrogel increases as the external pH, increases in the case of weakly acidic (anionic)
groups, but decreases if polymer contains weakly basic (cationic) groups (Fig.1.7).
Some of examples of that are delivered by pH sensitive method includes
Ciprofloxacin,
1
Indometacin,
35
Gatifloxacin
36
etc.

Fig.1. 7 Schematic representation of pH dependent in situ gels.
Ion sensitive in situ gels
Polymers may undergo phase transition in presence of various ions. Some of the
polysaccharides fall into the class of ion-sensitive ones.
38

Gellan gum (Gelrite) is a linear, (Fig.1.8) anionic hetero polysaccharide
secreted by the microbe Sphingomonas elodea (formerly known as Pseudomonas
elodea). The polysaccharide can be produced by aerobic fermentation and then
isolated from the fermentation broth by alcohol precipitation. The polymer backbone
consists of glucose, glucuronic acid and rhamnose in the molar ratio 2:1:1.
39
These are
linked together to give a tetra saccharide repeat unit .The native polysaccharide is
partially esterified with L-glycerate and acetate,
40
but the commercial product Gelrite


has been completely de-esterified by alkali treatment.
41
Formulations with the Gelrite
can be administered to ocular mucosa as a low viscosity solution. On contact with
cations in tear fluid the formulation will form a clear gel.
42
This is caused by cross
linking of the negatively charged polysaccharide helices by monovalent and divalent
cations (Na+, K+, Ca+). Several models have been presented to explain gellan gum
gelation.

Fig1.8 Structure of deacetylated gellan gum.
Mechanism involved in sol to gel transistion by gelrite is as follows, in an ion
free aqueous medium, Gelrite forms double helices at room temperature. This solution
has a viscosity close to that of water and the helices are only weakly associated with
each other (by van der waals attraction). When gel-promoting cations are present,
some of the helices associate into cation-mediated aggregates, which cross-link the
polymer. On heating the polysaccharide in an ion free environment, the
polysaccharide becomes a disordered coil. However, on heating the sample with
cations present, the non-aggregated helices melt out first and the aggregated helices
melt out at a higher temperature in a second transition. The divalent ions such as
magnesium or calcium were superior to monovalent cations in promoting the gelation


of the polysaccharide.
43
However the concentration of sodium in tears (2.6 g/l) is
quite sufficient to induce the gelation. Corneal contact time of formulations based on
gellan gum has been investigated using two main methods, which are fluorometry
44

and -scintigraphy.
45
Both techniques have demonstrated improved residence times
with Gelrite when compared with saline or various commercial solutions. Gelrite has
also provided corneal residence times superior to those of other hydrogel preparations
based on polymers such as cellulosic derivatives or xanthan gum.
Alginates being a family of unbranched binary copolymers, alginates consist
of (14) linked -D-mannuronic acid (M) and -L-guluronic acid (G) residues of
widely varying composition and sequence. By partial acid hydrolysis, alginate was
separated into three fractions.
46
Two of these contained almost homopolymeric
molecules of G and M respectively, while a third fraction consisted of nearly equal
proportions of both monomers and was shown to contain a large number of MG dimer
residues. It was concluded that alginate could be regarded as a true block copolymer
composed of homo polymeric regions of M and G, termed M and G-blocks,
respectively, inter spaced with regions of alternating structure. It was further shown
that alginates have no regular repeating unit and that the distribution of the monomers
along the polymer chain could not be described by bernoullian statistics. Knowledge
of the monomeric composition is hence not sufficient to determine the sequential
structure of alginates.
47, 48
Alginate with a high guluronic acid content will improve
the gelling properties and reduce the total polymer to be introduced into the eye.
The aim of this study is to prepare in situ ophthalmic gel of anti-infective drug
Levofloxacin hemihydrate to enhance ocular bioavailability and reduce dose
frequency and there by increasing patient compliance.

CHAPTER 2 AIMS AND OBJECTIVES

Department of pharmaceutics, Bharathi college of pharmacy 26

Most commonly available ophthalmic preparation are eye drops and ointments.
But these preparations when instilled in to the cul-de sac are rapidly drained away
from the ocular cavity due to the tear flow and naso-lachrymal drainage. Only small
amount is available for its therapeutic effect resulting in frequent dosing. When a drug
solution as dropped in to the eye, effective tear drainage and blinking results in
10-fold reduction of drug concentration in 4-20 minutes.
Ocular therapy could be significantly improved if the pre-corneal residence time
of drugs could be increased, several new preparations have been developed for
ophthalmic use not only prolong the contact time of the vehicle at ocular surface, but
also to slow down the elimination of the drugs. This problem can be overcome by
using in situ gel forming ophthalmic drug delivery systems prepared from polymers
that exhibit reversible phase transition and pseudoplastic behavior to minimize
interference with blinking. Such system can be formulated as liquid dosage form
suitable for administration by instillation in to the eye, which upon exposure to the
eye shift to the gel phase; gelation depends upon physiological pH, temperature and
ionic strength.
36, 49

The aim of present work is to prepare and evaluate in situ opthalmic gel of an
anti infective drug for sustained ocular delivery which is used for the treatment of
various infective diseases of the eye, to get better patient compliance by increasing
residence time and bioavailability.

2. AIMS AND OBJECTIVES
OF STUDY



The broad objective of the present work is
To develop an in situ ophthalmic gel of an anti infective drug- Levofloxacin
hemihydrate for sustained ocular delivery for the treatment of bacterial infections
of the eye.
To evaluate the prepared formulation for its
In vitro gelation and rheological studies,
Drug content uniformity,
In vitro release studies,
Sterility testing of the optimized in situ gel,
Stability studies,
Pharmacodynamic studies and
Pharmacokinetic release studies.
To provide a formulation with better residence time enhanced bioavailability after
topical administration and improved patient compliance.
2.1 PLAN OF WORK
1. Literature survey.
2. Experimental work.
Preformulation studies.
Interaction studies of polymers and drug before selection of the formulation by
FTIR.
Formulation of in situ gel by incorporating optimum polymer, excipents and
drug.
Optimization of ion exchange triggered in situ gelling system using different
polymeric concentrations of gelrite.



Preliminary evaluation of prepared in situ gel formulations by visual
appearance, clarity, pH, drug content, and in vitro gelation.
Estimation of drug by spectrophotometric method.
Evaluation studies of prepared in situ gel formulation for rheological
properties, in vitro release of the drug, antibacterial studies, sterility, stability
studies and pharmacodynamic studies (irritation study).
Comparative evaluation of in vitro drug release with marketed product.

CHAPTER 3 REVIEW OF LITERATURE


Eaga Chandra Mohan et al
1
., prepared in situ gels based on pH-triggered in situ
gelation, thermo reversible gelation and ion activated system. Poly acrylic acid
(Carbopol 940) was used as the gelling agent in combination of Hydroxy Propyl
methylcellulose, which acted as a viscosity-enhancing agent. (pH-triggered system).
Pluronic F-127 (14%) was used as the thermal reversible gelation in combination of
HPMC (1.5%) incorporation of HPMC was to reduce the concentration of pluronic
required for in situ gelling property, with 25% w/w pluronic F-127 reported to form
good gels. Gellan gum (Gelrite) is an anionic exocellular polysaccharide produced by
the bacterium pseudo monas elodea, having the characteristic property cation-induced
gelation (0.6%). The developed formulation was therapeutically efficacious, stable,
non irritant and provided sustained release of the drug over a 6 hours period, but
Gelrite formulation showing long duration of release followed by combination of
carbopol, HPMC and pluronic F-127 & HPMC. The developed system is thus a viable
alternative to conventional eye drops.
Chrystele Le Bourlais et al
6
., given recent advances in ophthalmic drug delivery
systems. Eye drops are the conventional dosage forms that accounts for 90% currently
accessible ophthalmic formulations. Despite the excellent acceptance by patients, one
of the major problems encountered is rapid precorneal drug loss. To improve ocular
drug bioavailability, in situ activated gel forming systems are preferred as they can be
delivered as drops, with sustained release properties.

3. REVIEW OF LITERATURE



Miyazaki S et al
26
., prepared in situ gelling Xyloglucan formulations for sustained
release ocular delivery of Pilocarpine HCL. They found that the degree of
enhancement of mitotic response following sustained release of Pilocarpine from 1.5
% w/w Xyloglucan gel was similar to that from a 25 % w/w Pluronic F127 gel.
Srividya B et al
33
., prepared sustained ophthalmic delivery of Ofloxacin from a pH
triggered in situ gelling system using Polyacrylic acid (Carbopol 940) as the gelling
agent in combination with HPMC E50LV. They observed that the developed
formulation was therapeutically efficacious, stable, non-irritating and provided
sustained release of the drug over an 8 h period.
Kumar S et al
34
., prepared a in situ forming gels by a combination of carbopol and
methylcellulose, they found that solution containing 1.5 % Methylcellulose, 0.3 %
carbopol have low viscosity and form a strong gel under simulated physiological
conditions. increase in concentration of either carbopol or methylcellulose results in
an increased in viscosity, shear stress among the compositions. They also studied the
rheological characterization of such a system at two different pH (4 and 7) and
temperatures (25 and 37C).
Thilek kumar M et al
35
., prepared pH induced in situ gelling system of
Indomethacin for sustained ocular delivery by using carbopol as the gelling agent in
combination with HPMC K15M. They observed that the carbopol solutions which are
acidic and less viscous, transform into stiff gels upon increase in pH by tear fluid of
the eye and produced sustained release of Indomethacin over 8 hour periods, which
made them an excellent candidate for in situ gelling ocular delivery system.
Doijad RC et al
36
., studied on preparation of in situ ophthalmic gels of Gatifloxacin
for the treatment of bacterial conjunctivitis, using sodium alginate as the gelling agent
with HPMC which acted as viscosity enhancing agent. The developed formulations


were therapeutically efficacious, stable, and non-irritant and provided sustained
release of drug over an eight hour period.
Rozier et al
41
., prepared novel ion activated in situ gels using Gelrite as a polymer
for ophthalmic vehicles and a 0.6 % Gelrite vehicle has been compared to an
equiviscous solution of hydroxy ethyl cellulose using Timolol Maleate as a drug
probe. They were observed that the formation of the gel prolonged precorneal
residence time and increased ocular bioavailability of Timolol.
Katarina Lindell et al
49
., showed in vitro release of Timolol maleate from an in situ
gelling polymer, cellulose ether [ethyl (hydroxy ethyl) cellulose] system. They
observed that the release of Timolol maleate was about equal for system with 1-2 %
w/w EHEC, implying that the release was controlled by a low concentration in the
gels and not by any drug-polymer interaction.
Kumar S et al
50
., studied modification of in situ gelling behaviour of carbopol
solutions by HPMC. They were found that in combination both HPMC and carbopol
form low viscosity liquid at pH 4 and transform into stiff gels with plastic rheological
behaviour and comparable viscosities upon increasing the pH up to 7.4 and HPMC-
PAA gels show slow in vitro release of incorporated Timolol Maleate.
Smadar Cohen et al
51
., developed a novel in situ forming ophthalmic drug delivery
system from alginate undergoing gelation in the eye. They demonstrated that an
aqueous solution of sodium alginate could gel in the eye, without the addition of
external calcium ions or other bivalent/polyvalent cations. Alginate with guluronic
acid contents of more than 65 %, such as Manugel DMB, instantaneously formed gels
upon their addition to simulated lachrymal fluid, while those having low guluronic
acid contents, such as Ketton LV, formed weak gels at a relatively slow rate and
hence it was indicated that the in situ gelling alginate system, based on polymers with


high guluronic acid contents was an excellent drug carrier for the prolonged delivery
of Pilocarpine.
Shulin Ding et al
52
., given recent development in ophthalmic drug delivery. Recent
research efforts in ophthalmic drug delivery have focused on systems in which drug
may be administered in the form of eye drops. As a result of these efforts, significant
advancements have made in the in situ forming gels.
Dimitrova et al
53
., developed a model aqueous ophthalmic solution of Indomethacin
using Pluronic F68 and Pluronic F127. They showed that both Pluronics acted very
similarly and were more effective as solublizers, created an appropriate viscosity and
formed reversible gels at higher temperature, ensured the Indomethacin chemical
stability and prolonged in vitro drug diffusion, and showed high physiological
tolerance on rabbit eyes.
Odile Sechoy et al
54
., developed a new long acting ophthalmic formulation of
Carteolol containing alginic acid. They observed that the alginic acid vehicle is an
excellent drug carrier, well tolerated and could be used for the development of a long
acting ophthalmic formulation of Carteolol. In vitro studies indicated that Carteolol
was released slowly from alginic acid formulation, suggesting an ionic interaction.
El-Kamel AH et al
55
., demonstrated in vitro/in vivo evaluation of Pluronic F127
based ocular delivery system for Timolol Maleate. He observed that the slowest drug
release was obtained from 15 % Pluronic F127 formulation containing 3 % methyl
cellulose. In vivo study showed that the ocular bioavailability of Timolol maleate
increased by 2.5 and 2.4 fold for 25 % Pluronic F127 gel formulation and 15 %
Pluronic F127 containing 3 % Methyl cellulose respectively, compared with 0.5 %
Timolol Maleate aqueous solution.


Naseem A Charoo et al
56
., prepared in situ forming ophthalmic gels of
Ciprofloxacin HCL for the treatment of bacterial conjuctivitis, using HPMC K15 M
and Carbopol 934, they concluded that the sol-to-gel system exhibited a zero-order
drug release pattern over 24 hr .
Sultana Y et al
57
., evaluated Carbopol-methyl cellulose based sustained release
ocular delivery system for Pefloxacin mesylate using rabbit eye model. It was found
that the optimum concentration of carbopol solution for in situ gel forming delivery
system was 0.3 % w/w and that for methyl cellulose solution was 1.5 % w/w. The
mixture of solutions showed a significant enhancement in gel strength in the
physiological condition. They observed that both in vitro and in vivo studies and these
studies indicated that the carbopol and methyl cellulose solution alone and mixture
can be used as an in situ gelling vehicle to enhance the ocular bioavailability of
Pefloxacin mesylate.
Kulkarni M.C et al
58
., prepared the ophthalmic in situ gelling formulation of
Flubiprofen sodium using Gellan gum. The formulation in gel form showed almost
complete release of drug. The formulation when subjected for accelerated stability
studies showed good physical and chemical stability and the in vivo studies of the
formulation in albino rabbits confirmed its in situ gelling capacity, non irritancy to
eyes as well as its non toxic nature.
Kugalur Ganesan Parthiban et al
59
., prepared pH dependent in situ ophthalmic
gels of Ketorolac tromethamine using polyacrylic acid (carbopol 940) which is used
as a gelling agent in combination with hydroxy propyl methyl cellulose (HPMC-
K15M, K4M) as a viscosity enhancer. Benzalkonium chloride at suitable
concentration was used as a preservative. The prepared formulations were evaluated
for clarity, pH measurement, gelling capacity, drug content, and in vitro diffusion


study. Under rheological investigation both solution and gel was found to be having
pseudo plastic behaviour. The selected formulations showed sustained release over a
period of 8hrs with increased residence times. Eye irritation test using the Draize test
protocol with cross over studies were preformed on selected formulations.
Shivanand swamy PH et al
60
., studied on formulation and evaluation of novel in
situ gum based ophthalmic drug delivery of Linezolid using various gum based
polymers. The results showed sustained release of Linezolid up to 6 hours.
Kaur IP et al
61
., studied on in situ ophthalmic preparation of Acetazolamide using
polymers such as poly vinyl alcohol, HPMC and Ethylene diamine tetra acetic acid as
a penetration enhancer to increase the absorption of the drug. Formulations were
evaluated for their in vitro release pattern. The effect of these formulations on the
intra ocular pressure in rabbits was investigated. These formulations were found to
maximize the therapeutic effects with decreased frequency of administration.
Divyesh HS et al
62
., studied on mucoadhesive ophthalmic in situ hydrogel of
Moxifloxacin Hcl using combination of poloxamer 407 and poloxamer 188 with
mucoadhesive polymers like Gelrite, xantum gum and sodium alginate with a view
for enhancing bioavailability of the drug and developed formulations showed
sustained release of 10 hours.
Sindhu Abraham et al
63
., studied on in situ ion activated gelling system of
Ofloxacin using sodium alginate as gelling agent and Hydroxyl propyl cellulose
(HPC) as viscosity enhancing agent, in vitro release showed combination of HPC and
alginate solution better retained the drug than HPC and alginate alone, the prepared
formulations followed first order diffusion controlled release kinetics.



Sirish Vodithala et al
64
., prepared in situ ophthalmic gel of Ketorolac tromethamine
using gelrite as a polymer. The formulations were evaluated for clarity, pH, gelling
capacity, drug content, rheological study, in vitro drug release, ocular irritancy studies
(as per Draize test) and in vivo corneal permeation studies using isolated goats cornea.
The developed formulations showed sustained release of drug up to 6 hrs. The
formulations were found to be non irritating with no ocular damage.
Johan Carlfors et al
65
., studied the rheology of in situ gels prepared by using gelrite
as a polymer, he also performed a complementary in vivo study for determining
precorneal contact times in humans and in rabbits. He concluded that the elastic
moduli of the gels increased with increasing concentration of electrolytes. At
physiological concentration of the electrolytes, the elasticity of the gels was
independent of Gelrite concentration. The human contact times increased up to 20
hours with decreasing osmolality of the formulations. The results indicate that a high
rate of the sol / gel transition results in long contact times.
Coquelet C et al
66
., evaluated association between benzalkonium chloride and a poly
acrylic acid in gels by microfiltration and membrane dialysis. The interaction between
benzalkonium chloride and the respective polymers, the related availability
benzalkonium chloride in the corresponding solutions, were studied for aqueous
preparation of hydroxy ethyl cellulose, polyvinyl alcohol and cross linked poly acrylic
acid. The study was performed by means of a cross flow filtration process with an
alumina membrane. In the presence of the poly acrylic acid gel, the rejection rate of
benzalkonium chloride is much higher than with hydroxy ethyl cellulose and
polyvinyl alcohol. These results can be explained by the association of the
benzalkonium cation with the negative carbohydrate group of the poly acrylic acid.
This effect, which was confirmed by dialysis experiment, leads to trapping of


benzalkonium cation inside the polymer network and could be of interest in the
reduction of the harmful effects of benzalkonium chloride observed in the treatment
of eye diseases.
Furrer et al
67
., given application of in vivo confocal microscopy to the objective
evaluation of ocular irritation induced by surfactants. An ocular irritation test using
confocal laser scanning ophthalmoscopy has been developed in which corneal lesions
subsequent to instillation of surfactants are specifically marked by fluorescein and
assessed by digital image processing. Benzalkonium chloride, a cationic surfactant at
a concentration range of 0.01 to 0.5% was tested. The cornea was evaluated for
in vivo ocular tolerance by confocal microscopy. In both rabbits and mice, the test
revealed following irritation ranking: cationic > anionic > nonionic surfactants. In
both animal models, the ocular damage increased with the concentration of
benzalkonium. The test was sensitive enough to detect ocular micro lesions at
concentration of surfactants as low as 0.01% for benzalkonium.



3.1.1LEVOFLOXACIN HEMIHYDRATE
68
Levofloxacin hemihydrate is member of the fluoroquinolone class of
antimicrobial drugs. It is active against a wide range of Gram +ve and Gram -ve
organisms.
Structural Formula

Molecular Formula :C
18
H
20
FN
3
O
Generic name : Levofloxacin.
Nomenclature :(-)-(S)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-
1piperazinyl)7-oxo7Hpyrido [1, 2, 3-de]-1,
4benzoxazine-6-carboxylicacid hemihydrate.
Molecular Weight : 361.3675 Daltons.
Melting Range : 226C.
log p value : 2.1.
Half life : 6 to 8 hours.

3.1 DRUG PROFILE


Description
A synthetic fluoroquinolone (fluoroquinolones) antibacterial agent that inhibits
the super coiling activity of bacterial DNA gyrase, halting DNA replication.
Appearance
Levofloxacin hemihydrate is pale yellow coloured amorphous solid.
Solubility
Levofloxacin hemihydrate is in soluble in water but its predicted water
solubility is 1.44e+00 mg/ml, it is soluble in chloroform and other organic solvents,
solubility of Levofloxacin hemihydrate can be increased by using co solvents such as
glycerine and propylene glycol.
It is considered to be freely soluble in this pH range, as defined by USP
nomenclature. Above pH 5.8, the solubility increases rapidly to its maximum at pH
6.7 (272 mg/ml) and is considered freely soluble in this range. Above pH 6.7, the
solubility decreases and reaches a minimum value (about 50 mg/mL) at a pH of
approximately 6.9.
Bio pharmaceutics
Mechanism of Action as Antibacterial Agent
Levofloxacin inhibits bacterial type II topoisomerases, topoisomerase IV and
DNA gyrase. Levofloxacin, like other fluoroquinolones, inhibits the A subunits of
DNA gyrase, two subunits encoded by the gyrA gene. This results in strand breakage
on a bacterial chromosome, supercoiling and resealing; DNA replication and
transcription are inhibited.
Pharmacokinetics
Absorption of Levofloxacin after single or multiple doses of 200 to 400 mg is
predictable and the amount of drug absorbed increases proportionately with the dose.


The peak and trough plasma concentrations attained following multiple once daily
oral 500 mg regimens were approximately 5.7 g/ml and 0.5 g/ml respectively.
There is no clinically significant effect of food on the extent of absorption of
Levofloxacin.
Adverse Effects
The most frequently reported drug-related adverse reaction was transient.
Ocular burning or discomfort. Other reported reactions include stinging, redness,
itching, chemical conjunctivitis / keratitis, blurred vision, dryness, and eye pain.
Precautions and Warnings
If an allergic reaction to Levofloxacin occurs, discontinue the drug. Serious
acute hypersensitivity reactions may require immediate emergency treatment, oxygen
and airway management, including incubation should be administered as clinically
indicated. As with other anti-infective, prolonged use may result in overgrowth of non
susceptible organisms, including fungi. If super infections occur discontinue use and
institute alternative therapy. Levofloxacin should be discontinued at the first
appearance of a skin rash or any other sign of hypersensitivity reaction.
Usage and Administrations
Topically
Levofloxacin as 0.5 % eye drops is used for treatment of Bacterial infections.
Orally
Levofloxacin is also used for the treatment of susceptible infections of skin,
lungs, ears, airways, bones, and joints caused by susceptible bacteria. Levofloxacin
also is frequently used to treat urinary infections, including those resistant to other
antibiotics, as well as prostatitis. Levofloxacin is effective in treating
infectious diarrhoea caused by E.coli, Shigella and Campylobacter jejuni.


Levofloxacin also can be used to treat various obstetric infections, including mastitis
(infection of the breast).
3.2 PROFILE OF EXCIPENTS.
3.2.1 GELRITE
69
Non-proprietary Names : Gellan gum.
Functional Category : Gelling agent alternative to agar.
Synonyms : gellan gum.
Chemical composition : Polysaccharide comprising glucuronic acid,
Rhamnose and glucose.
Physical state : Dry powder.
Description : Gelrite is a White to tan coloured solid.
pH : The pH of 1 % w/v aqueous dispersion is 7.
Solubility : Soluble in water forming viscous solution
becoming a paste at concentration >5% gels
if heated and cooled.
Materials to avoid : Strong acids, strong bases.
Decomposition products : May include CO
2
and CO.
Hazardous polymerization: None.
Stability and Reactivity
Storage and handling procedures should follow the normal practices
recognized as desirable for naturally derived polymeric gelling agents. The gel
strength of the dry powder is retained, even after prolonged storage at 50 C
(122
0
F) and will recover from freezing. However, as with any polysaccharide,
such changes in temperature may tend to reduce its stability and should be


avoided. Gellan gum should be stored tightly closed, in a cool (<60 F) dry place
for maximum shelf life.
Toxicity
Gelrite (gellan gum) caused no oral toxicity in rats fed the pure product at a
single dose of 5000 mg/kg. From the results of eye irritation tests, Gelrite is not
considered to be an eye irritant. During dust inhalation tests, no toxic symptoms were
exhibited by rats exposed to concentrations averaging 6.09 mg/litre for a 4-hour
period. The lungs of these necropsied rats appeared normal. Mutagenicity tests show
Gelrite to be negative in the Ames test. No special precautions are required to handle
Gelrite other studies on the safety of gellan gum are in progress.
Applications in Pharmaceutical Formulation or Technology
As an in situ ion exchange and thermo reversible gelling agent.
3.2.2 PROPYLENE GLYCOL
70
Non-proprietary Names : Propylene glycol.
Synonyms : Methyl glycol.
Chemical Name and CAS Registry Number : 1, 2-Propanediol [57-
55-6]

Empirical Formula and Molecular Weight : C
3
H
8
O
2
, 76.09.
Boiling point : 188C.
Density : 1.038 g/cm3 at 20C.
Flammability : Upper limit -12.6%v/v,
Lower limit-2.6%v/v.
Flash point : 99C (open cup).
Heat of combustion : 1803.3 kJ/mol.


Heat of vaporization : 705.4 J/g at B.P.
Melting point : 59C.
Refractive index : 1.4324.
Description
Propylene glycol is a clear, colourless, viscous, and practically
odourless Liquid, with a sweet, slightly acrid taste resembling that of
glycerine.
Functional Category
Anti microbial preservative, co solvent, disinfectant, humectant,
plasticizer, stabilizing agent.
Applications in Pharmaceutical Formulation
As humectant in topicals up to 15%.
As preservative in solutions and semisolids up to 1530%.
As solvent or co solvent in aerosol solutions 1030%, oral solutions
1025%, Parenterals 1060%, topicals 580%.
Stability and Storage Conditions
At cool temperatures, propylene glycol is stable in a well closed
container, but at high temperatures, it tends to oxidize, giving rise to products
such as propionaldehyde, lactic acid, pyruvic acid and acetic acid. Propylene
glycol is chemically stable when mixed with ethanol (95%), glycerine,
aqueous solutions may be sterilized by autoclaving.



3.2.3 BENZOLKONIUM CHLORIDE
70
Non proprietary Name : Benzalkonium Chloride.
Synonyms : Benzalkonii chloridum.
Chemical Name and CAS Registry Number : Alkyl dimethyl (phenyl
methyl) ammonium
chloride [8001-54-5].
Description
Benzalkonium chloride occurs as a white or yellowish-white amorphous
powder, a thick gel, or gelatinous flakes. It is hygroscopic, soapy to the touch and has
a mild aromatic odour and very bitter taste.
Empirical Formula and Molecular Weight
The USP32NF27 describes benzalkonium chloride as a mixture of Alkyl
benzyl di methyl ammonium chlorides of the general formula [C6H5CH2N (CH3)2R]
Cl, where R represents a mixture of alkyls, including all or some of the group
beginning with n-C
8
H
17
and extending through higher homologs, with n-C12H25, n-
C
14
H
29
,a n-C
16
H
33
comprising the major portion. The average molecular weight of
benzalkonium chloride is 360.
Functional Category
Antimicrobial preservative, antiseptic, disinfectant, solubilising agent and can
also be used as wetting agent.
CHAPTER 4 MATERIALS AND METHODS


4.1 MATERIALS
Table 4.1 List of chemicals used with grade and supplier.
SL.No Chemicals Supplier
1 Levofloxacin hemihydrate KAPL, Bangalore
2 Gelrite KAPL, Bangalore
3 Propylene glycol KAPL, Bangalore
4 Benzalkonium chloride KAPL, Bangalore
5 Sodium chloride KAPL, Bangalore
6 Calcium chloride dihydrate KAPL, Bangalore
7 Sodium bicarbonate KAPL, Bangalore
8 Hydrochloric acid KAPL, Bangalore
9 Sodium hydroxide KAPL, Bangalore

4. MATERIALS AND METHODS



Table 4.2 List of instruments used.
SL.
No
Instrument Manufacturer
1.
U V visible spectrophotometer Shimadzu (UV 1601)
2.
FTIR spectrophotometer Shimadzu 8300
3.
Magnetic stirrer Remi motors, Ahmadabad.
4.
Mechanical stirrer Remi motors, Ahmadabad
5.
Electronic balance Sartorius
6.

Digital pH meter

Digisun, Hyderabad
7.
Digital melting point apparatus
CL 725/726,
Microcontroller based
melting point apparatus
8.
Stabality chamber Neutronics
9.
Franz diffusion cell Molded

4.2 METHODS
4.2.1 PREFORMULATION STUDIES
Preformulation testing is the first step in the rationale development of dosage
forms of a drug substance. It can be defined as an investigation of physical and
chemical properties of a drug substance alone and when combined with excipients.
The overall objective of preformulation testing is to generate information useful to the
formulator in developing stable and bioavailable dosage forms, which can be mass
produced.


Identification of Levofloxacin hemihydrate.
Identification of LEV was carried out by FTIR spectrophotometer.
Melting point determination
Melting point of LEV was determined by open capillary method.
Solubility
Solubility of LEV was determined based on co solvency method using
propylene glycol, glycerin and water.
Determination of
max

A solution of LEV containing the concentration 10 g/ ml was prepared in
STF pH 7.4 and UV spectrum was taken using Shimadzu (UV-1601) double beam
spectrophotometer. The solution was scanned in the range of 200 400 nm.
4.2.2 Preparation of in situ gels of LEV.
Table 4.3 Formulation of in situ gels of LEV

SL. No

Ingredients

Concentration (% w/v)
F1 F2 F3 F4 F5 F6

1

Levofloxacin
hemi hydrate

0.512

0.512

0.512

0.512

0.512

0.512

2

Gelrite

0.2

0.3

0.4

0.5

0.6

0.7

3

Propylene glycol

8

8

8

8

8

8

4

Benzalkonium
chloride

0.01

0.01

0.01

0.01

0.01

0.01
5 De ionized water 100 100 100 100 100 100



Procedure for preparation of in situ gels of LEV
65, 71

Polymer solution was prepared by dispersing Gelrite in de ionized water by
heating up to 90
0
c for 20 minutes followed by cooling to room temperature, drug
solution was prepared by dissolving Levofloxacin hemihydrate in mixture of
propylene glycol and water (100:8), drug solution was mixed with polymer solution
using a magnetic stirrer, Benzalkonium chloride in concentration of 0.01% was added
which acts as preservative. The prepared in situ gels were filled in glass vials closed
with rubber closures and sealed with aluminium caps and sterilized by autoclave at
121
0
C 15 psi for 20 minutes.
4.2.3 Evaluation of in situ gels of Levofloxacin hemihydrate.
Interaction studies
72

IR spectra were taken by using Fourier transform infrared spectrophotometer
(840, Shimadzu, Japan). The pellets of drug and potassium bromide were prepared by
compressing the powders at 20 psi on KBrpress and the spectra was scanned in the
wave number range of 4000 600 cm
1
. FTIR study was carried on pure drug, physical
mixture of drug and polymers, formulations to confirm the compatibility of drug with
other excipients used in the preparation of in situ gels (Fig. 5.1-5.4).
Visual Appearance and Clarity
33

Visual appearance and clarity was checked under fluorescent light against a
white and black back ground for presence of any particulate matter (Table 5.2).
pH
36

The pH of the prepared in situ gelling system after addition of all the ingredients
was measured using pH meter (Table 5.2).



I n vitro gelation
36
Gelling capacity of formulations was evaluated in order to identify the
formulations suitable for use as in situ gelling systems. Gelling capacity was
determined by mixing the formulation with simulated tear fluid in the proportion 25:7
and examined visually.
The composition of simulated tear fluid was sodium chloride (0.670 g), sodium bi
carbonate (0.2g), calcium chloride dihydrate and bi-distilled water quantity sufficient
up to100 g. Physiological pH (7.40.2) was adjusted by adding the required amount
of 0.1 N HCL (Table 5.3).
Rheological Studies
73
Viscosity of the instilled formulation is an important factor in determining
residence time of drug in the eye. The prepared solutions were allowed to gel in the
simulated tear fluid and then the viscosity determination were carried out by using
Brooke field viscometer RVT model in spindle no S-34, angular velocity ran from
10-100 rpm.
9
Viscosity of the formulations increased with increase in polymer
concentration. The hierarchy of shear rate was reversed and average of two readings
was used to calculate viscosity (Tables 5.4-5.5 and Fig. 5.5-5.6).
Sterility Testing
74
Sterility testing is intended for detecting the presence of viable form of
microorganisms and was performed for aerobic and anaerobic bacteria and fungi by
using fluid thioglycolate medium and soyabean casein digest medium, respectively as

per the indian pharmacopoeia.
Preparation of media
Fluid thioglycolate medium and Soyabean casein digest medium were prepared
by suspending all ingredients in 1000 ml of distilled water, separately boiled until it


dissolves completely. Then it was sterilized by autoclaving at 15 lbs pressure, 121
0
C
for 15 minutes and cooled. After cooling 25 ml of both the medium were transferred
to the test tubes.
Preparation of Samples
The sterile formulations were taken into laminar airflow. Sterile formulation
was removed from the vials by help of syringe. This solution was passed through the
membrane filter of 0.45m size with the help of vacuum pump. After filtration, the
filter paper was removed from funnel and it was cut into two half. One half was
dropped in bacterial media (Fluid thioglycolate) and the other half was dropped in the
fungal media (Soyabean casein digest). The media were kept for incubation for 7 days
at 37
0
C. Both the media were observed every day for any microbial contamination
and compared with a positive and negative control (Table 5.6).
Drug Content Analysis
62

Estimation of Levofloxacin hemihydrate by Spectrophotometric Method
A simple and rapid method for estimation of Levofloxacin hemihydrate by UV
spectrophotometric method was developed in simulated tear fluid (STF).
Levofloxacin hemihydrate in simulated tear fluid of pH 7.4 shows
max
at 287.5 nm.
Preparation of simulated tear fluid
Dissolve 0.670g of sodium chloride, 0.2g of sodium bicarbonate and 0.008g
calcium chloride di hydrate in 100 ml of de ionized water and adjust the pH to 7.4
using 0.5 M sodium hydroxide and 0.5 M hydrochloric acid.
Preparation of Standard Stock Solution
The standard stock solution was prepared by dissolving 100 mg Levofloxacin
hemihydrate of in 100 ml of simulated tear fluid, to get the 1mg/ml concentration of solution.



Working Standard Solution
From above stock solution, 2 ml was pipetted out in to a 10 ml volumetric flask
and made up to 10 ml with simulated tear fluid to give a concentration of 20 /ml,
respectively.
Procedure for Calibration of LEV using STF at
max
287.5 nm
Above working standard solution 1-6 ml was taken and was diluted to 10 ml to
get 2-12 /ml and absorbance was taken at
max
287.5 nm. The obtained data is given
in Table 5.7 and standard plot of absorbance versus concentration was plotted, which
is given in Fig. 5.7.
The vials containing formulation were properly shaken for 2-3 min. One ml of
the formulation was transferred into 100 ml volumetric flask with 1 ml calibrated
graduated pipette, 50 ml of simulated tear fluid with pH 7.4 was added gel was
completely crushed with the help of glass rod followed by vigorous shaking until the
formed gel gets completely dispersed to give clear solution. Final volume was
adjusted to 100 ml with STF, aliquot of 1ml was taken and further diluted to 10 ml
with STF, obtained solution was filtered through 0.45m filter membrane and the
drug concentration was determined by UV Visible spectrophotometer at 287.5 nm
(Table 5.7).
I n vitro release studies
75
In vitro drug release from the formulations was studied by the diffusion cell.
Here the pH of the Lacrimal fluid and the blinking rate of the eye were taken into
consideration and were simulated. The procedure for standard calibration is same as
mentioned under drug content determination.



Procedure
In vitro release studies were carried out using bichambered donor receiver
compartment model (Franz diffusion cell) using cellophane membrane soaked
overnight in the receptor medium (simulated tear fluid, pH 7.4). The diffusion
medium was 100ml of simulated tear fluid stirred at 50rpm at 37
0
C 0.5
0
C. One end
of the diffusion tube was covered by a cellophane membrane. The 1ml formulation
were spread on the cellophane membrane and membrane was placed such that it just
touches the diffusion medium (STF) present in receptor compartment. The drug
samples were withdrawn at the interval of one hour for the period of 8 hrs from
diffusion medium and analyzed by a UV spectrophotometer at 287.5nm using
simulated tear fluid as blank.
Comparative evaluation of marketed products with prepared in situ gels
75
In vitro release studies of marketed formulation was carried out using
bichambered donor receiver compartment model (Franz diffusion cell) using
cellophane membrane soaked overnight in the receptor medium (simulated tear fluid,
pH 7.4). The diffusion medium was 100ml of simulated tear fluid stirred at 50rpm at
37
0
C 0.5
0
C. One end of the diffusion tube was covered by a cellophane membrane.
The 1ml formulation were spread on the cellophane membrane and membrane was
placed such that it just touches the diffusion medium (STF) present in receptor
compartment. The drug samples were withdrawn at the interval of one hour for the
period of 8 hrs from diffusion medium and analyzed by a UV spectrophotometer at
287.5nm using simulated tear fluid as blank (Table 5.8-5.9 and Fig. 5.8-5.9).



Pharmacokinetic Release Studies
6

All the optimized formulations were subjected to study the release kinetics and
the best fit kinetic model was determined for the optimized formulations using
analysis software PCP Disso V2. (Table 5.10-5.14, Fig. 5.10-5.13)
Antimicrobial Efficacy Studies
76
The Antimicrobial efficacy studies were carried out to ascertain the biological
activity of the optimized formulations. Staphylococcus aureus, Pseudomonas
aeruginosa and E.coli were used as the test organisms. Anti microbial efficiency was
determined by agar diffusion test employing Cup-Plate method. Sterile solutions of
Levofloxacin hemihydrate (standard solution) and the developed formulations were
diluted

at different concentration (test solutions) these solutions were

poured in to
cups bored into sterile nutrient agar previously seeded

with test organisms
(Pseudomonas aeruginosa, E.coli and Staphylococcus

aureus), after allowing diffusion
of the solutions for 2 hours, the

agar plates were incubated at 37
0
C for 24hrs. The zone
of inhibition

(ZOI) measured around each cup and was compared with that of control.

The entire operation except the incubation was carried out in a laminar

airflow unit.
Both positive and negative controls were maintained

during the study (Table 5.15).
Ocular Irritancy Studies
77, 78
In developing a novel ophthalmic delivery system, an injury to the eye was
taken into consideration. Since, eye being a sensitive, most delicate and yet most
valuable of the sense organs, the injuries to the cornea, conjunctiva and iris were
measured according to Draize test.
The prepared in situ gels was used for in vivo studies, the protocol was approved
by college ethical committee with registration number BCP/IAEC/PCU-01.The
Draize technique designed for testing ocular irritation potential of the ophthalmic


product prior to marketing was used. According to the Draize test, the amount of
substance applied to the eye is normally 100l placed into the lower cul-de-sac with
observation of the various criteria made at a designed time interval of 1hr, 24hrs,
48hrs, 72hrs and 1week after administration. Three male rabbits weighing 1.5 to 2kg
were used for the present study. The sterile formulation was instilled twice a day for a
period of 7 days and a cross-over study was carried out ( A 3 day washing period with
saline was carried out before the cross-over study).Rabbits were observed periodically
for redness, swelling, watering of the eye.
Accelerated Stability Studies
1
Stability is defined as the extent, to which a product retains with in specified
limits and through out its period of storage and use ie, shelf life. Stability studies were
carried out on optimized formulations according to international conference on
harmonization (ICH) guidelines.
A sufficient quantity of formulations in previously sterilized vials was stored in
desiccators containing a saturated solution of sodium chloride, which gives a relative
humidity of 755 %.The desiccators were placed in a hot air oven maintained at a
temperature 40
0
C0.5
0
C and at room temperature. Samples were withdrawn at 7 days
interval for 42 Days. Percent drug remaining was calculated and plotted against time
in days (Tables 5.16-5.23 and Fig. 5.14-5.15).

CHAPTER 5 RESULTS


5.1 PREFORMULATION STUDIES
Melting point determination
The melting point of LEV was found to be 226.2
o
C.
Solubility study
Solubility of Levofloxacin hemihydrate was found to be dependent on pH.
LEV was soluble in co solvent mixture of propylene glycol and water, Glycerin
and water, it was also found soluble in organic solvents like DMSO.
Determination of
max

max
of LEV was found to be 287.5 in STF pH 7.4.
5.2 EVALUATION OF PREPARED IN SITU GELLING SYSTEM
Interaction Studies
The prepared in situ gelling systems were evaluated for interaction studies
to ensure that there is no interaction occurred in between drug and polymers. For
confirmation of stability of drug in the prepared formulations the IR spectra was
taken and compared with that of pure drug. The result of these studies revealed
that there were no definite changes obtained in the bands of drug with respect to
pure drug. (Fig .5.1-5.4).

5. RESULTS

CHAPTER 5 RESULTS


Fig. 5.1 IR spectra of pure LEV.

Fig. 5.2 IR spectra of Gelrite.

CHAPTER 5 RESULTS


Fig. 5.3 IR spectra of physical mixture of LEV and Gelrite.

Fig. 5.4 IR spectra of in situ gel of Levofloxacin hemihydrate.
CHAPTER 5 RESULTS


TABLE 5.1 Interpretations of IR spectra.
SL.
NO
IR SPECTRUM GROUPS PEAKS
(CM
-1
)

STRETCHING
/DEFORMATION

1

Levofloxacin
hemihydrates
OH 3264.63 Stretching
C=O
(Carboxylic
acid)
1724.42 Stretching
C=O
(Aromatic)
1620.26 Stretching
CH
3
1341.54 Stretching
C-F 1057.99 Stretching

2

Physical mixture of
LEV and gelrite
C=O
(Carboxylic
acid)
1722.49 Stretching
C=O
(Aromatic)
1622.19 Stretching
CH
3
1340.57 Stretching

3

In situ gel
C=O
(Carboxylic
acid)
1720.32 Stretching
C=O
(Aromatic)
1624.18 Stretching
CH
3
1340.56 Stretching

CHAPTER 5 RESULTS


Evaluation of Visual appearance, Clarity, pH, and Drug Content
All the prepared in situ gelling systems were evaluated for preliminary
steps such as visual appearance, clarity, pH, and drug content. These formulations
were transparent and clear. The pH of the formulations was found to be 7.1 to 7.4,
and drug content was in between 92-98% (Table 5.2).
Table 5.2 Preliminary evaluation of visual appearance, clarity, pH, and drug content.

Formulation
Code

Visual
appearance

Clarity

pH

Drug content
F1 Transparent Clear 7.12 98.01

I n vitro Gelation
Prepared in situ gelling systems were evaluated for the in vitro gelation
capacity. All the formulations gave satisfactory results (Table 5.3).

CHAPTER 5 RESULTS


Table 5.3 Evaluation of gelling capacity.
Formulations Gelling Capacity
F1 ++
F2 +++
F3 +++
F4 +++
F5 +++
F6 +++
Note: ++ gelation immediate and remains for few hours, +++ shows gelation
immediate and remains for extended period.
Rheological Studies
For the development of optimum in situ gelling system, two major prerequisites
viscosity and gelling capacity should be taken in consideration, since the ocular shear
rate is very high ranging from 0.03 S
-1
during inter-blinking periods to 4250-28500 S
-1

during blinking, viscoelastic fluid with a viscosity that is high under low shear rate
condition and low under high shear rate condition, which is called Pseudo plastic
fluid, is often preferred, so dynamic viscosity of formulations were measured as the
change of shear rate before and after gelation (Tables 5.4-5.5 and Fig. 5.5-5.6).

CHAPTER 5 RESULTS


Table 5.4 Rheological studies of in situ gels before gelation.
Shear
rate(RPM)
Viscosity of the formulation
F1 F2 F3 F4 F5 F6
10 550 825 1120 1380 1635 1855
20 380 640 790 860 1190 1270
50 260 420 440 485 655 850
100 190 240 260 280 320 485

Fig. 5.5 Rheological studies of in situ gels before gelation.

CHAPTER 5 RESULTS


Table 5.5 Rheological studies of in situ gels after gelation.
Shear
rate(RPM)
Viscosity of the formulation
F1 F2 F3 F4 F5 F6
10 1320 1405 2600 2020 3280 4180
20 710 1110 1320 1545 2190 2270
50 460 680 735 890 1080 1530
100 320 405 440 500 560 620

Fig. 5.6 Rheological studies of in situ gels after gelation

CHAPTER 5 RESULTS


Sterility Testing
All the prepared in situ gelling systems were evaluated for the sterility. After 7
days of incubation the results showed no microbial growth in all formulations
(Table 5.6).
Table 5.6 Test of Sterility.
Formulation
code
Days of incubation
1 2 3 4 5 6 7
F1 _ _ _ _ _ _ _
F2 _ _ _ _ _ _ _
F3 _ _ _ _ _ _ _
F4 _ _ _ _ _ _ _
F5 _ _ _ _ _ _ _
F6 _ _ _ _ _ _ _

Wheresign indicate the no growth.
Estimation of Levofloxacin hemihydrate by Spectrophotometric method
A simple Spectrophotometric method for estimation of Levofloxacin
hemihydrate was developed in Simulated Tear Fluid, which exhibited
max
at 287.5
nm in Beers range of 2-12 g/ml. Results are shown in Table 5.7. and Fig. 5.7.

CHAPTER 5 RESULTS


Table 5.7 Standard calibration data of LEV.
SL.
No
Concentration
(g/ml)
Absorbance (nm)
1 0 0.000
2 2 0.143
3 4 0.267
4 6 0.422
5 8 0.557
6 10 0.704
7 12 0.841

Fig. 5.7 Calibration curve of Levofloxacin hemihydrate in STF.
I n vitro release studies
The in vitro release of Levofloxacin hemihydrate from the prepared
formulations was studied through cellophane membrane using diffusion cell. The
release studies of prepared in situ gelling systems were carried out up to 8 hours.
In vitro release studies of marketed eye drops (Levobact) was done through
cellophane membrane using diffusion cell and the release marketed product was up to
3 hours (Table 5.8-5.9and Fig. 5.8-5.9).
CHAPTER 5 RESULTS


Table 5.8 In vitro release data of marketed eye drops.
SL. No Time (Min)

(%)CDR SD

1 30 220.57
2 60 49.30.59
3 90 59.90.61
4 120 74.730.66
5 150 84.80.40
6 180 95.180.58
SD=Standard deviation (n3)

Fig. 5.8 In vitro release profile of marketed eye drops.
CHAPTER 5 RESULTS


Table 5.9 Comparative in vitro release data of marketed eye drops and prepared in
situ gels.

Time
(HRS)
% Cum. drug release

F1SD

F2SD

F3SD
F4SD

1
30.420.502 20.480.528 17.550.349
14.340.222
2
42.510.311 31.760.733 32.540.508
25.530.536
3
51.430.375 43.730.797 40.230.646
34.20.337
4
66.150.830 54.270.610 48.20.492
43.850.670
5
74.350.971 62.400.555 60.10.618
53.470.740
6
79.320.884 69.480.515 68.920.697
61.350.947
7
87.560.681 79.150.684 73.430.421
67.140.883
8
90.370.260 84.560.655 78.270.517
74.990.875
% Cum. drug release
F5SD

F6SD

LevobactSD
13.280.505 12.290.353
49.3 0.59
28.110.717 21.660.785
74.73 0.66
36.961.005 35.331.175
95.18 0.58
43.680.890 42.090.775

55.950.621 51.290.657

64.770.854 58.441.171

68.551.066 65.191.111

72.870.875 71.020.411


CHAPTER 5 RESULTS


Release kinetics of in situ gels.
Table 5.10 Comparative Zero order release kinetics data of in situ gels.

SL.No.

Time (HRS)
% Cum. drug release

F1SD

F2SD

F3SD
1
1
30.420.502 20.480.528 17.550.349
2
2
42.510.311 31.760.733 32.540.508
3
3
51.430.375 43.730.797 40.230.646
4
4
66.150.830 54.270.610 48.20.492
5
5
74.350.971 62.400.555 60.10.618
6
6
79.320.884 69.480.515 68.920.697
7
7
87.560.681 79.150.684 73.430.421
8
8
90.370.260 84.560.655 78.270.517
% Cum. drug release
F4SD

F5SD

F6SD

14.340.222 13.280.505 12.290.353
25.530.536 28.110.717 21.660.785
34.20.337 36.961.005 35.331.175
43.850.670 43.680.890 42.090.775
53.470.740 55.950.621 51.290.657
61.350.947 64.770.854 58.441.171
67.140.883 68.551.066 65.191.111
74.990.875 72.870.875 71.020.411

CHAPTER 5 RESULTS


Table 5.11 Comparative first order release kinetics data of in situ gels.

SL.No.

Time (HRS)
Log% Cumulative drug remained to be released

F1SD

F2SD

F3SD
1 1
2 2 2
2 2
1.8420.003 1.8420.003 1.8420.003
3 3
1.7600.002 1.7600.002 1.7600.002
4 4
1.6860.003 1.6860.003 1.6860.003
5 5
1.5290.010 1.5290.010 1.5290.010
6 6
1.4090.016 1.4090.016 1.4090.016
7 7
1.3150.018 1.3150.018 1.3150.018
8 8
1.0940.023 1.0940.023 1.0940.023
Log% Cumulative drug remained to be released

F4SD

F5SD

F6SD
2 2 2
1.9330.001 1.9380.002 1.9430.017
1.8720.003 1.8570.004 1.8940.004
1.8180.002 1.8000.006 1.8110.007
1.7490.005 1.7510.006 1.7030.005
1.6680.006 1.6440.006 1.6880.005
1.5870.010 1.5470.010 1.6180.012
1.5170.011 1.4970.014 1.5420.013

CHAPTER 5 RESULTS


Table 5.12 Comparative higuchi release kinetics data of in situ gels .

SL.No.
T
% Cum. drug release

F1SD

F2SD

F3SD
1 0 0 0 0
2 1.0 30.420.502 20.480.528 17.550.349
3 1.41 42.510.311 31.760.733 32.540.508
4 1.73 51.430.375 43.730.797 40.230.646
5 2.0 66.150.830 54.270.610 48.20.492
6 2.23 74.350.971 62.400.555 60.10.618
7 2.449 79.320.884 69.480.515 68.920.697
8 2.646 87.560.681 79.150.684 73.430.421
% Cum. drug release

F4SD

F5SD

F6SD
0 0 0
14.340.222 13.280.505 12.290.353
25.530.536 28.110.717 21.660.785
34.20.337 36.961.005 35.331.175
43.850.670 43.680.890 42.090.775
53.470.740 55.950.621 51.290.657
61.350.947 64.770.854 58.441.171
67.140.883 68.551.066 65.191.111

CHAPTER 5 RESULTS


Table 5.13 Comparative peppas release kinetics data of in situ gels.

SL.No.
Log T
Log% Cum. drug release
F1SD F2SD F3SD
1 0 0 0 0
2 0 1.4830.007 1.3110.01 1.2440.008
3 0.30 1.6280.003 1.5020.01 1.5120.099
4 0.47 1.7110.003 1.6410.007 1.6050.186
5 0.60 1.8210.005 1.7350.001 1.6830.237
6 0.69 1.8710.005 1.7950.004 1.7790.286
7 0.77 1.8990.004 1.8420.003 1.8380.004
8 0.84 1.9420.003 1.8980.003 1.8660.002
Log % Cum. drug release
F4SD F5SD F6SD
0 0 0
1.1570.006 1.3460.012 1.0890.016
1.4070.009 1.5470.015 1.3360.011
1.5340.004 1.6520.010 1.5480.011
1.6420.006 1.7460.007 1.6240.008
1.7280.005 1.8150.005 1.7100.004
1.7880.006 1.8590.008 1.7670.005
1.8270.005 1.9020.007 1.8140.006

CHAPTER 5 RESULTS


Fig. 5.9 Comparative in vitro release of marketed eye drop and in situ gels.

Fig. 5.10 Comparitive zero order release kinetics of in situ gels.

CHAPTER 5 RESULTS


Fig. 5.11 Comparitive first order release kinetics of in situ gels.

Fig. 5.12 Comparative higuchi release kinetics of in situ gels.
CHAPTER 5 RESULTS


Fig. 5.13 Comparative peppas release kinetics of in situ gels.
Table 5.14 Regression co-efficient (r2) values of different kinetic models.
Formulation

Zero
order

First
order
Higuchi
Matrix
Peppas plot
r
2
value n value
F1 0.927 0.988 0.994 0.992 0.546
F2 0.973 0.982 0.979 0.998 0.698
F3 0.968 0.990 0.976 0.992 0.718
F4 0.987 0.990 0.961 0.999 0.797
F5 0.972 0.993 0.965 0.998 0.654
F6 0.985 0.996 0.957 0.993 0.857

Antimicrobial Efficacy Studies
The optimized in situ gelling formulations showed antimicrobial activity when
tested microbiologically by the Cup-Plate technique. Clear zones of inhibition were
obtained in all the formulations. The diameter of zone of inhibition produced by
formulations against all test microorganisms is given in Table 5.15.
CHAPTER 5 RESULTS


Table 5.15 Antimicrobial activity of in situ gels.
Test Micro
organisms
Diameter of the Zone of Inhibition Produced By in situ Gels
(mm)
F1 F2 F3 F4 F5 F6 LEV
Staphylococcus
Aureus
24 25 25 24 23 25 26
Pseudomonas
Aeruginosa
32 27 29 30 29 31 33
E.coli

25 24 26 24 24 26 27

Ocular Irritancy Studies
Prepared in situ gelling systems were subjected for ocular irritancy studies. A
total four albino rabbits (male) weighing 1.5-2 kg was used for the present study. The
sterile formulations were instilled twice a day for a period of 7 days. Rabbits were
observed periodically for redness, swelling, watering of the eye. The evaluation was
made according to the Draize test protocol. All the formulations were found to be
non-irritating with no ocular damage or abnormal clinical signs to the cornea, iris, and
conjunctiva.
Accelerated Stability Studies

According to ICH guideline, the accelerated stability studies were carried for
prepared in situ gelling systems. All the Formulations were analyzed for visual
appearance, clarity, pH and drug remaining. 6 weeks of stability studies reveal that
there was no change in visual appearance and clarity. All the formulations showed
slight changes in pH, but it were in acceptable limits ( 0.5).Study of %drug
CHAPTER 5 RESULTS


remaining in all formulations reveals that there were no definite changes observed to
justify for drug degradation. (Tables 5.16-5.23 and Fig. 5.14-5.15).
Table 5.16 Stability studies of Formulation F1.
SL.
No
Number
of Days
Visual Appearance Clarity Ph
RT 40C RT 40C RT 40C
1 0 Transparent Transparent Clear Clear 7.12 7.12

SL.
No
Number
of Days
Visual Appearance Clarity Ph

CHAPTER 5 RESULTS


SL.
No
Number
of Days
Visual Appearance Clarity pH

SL.
No
Number
of Days

CHAPTER 5 RESULTS


SL.
No
Number
of Days

SL.
No
Number
of Days

CHAPTER 5 RESULTS


Table 5.22 Stability studies of all formulations at room temperature.
SL.No Number
of weeks
%DRUG REMAINING
F1 F2 F3 F4 F5 F6
1 0 98.01 97.66 96.08 95.29 93.43 92.97
2 1 98.01 97.66 96.02 95.16 93.23 92.85
3 2 97.95 97.56 95.92 95.03 93.12 92.73
4 3 97.78 97.34 95.83 94.97 93.01 92.61
5 4 97.67 97.22 95.70 94.86 92.87 92.45
6 5 97.57 97.02 95.45 94.72 92.46 92.12
7 6 97.24 96.78 95.23 94.44 92.34 91.87

Table 5.23 Stability studies of all formulations at 40
0
c.
SL.No Number
of weeks
%DRUG REMAINING
F1 F2 F3 F4 F5 F6
1 0 98.01 97.66 96.08 95.29 93.43 92.97
2 1 98.01 97.66 96.02 95.16 93.23 92.85
3 2 97.95 97.56 95.92 95.03 93.12 92.73
4 3 97.69 97.24 95.72 94.87 92.98 92.54
5 4 97.58 97.01 95.55 94.73 92.77 92.31
6 5 97.34 96.79 95.39 94.46 92.32 92.16
7 6 97.12 96.45 95.07 94.23 92.21 91.67

CHAPTER 5 RESULTS


Fig. 5.14 Stability studies of in situ gels at room temperature.

Fig. 5.15 Stability studies of in situ gels at 40
0
C.
CHAPTER 6 DISCUSSION


Ocular therapy could be significantly improved if the pre-corneal residence time
of drugs could be increased; several new preparations have been developed for
ophthalmic use not only to prolong the contact time of the vehicle at ocular surface
but also to slow down the elimination of the drugs.
Conventional ophthalmic solution dosage forms have advantage such as, ease of
instillation and proper dosage administration. Beside ophthalmic ointments have the
advantages of increased contact time. By utilizing these advantages of different
dosage forms the newer approach, in situ gelling system was developed. These gels
exhibit a unique property of sol-to-gel transition when a change in their
physicochemical property takes places. This type of novel ocular drug delivery can
provide increased bioavailability by increasing residence time of gel formed and
better patient compliance due to ease of administration.
The aim of the present work envisaged Preparation and evaluation of in situ
opthalmic gel of an anti infective drug for sustained ocular delivery for the treatment
of various bacterial diseases of eye by providing comfortness, compliance to the
patients and improved therapeutic performance of the drug over conventional ocular
dosage forms.
Preparation of in situ gelling Systems
In the present work the in situ gelling systems were prepared by ion exchange
and temperature dependent methods with the help of gelling agent Gelrite and
humectant propylene glycol.

6. DISCUSSION



Evaluation of in situ gelling systems
For the conformation of the intactness of the drug in formulations all
formulations were subjected to IR study and compared to IR absorption spectra of
pure drug. Studies revealed that there was no definite changes in bands were observed
with respect to pure drug. So it was confirmed that formulations did not have any drug
polymer interactions.
Optimized in situ gels were subjected for preliminary evaluation such as visual
appearance, clarity, pH and drug content. All formulations were found transparent and
clear, pH of the formulations was within 7.1 to 7.4 and drug content was found within
92-98% in all optimized in situ gelling systems.
Using simulated tear fluid a simple spectrophotometric method for estimation of
Levofloxacin hemihydrate was developed. The absorption maxima by UV
spectrophotometer were obtained at 287.5 nm in Beers range of 2-12 g/ml.
During blinking the shear rate on the preparation is large. If the viscosity is too
high, this will result in irritation. On the other hand, if the viscosity is too low, it will
give rise to increased drainage. So the formulation should have optimum viscosity for
easy instillation into the eye as liquid, which will undergo a rapid sol-to-gel transition,
hence the good gelling capacity. But administration of the formulation should
influence the pseudoplastic character of precorneal film. In order to evaluate the
rheological behavior, viscosity of the formulations before and after addition of STF
was evaluated using Brook Field viscometer (RVT MODEL). It showed that viscosity
of all formulations decreased as the shear rate increased, which indicates the character
of pseudoplastic fluid.


Further, all formulations were subjected for sterility testing using nutrient agar
media and incubated for 7 days under daily observation. This study showed that
formulations did not having any microbial contamination and was sterile.
The in vitro release studies were carried out for all formulations using
cellophane membrane and STF as the medium. Release kinetic studies of prepared in
situ gels showed that the in situ gels followed first order drug release mechanism.
Higuchi matrix equation confirmed the release by diffusion controlled mechanism.
Korsemeyer-Peppas n value of prepared in situ gels was found to be above 0.5 this
indicated that the formulation followed non-fickian diffusion controlled mechanism.
Obtained results indicated that F6 showed better sustaining effect amongst all
formulations. This may be due to the higher concentration of gelrite.
Antimicrobial efficacy study carried out by using Staphylococcus Aureus,
Pseudomonas Aeruginosa and E.coli as test microorganisms. After incubation up to
24 hours, it was found that all formulations were having effective anti microbial
action.
Lastly, formulations were evaluated for the stability studies (at RT and 34C,
755 % RH) for 42 days. Results reveal that no changes were found in visual
appearance, clarity and pH. These formulations were also analyzed for % drug
remaining. This study showed that there was no definite change observed in the
intactness of the drug after accelerated study of 42 days.
Hence from the above results we can conclude that it is possible to formulate in
situ ophthalmic gels of Levofloxacin hemihydrate using Gelrite for treatment of
various bacterial infections.

CHAPTER 7 CONCLUSION


In this study in situ opthalmic gel of Levofloxacin hemihydrate, which is broad
spectrum anti bacterial agent used in the treatment of ocular infections was prepared
by using gelrite as a release retardant, it was found that increase concentration of
gelrite decreased the decreased the drug release.
Optimized formulations F6 (0.6 % Gelrite and Propylene glycol 8%), F5 (0.5 %
Gelrite and Propylene glycol 8%), F4 (0.4 % Gelrite and Propylene glycol 8%) and F3
(0.3 % Gelrite and Propylene glycol 8%) were liquid before instillation in to eye and
underwent rapid gellation upon instillation in to eye, the formulations were found to
be clear, having good in situ gelling capacity , having drug content 92-98%, optimized
formulations were sterile and showed sustained drug release over 8 hours period as
compared to marketed eye drop, release kinetic study showed that the formulation
followed first order diffusion controlled and non fickian release mechanism, the
optimized formulations was having good antibacterial efficacy, as per the Draize test
protocol the ocular irritancy studies were carried out, results showed that formulations
were non irritant. As per ICH guidelines the stability study of formulations were
carried out results showed that formulations were stable (transparent and clear) at
room temperature as well as at 40C.

7. CONCLUSION

CHAPTER 8 SUMMARY


The aim of the present work envisaged Preparation and evaluation of in situ
opthalmic gel of an anti infective drug for sustained ocular delivery for the treatment
of various bacterial diseases of eye, by providing comfortness, compliance to the
patients and improved therapeutic performance of the drug over conventional ocular
dosage forms.
Formulation containing Gelrite (0.2-0.7 %) was prepared along with propylene
glycol a water miscible co solvent and humectant, as adjuvant in order to improve the
solubility of Levofloxacin hemihydrate.
Optimized formulations F6 (0.6 % Gelrite and Propylene glycol 8%), F5 (0.5 %
Gelrite and Propylene glycol 8%), F4 (0.4 % Gelrite and Propylene glycol 8%) and F3
(0.3 % Gelrite and Propylene glycol 8%) were liquid before instillation to the eye and
underwent rapid gelation upon instillation to the eye.
FTIR study of physical mixture of drug and polymer, prepared in situ gels was
carried out and were compared with IR absorption spectra of pure drug. Studies reveal
that there were no definite changes in bands observed with respect to pure drug. So it
was confirmed that formulations do not have any drug polymer interactions.
Optimized in situ gels were subjected for preliminary evaluation such as visual
appearance, clarity, pH and drug content. All formulations were found transparent and
clear, pH of the formulations was within 7.1 to7.4, drug content was found within
92-98% in all optimized in situ gelling systems.
In order to evaluate the rheological behavior, viscosity of the formulations
before and after addition of STF was evaluated using Brook Field viscometer. It
8. SUMMARY

CHAPTER 8 SUMMARY


showed that viscosity of all formulations decreased as the shear rate increased, which
indicates the character of pseudoplastic fluid.
Sterility testing was done by using nutrient agar media and incubated for 7 days
under daily observation. This study showed that formulations do not having any
microbial contamination and was sterile.
In vitro release of Levofloxacin hemihydrate from the selected formulations
was studied through diffusion cell using cellophane membrane for 8 hours. It was
compared with the marketed eye drop. Results reveal that all formulations exhibited
sustained release of the drug from gelrite polymeric network over 8 hours.
Release kinetic studies showed that the in situ gels followed first order drug
release mechanism. Higuchi matrix equation confirmed the release was diffusion
controlled. Korsemeyer-Peppas n value of 0.55-0.85 indicated that the formulation
followed non-fickian diffusion controlled release mechanism.
Antimicrobial efficacy study carried out by using Staphylococcus Aureus,
Pseudomonas Aeruginosa and E.coli as test microorganisms. After incubation up to
24 hours, it was found that all formulations had effective anti microbial action.
The stability study was carried out for all optimized formulations up to 42 days.
Results reveal that no changes were found in visual appearance, clarity and pH. These
formulations were also analyzed for % drug remaining; this study showed that there
were no definite changes observed in the intactness of the drug after accelerated
stability study of 42 days.

CHAPTER 9 BIBLIOGRAPHY


1. Mohan EC, Jagan Mohan K, Venkatesham A. Preparation and evaluation of in
situ gels for ocular drug delivery. J Pharm Res 2009;2(6):1089-94.
2. www.google.com Primary care ophthometry news, a guide to Topical anti
infectives.
3. Martindale. The complete drug reference, 34th ed. Pharmaceutical press. 1996.
p.127-227.
4. Hill JM, Callaghan O, Hobden RJ, Kaufman E, Mitra AK. Ophthalmic drug
delivery systems. New York, Marcel Dekker Inc; 1993. vol 58. p. 1-204.
5. Chein YW, Novel drug delivery systems, 2nd ed. New York, Marcel Dekker
Inc; 1993. vol 29. p. 269-300.
6. Bourlais CL, Acar L, Zia H, Sado PA, Needhan S, Leverge R. Ophthalmic
drug delivery systems Recent Advances. Progress in Retinal and Eye
Research 1998;17(1):33-58.
7. Lieberman HA, Rieger MM, Banker GS. Pharmaceutical dosage forms:
disperse system, 2nd ed. New York, Marcel dekker Inc; vol 2. p. 357-97.
8. Geeta MP, Madhubhai MP, Recent advances and challenges in ocular drug
delivery systems, Pharma Times 2007;39(1):21-25.
9. Peppas NA, Bures P, Leabandung W, Ichikawa H. Hydrogels in
pharmaceutical formulations, Eur J Pharm and Biophar 2000;50:27-46.
10. Sreenivas SA, Hiremath SP, Godbole AM. Ofloxacin ocular inserts: Design,
formulation and evaluation. Iranian J pharmcol 2006;5(2):159-62.
9. BIBLIOGRAPHY



11. Sasaki H, Tei C, Nishida K, Nakamura J. Drug release from an ophthalmic
insert of a beta-blocker as an ocular drug delivery system. J Contr Rel
1993;27:127-37.
12. Bawa R. Ophthalmic drug delivery systems. New York Marcel Dekker, Inc;
1993. vol 58. p. 224-48.
13. Robinson JR. Ocular evaluation of polyvinyl alcohol vehicle in rabbits. J
Pharm Sci 1975;64:1312-16.
14. Felt O, Baeyens V, Zignani M, Buri P,Gurny R. Mucosal drug delivery ocular
Encyclopaedia of controlled drug delivery. Geneva, University of Geneva.
1999. vol 2.p. 60522.
15. Soppinath KS, Aminabhavi TM, Dave AM, Kumar SG, Rudzinski WE.
Stimulus responsivesmart hydrogels as novel drug delivery systems. Drug
Dev Ind Pharm.2002;28:957-74.
16. Van M. Biopharmaceutics of ocular drug delivery, P. Edman ed, Boca Raton,
CRC press, 1993, p.27-42.
17. Wichterle O, Lim D. Hydrophilic gels for biological use. Nature
1960;185:117-18.
18. Miller SC, Donovan MD. Effect of Poloxamer 407 gel on the miotic activity
of pilocarpine nitrate in rabbits. Int J Pharm 1982;12:14752.
19. Gurny R, Boye T, Ibrahim H. Ocular therapy with nanoparticulate systems for
controlled drug delivery. J Contr Rel 1985;2: 35361.
20. Moorhouse R, Colegrove GT, Sandford PA, Baird JK, Kang KS. A new gel
forming polysaccharide, Solution Properties of Polysaccharides. ACS
Symposium series, Washington-DC 1981;11124.


21. Hoffman AS, Afrassiabi A, Dong A. Thermally reversible hydrogels: II
Delivery and selective removal of substances from aqueous solutions. J
Control Release 1986;4:21322.
22. Gariepy E.R, Leroux J.C. In situ forming hydrogels review of temperature-
sensitive systems. Eur J Pharm. Biopharm 2004;58:40926.
23. Cabana A, Ait-Kadi A, Juhasz J. Study of the gelation process of polyethylene
oxide-polypropylene oxide polyethylene oxide copolymer (Poloxamer 407)
aqueous solutions. J Colloid Interface Sci 1997;190:30712.
24. Harrington WF, Von Hippel WF. The structure of collagen and gelatin. Adv.
Protein Chem 1961;1:1138.
25. Shirakawa M, Yamatoya K, Nishinari K. Tailoring xyloglucan properties
using an enzyme. Food Hydrocoll 1998;22:258.
26. Miyazaki S, Suisha F, Kawasaki N, Shirakawa M, Yamatoya K, Attwood D.
Thermally reversible xyloglucan gels as vehicles for rectal drug delivery.
J Contr Rel 1998;56:7583.
27. Gariepy ER, Leroux JC. In situ forming hydrogels reviews of temperature-
sensitive systems. Eur J Pharm. Biopharm 2004;58:40926.
28. Lin HR, Sung KC. Carbopol/pluronic phase change solutions for ophthalmic
drug delivery. J Contr Rel 2000;69:379.
29. Boye T, Gurny R, Ibrahim H. Ocular therapy with nanoparticulate systems for
controlled drug delivery. J Contr Rel 1985;2:353-61.
30. Le Bourlais CA, Treupel-Acar L, Rhodes CT, Sado PA, Leverge R. New
ophthalmic drug delivery system. Drug Dev Ind Pharm 1995;21:19-59.
31. Hui HW, Robinson JR. Ocular drug delivery of progesterone using of
bioadhesion polymer. Int J Pharmaceut Sci 1985;26:203-13.


32. Davis NM, Farr SJ, Hadgraft J, Kellaway IW. Evaluation of mucoadhesive
polymers in ocular drug delivery: Part 1, viscous solution. Pharm Res
1991;8(8):1039-43.
33. Srividya B, Cardoza RM, Amin PD. Sustained ophthalmic delivery of
ofloxacin from a pH triggered in situ gelling system. J Contr Rel
2001;73:205-11.
34. Kumar S, Haglund BO, Himmelstein KJ. In situ forming gels for ophthalmic
drug delivery. J Ocul Pharmacol 1994;10:47-56.
35. Thilak kumar M, Bharathi D, Balasubramaniam J, Kant S, Pandit JK. pH

induced in situ gelling of Indometacin for Sustained ocular delivery. Ind J
Pharm Sci 2005;67(3):327-33.
36. Doijad RC, Manvi FV, Malleswara VSN, Prajakta Alase. Sustained
ophthalmic delivery of Gatifloxacin from in situ gelling system. Ind J Pharm
Sci 2006;68(6):809-14.
37. Bhardwaj TR, Kanwar M, Lal R, Gupta A. Natural gums and modified natural
gums as sustained release carriers. Drug Dev Ind Pharm 2000;26:1025-38.
38. Jansson PE, Lindberg B. Structural studies of gellan gum, an extracellular
polysaccharide elaborated by Pseuomonas elodea. Carbohydr. Res
1983;124: 13539.
39. Kuo MS, Mort AJ, Dell A. Identification and location of L-glycerate, an
unusual acyl substituent in gellan gum. Carbohydr. Res 1986;156: 17387.
40. Morris VJ, Stephen AM. Bacterial polysaccharides, in food polysaccharides
and their application. Marcel Dekker, New York, 1995; 34175.


41. Rozier A, Mazuel C, Grove J, Plazonnet B. Gelrite: A novel ion activated in
situ gelling polymer for ophthalmic vehicles. Effect on bioavailability of
timolol. Int J Pharm 1989;57:16368.
42. Kang KS, Veeder GT, Mirrasoul PJ, Kaneko T, Cottrell IW, Agarlike.
Polysaccharide produced by a Pseudomonas species: production and basic
properties. Appl Environ Microbiol 1982;43:108691.
43. Maurice DM, Srinivas SP. Use of fluorometry in assessing the efficacy of a
cation-sensitive gel as an ophthalmic vehicle: comparison with scintigraphy.
J Pharm Sci 1992;81:61518.
44. Meseguer G, Gurny R, Buri P, Rozier A, Plazonnet B. Gamma scintigraphic
study of precorneal drainage and assessment of miotic response in rabbits of
various ophthalmic formulations containing Pilocarpine. Int J Pharm.
1993;95:22934.
45. Haug A, Larsen B, Smidsrod OA. study of the constitution of alginic acid by
partial hydrolysis. Acta Chem Scand 1966;20:18390.
46. Painter TJ, Smidsrod O, Haug A. A computer study of the changes in
composition distribution occurring during random depolymerisation of a
binary linear heteropolysaccharide. Acta Chem Scand 1968;22:163748.
47. Larsen B, Smidsrod O, Painter TJ, Haug A. Calculation of the nearest
neighbor frequencies in fragments of alginate from the yields of free
monomers after partial hydrolysis. Acta Chem Scand 1970;24:72628.
48. Brittain HG. Analytical profiles of drug substances and excipients. Elsevier
23:321-61.
49. Lindell K, Engstrom S. In vitro release of Timolol from an in situ gelling
polymer system. Int J Pharm 1993;95(1-3):219-28.


50. Kumar S, Himmelstein KJ. Modification of in situ gelling behavior of
carbopol solutions by HPMC. J Phar Sci 1995;84(3):344-8.
51. Cohen S, Lobel E, Trevgoda A, Peled Y. A Novel in situ forming ophthalmic
drug delivery system from alginates undergoing gelation in the eye. J contr rel
1997;44(2-3):201-8.
52. Ding S. Recent Developments in ophthalmic drug delivery. Pharmaceutical
sciences and technology Today 1998;1(8):328-35.
53. Dimitrova E, Bogdanova SV, Mitcheva M, Tanev I, Minkov E. Development
of model aqueous ophthalmic solution of indomethacin. Drug Devel Ind
Pharm 2000;26(12):1297-01.
54. Sechoy O, Tissie G, Sebastian C, Maurin F, Driat J, Trinquand C. A new long
acting ophthalmic formulation of carteolol containing alginic acid. Int J Pharm
2000;207:109-16.
55. El-Kamel AH. In vitro and in vivo evaluation of pluronic F127-based ocular
delivery system for timolol maleate. Int J Pharm 2000;241:47-55.
56. Charoo NA, Kohil K, Ali A. Preparation of in situ forming ophthalmic gels of
ciprofloxacin HCL for the treatment of conjunctivitis: in vitro and in vivo
studies. J Phar Sci 2002;92(2):407-13
57. Sultana Y, Aquil M, Ali A, Zafar S. Evaluation of carbopol-methyl cellulose
based sustained release ocular delivery for pefloxacin mesylate using rabbit
eye model. Pharma Dev Technol 2006;11(3):313-9.
58. Kulkarni MC, Damle AV. Development of ophthalmic in situ gelling
formulation of flurbiprofen sodium using gellan gum. Ind Drugs
2007;44(5):373-7.


59. Kugalur Ganesan Parthiban, Rangasamy Manivannan, Balasubramanium
Senthil Kumar, Mirza Beg Ahasan. Formulation and evaluation of Ketorolac
ocular pH-triggered in situ gel Int J Drug Dev and Res 2010;2(2):379-87.
60. Shivanand Swamy PH, Fatima sanjeri dasankoppa, Abidabegum N, Vilas GJ.
Formulation and evaluation of novel in situ gum based ophthalmic drug
delivery of Linezolid. Sci Pharm 2008;76:515-32.
61. Kaur IP, Singh M, Kanwar M. Formulation and evaluations of ophthalmic
preparations of Acetazolamide. Int J Pharm Sci 2000;199(2):119-27.
62. Divyesh HS, Shailesh T, Prajapathi, Rajesh K, Parikh, Lakshmanbhai D Patel.
Studies on poloxamer based muco adhesive ophthalmic in situ hydrogel of
Moxifloxacin Hcl. Int J Pharm Res 2009;1(3):77-86.
63. Sindhu Abraham, Sharoon furtado, Bharath S, Basavaraju BV, Devaswaran K,
Madavan V. Sustained ophthalmic delivery of Ofloxacin from an ion activated
in situ gelling system. Pak J Pharm Sci 2009;22(2):175-79.
64. Sirish Vodithala, Sadhna Khatry, Nalini Shastri M. Sadanandam. Formulation
and evaluation of ion activated ocular gels of Ketorolac tromethamine. Int J
Curr Pharm Res 2010;2(3):31-8.
65. Carlfors j, Edsman K, Peterson R, Jornving K. Rheological evaluation of in
situ gels. Eur j pharm sci 1998;6:113-19.
66. Coquelet C, Lakhchaf N, Persin M, Rao LS, Sarrazin J. Association between
benzalkonium chloride and poly (Acrylic Acid) gel study by microfiltration
and membrane dialysis. J Membrane Sci 1996;120(2):287-93.
67. Furrer P, Plazonnet B, Mayer JM, Gurny R. Application of in vivo confocal
microscopy to the objective evaluation of ocular irritation induced by
surfactants. Int J Phar 2000;207:89-98.


68. www. Drug bank.com.
69. www.gold bio.com.
70. Raymond C Rowe, Paul J Sheskey. Handbook of pharmaceutical excipients,
Pharmaceutical press London. 2009. vol 6.p.568-94.
71. Amal El-kamel, Heba Al-Dosari, Fahad Al-Jenoobi. Environmentally
responsive ophthalmic gel formulation of carteolol hydrochloride. Drug Del
2006;13:55-9.
72. Farah Siddiqui, Abul Kalam, Nayyar Parvez, Suman Yadav, Yasmin Sultana,
Asgar Ali. Gellan-based systems for sustained ophthalmic delivery of
ofloxacin. Continental J Pharmaceutical Sciences 2008;2:114.
73. Bothner H, Waaler T, Wik O. Rheological characterization of tear substitutes.
Drug Dev Ind Pharm 1990;16:75568.
74. Controller of Publication. Indian Pharmacopoeia, Ministry of Health and
Family Welfare, Government of India, New Delhi1996. vol2. p. A117-47.
75. Padma Preetha J, Karthika K, Rekha NR, Khalid Elshafie. Formulation and
evaluation of in situ ophthalmic gels of Diclofenac sodium. J Chem Pharm
Res 2010;2(3):528-35.
76. Unlu N, Ludwig A, Van OM, Hincal AA. Formulation of carbopol 940
ophthalmic vehicles and in vitro evaluation of the influence of simulated
lacrimal fluid on their physico chemical properties. Pharmazie 1991;
46(11):784-8.
77. Draize J, Woodward G, Calvery O. Methods for the study of irritation and
toxicity of substance applied topically to the skin and mucous membrane. J
Pharmacol Exp Ther 1994;82:37790.


78. Michael H, Mostafa H, Mehdi J, Taravat G. Draize Rabbit eye test
compatibility with eye irritation threshold in humans: A quantitative
structural-Activity relationship analysis. Toxicol Sci 2003;76:38491.

CHAPTER 10 ANNEXURES

LIST OF PUBLICATIONS
Review article
1) Nittur Jayaprakash Rajas*. Kunchu Kavitha, Theetha Gounder Tamizh Mani.
In situ opthalmic gels: a developing trend. International journal of pharmaceutical
research and review (Accepted).
Research articles
2) Kavitha K, Rajas N.J*. Sustained Ophthalmic Delivery of Levofloxacin
hemihydrate from An Ion Activated in Situ Gelling System .International journal
of Pharmatech research (Communicated).
3) Kavitha K, Rajas N.J*. More mangesh rajendra, Rupesh kumar M, Tamizh mani
T. Environmentally responsive in situ ophthalmic gel containing Levofloxacin
hemihydrate. Drug development and industrial pharmacy (Communicated).

10. ANNEXURES

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