Thermo-Reversible Subcutaneous PH.D Thesis

DEVELOPMENT AND EVALUATION OF
THERMO-REVERSIBLE SUBCUTANEOUS AND

OPHTHALMIC DRUG DELIVERY SYSTEM
Ph.D Thesis
By
FAZLI NASIR
DEPARTMENT OF PHARMACY
UNIVERSITY OF PESHAWAR
2012
DEVELOPMENT AND EVALUATION OF
THERMO-REVERSIBLE SUBCUTANEOUS AND
OPHTHALMIC DRUG DELIVERY SYSTEM
Ph.D Thesis
By
FAZLI NASIR
THESIS SUBMITTED TO UNIVERSITY OF PESHAWAR IN

PARTIAL FULFILLMENT FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY IN PHARMACY
DEPARTMENT OF PHARMACY
UNIVERSITY OF PESHAWAR
2012
Table of contents
ABSTRACT ................................................................................................................................... 1
1. INTRODUCTION ................................................................................................................ 4
2. BACK GROUND ................................................................................................................... 9
2.1 Drug Delivery System ............................................................................................................... 9
2.2 Delayed Release ......................................................................................................................10
2.3 Extended Release ....................................................................................................................11
2.4 Site Specific Targeting..............................................................................................................11
2.5 Receptor Targeting ..................................................................................................................11
2.6 Fast Dissolve Drug Delivery System (Flash)...............................................................................12
2.7 Drug Modification ...................................................................................................................13
2.8 Dosage Form Modification.......................................................................................................13

2.8.1 Reservoir Devices .........................................................................................................................13
2.8.2 Matrix Devices .............................................................................................................................13
2.8.3 Hybrid System ..............................................................................................................................14
2.9 Ophthalmic Drug Delivery:.......................................................................................................15

2.9.1 Eye ...............................................................................................................................................15
2.9.1.1 The Outer Layer...................................................................................................................16
2.9.1.2 The Inner Layer ...................................................................................................................18
2.9.1.3 Drug Kinetics through Eye ...................................................................................................18
2.10 Ocular Routes ..........................................................................................................................19

2.10.1 Topical Ocular ..........................................................................................................................19
2.10.2 Sub-conjunctival ......................................................................................................................20
2.10.3 Intra-vitreal ..............................................................................................................................20
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2.11 Challenges in Ocular Drug Delivery ..........................................................................................21
2.11.1 Ophthalmic Dosage Forms ...................................................................................................... 23
2.11.1.1 Aqueous Solutions...............................................................................................................23
2.11.1.2 Suspensions .........................................................................................................................23
2.11.1.3 Ointments ...........................................................................................................................24
2.11.1.4 Pre-Formed Gels and Muco-adhesive Polymer Systems.....................................................24
2.11.1.5 Gel Forming Liquids.............................................................................................................24
2.11.1.6 Non-erodible Ocular Inserts ................................................................................................25
2.11.1.7 Prodrugs ..............................................................................................................................25
2.11.1.8 Microspheres and Nano-particles .......................................................................................26
2.12 Subcutaneous Drug Delivery .................................................................................................... 26
2.13 Parenteral Depot System .........................................................................................................29

2.13.1 Reasons for development of PDS (Parenteral Depot System).................................................29
2.13.1.1 Advantages:.........................................................................................................................29
2.13.1.2 Disadvantages: ....................................................................................................................30
2.13.1.3 Types of Depot Formulations: .............................................................................................30
2.13.2 Injectable Drug Delivery System.............................................................................................. 30
2.13.2.1 Emulsions ............................................................................................................................30
2.13.2.2 Liposomes ...........................................................................................................................30
2.13.2.3 Microspheres: .....................................................................................................................31
2.13.2.4 Micelles ...............................................................................................................................32
2.13.2.5 Thermoplastic Pastes .......................................................................................................... 32
2.13.2.6 Thermosets .........................................................................................................................33
2.13.3 Stimuli Sensitive Solution to Gel..............................................................................................34
2.13.3.1 Temperature Sensitive Hydrogels .......................................................................................34
2.13.3.2 pH Sensitive Hydrogels........................................................................................................ 34
2.13.3.3 In situ Solidifying Organogels ..............................................................................................35
2.13.4 Polymers in Drug Delivery System ...........................................................................................35
2.13.4.1 Classification of Polymers ................................................................................................... 36
2.13.4.2 Properties of Polymers as Candidate for Controlled Release .............................................40
2.14 Hydrogels ................................................................................................................................41

2.14.1 Environmental Sensitive Hydrogels .........................................................................................42
2.14.1.1 Temperature sensitive hydrogels ........................................................................................42
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2.14.1.2 pH Sensitive Hydrogels........................................................................................................ 42
2.14.1.3 Electric Signal Sensitive Hydrogels ......................................................................................42
2.14.1.4 Light Sensitive Hydrogels .................................................................................................... 42
2.14.1.5 Ion Sensitive Hydrogels ....................................................................................................... 42
2.14.1.6 Pressure Sensitive Hydrogels ..............................................................................................42
2.14.1.7 Temperature Sensitive Hydrogels .......................................................................................43
2.14.2 Pluronic (Poloxamers) .............................................................................................................44
2.14.3 Methyl Cellulose (MC) .............................................................................................................50
2.14.3.1 Biodegradation ....................................................................................................................51
2.14.3.2 Thermo-gelation..................................................................................................................51
2.14.3.3 Applications of Methyl Cellulose.........................................................................................52
2.14.4 Hydroxy propyl methyl cellulose (HPMC) ................................................................................53
2.14.4.1 Thermo-gelation..................................................................................................................54
2.14.4.2 Applications of Hydroxy propyl methylcellulose (HPMC) ...................................................55
2.14.5 Diclofenac Sodium ...................................................................................................................56
2.14.5.1 Chemical Name ...................................................................................................................56
2.14.5.2 Molecular Formula and Structure .......................................................................................56
2.14.5.3 Mechanism of Action .......................................................................................................... 56
2.14.5.4 Uses .....................................................................................................................................56
2.14.5.5 Dosage and Administration .................................................................................................57
2.14.5.6 Pharmacokinetics ................................................................................................................57
2.14.6 Timolol Maleate.......................................................................................................................60
2.14.6.1 Chemical Formula and Structure.........................................................................................60
2.14.6.2 Molecular Weight................................................................................................................60
2.14.6.3 Description ..........................................................................................................................60
2.14.6.4 Melting Point.......................................................................................................................60
2.14.6.5 Solubility..............................................................................................................................61
2.14.6.6 pH ........................................................................................................................................61
2.14.6.7 Uses .....................................................................................................................................61
2.14.6.8 Dosage.................................................................................................................................61
2.14.6.9 Pharmacokinetics ................................................................................................................61
2.14.6.10 Contraindication ............................................................................................................. 62
2.14.6.11 Side Effects......................................................................................................................62
2.14.6.12 Drug interactions ............................................................................................................ 62
2.14.7 Insulin ......................................................................................................................................63
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2.14.7.1 Insulin Production and Secretion ........................................................................................63
2.14.7.2 Effect of Insulin ...................................................................................................................64
2.14.7.3 Insulin Regulation................................................................................................................65
3. MATERIALS AND METHODS ........................................................................................ 68
3.1 Materials .................................................................................................................................68
3.2 Instrumentation ......................................................................................................................68
3.3 Study Design ...........................................................................................................................69

3.3.1 Preparation of Polymer Solutions ................................................................................................70
3.3.2 Measurement of the Sol–Gel Transition Temperature (Tsol-gel) ................................................72
3.3.3 Measurement of Steady Shear Viscosity .....................................................................................72
3.3.4 Clarity of the Formed Gel .............................................................................................................73
3.3.5 Autoclaving ..................................................................................................................................73
3.3.6 Preparation of Drug Polymer Solutions .......................................................................................73
3.3.7 Preparation of Diclofenac Sodium In-situ Gel Formulations .......................................................73
3.3.8 Preparation of Timolol Maleate In-situ Gel Formulations ...........................................................74
3.3.9 Preparation of Human Insulin (recombinant) In-situ Gel Formulations ......................................75
3.3.10 Measurement of Tsol- gel DS Formulations ............................................................................76
3.3.11 Measurement of Steady Shear Viscosity of Diclofenac Sodium Formulations ........................77
3.3.12 Clarity of the Formed Gel of DS Formulations .........................................................................77
3.3.13 Drug Content of DS Formulations............................................................................................77
3.3.14 Determination of In-vitro of Drug Release from DS Formulations ..........................................77
3.3.15 Measurement of Tsol- gel of TM Formulations .......................................................................78
3.3.16 Measurement of Steady Shear Viscosity of TM Formulations ................................................78
3.3.17 Clarity of the Formed Gel of TM Formulations........................................................................79
3.3.18 Drug Content of TM Formulations ..........................................................................................79
3.3.19 Determination of In-vitro of Drug Release from TM Formulations .........................................79
3.3.20 Measurement of Tsol- gel Insulin Preparations ......................................................................80
3.3.21 Measurement of Steady Shear Viscosity of Insulin Preparations ............................................80
3.3.22 Clarity of the Formed Gel of Insulin Preparations ...................................................................81
3.3.23 Drug Content of Insulin Preparations ......................................................................................81
3.3.24 Determination of In-vitro Drug Release from Insulin Preparations ........................................81
3.3.25 Drug Release Kinetics ..............................................................................................................82
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3.3.25.1 Zero Order Kinetic Model....................................................................................................82
3.3.25.2 First Order Kinetic Model .................................................................................................... 82
3.3.25.3 Higuchi Model .....................................................................................................................83
3.3.25.4 Hixson –Crowell Model .......................................................................................................83
3.3.25.5 Korsmeyer-Peppas Model ...................................................................................................83
3.3.26 In Vivo Evaluation of Formulations..........................................................................................84
3.3.26.1 Animal Handling ..................................................................................................................84
3.3.26.2 Diclofenac Sodium Administration and Sampling ...............................................................84
3.3.26.3 Timolol Maleate Administration and Sampling...................................................................85
3.3.26.4 Insulin Administration and Sampling ..................................................................................85
3.3.27 Pharmacokinetic Parameters ..................................................................................................86
3.3.28 HPLC analysis method .............................................................................................................86
3.3.28.1 Materials and methods .......................................................................................................86
3.3.28.2 Preparation of Standard Stock Solutions ............................................................................87
3.3.28.3 Sample Preparation.............................................................................................................87
3.3.28.4 Liquid-Liquid Extraction ...................................................................................................... 88
3.3.28.5 Chromatographic Conditions ..............................................................................................89
3.3.28.6 Chromatographic Conditions and Experimental Parameters Optimization ........................90
3.3.28.7 Validation of the Method ....................................................................................................91
4. RESULTS AND DISCUSSION ......................................................................................... 97
4.1 HPLC –UV Method for Analysis of Diclofenac Sodium and Timolol Maleate ..............................97
4.1.1 Optimization of HPLC Experimental Parameters .........................................................................98
4.1.1.1 Mobile Phase.......................................................................................................................98
4.1.1.2 Stationary Phase .................................................................................................................99
4.1.1.3 Flow Rate.............................................................................................................................99
4.1.1.4 Column Oven Temperature...............................................................................................100
4.1.1.5 pH ......................................................................................................................................100
4.1.1.6 Wave Length .....................................................................................................................101
4.1.1.7 Internal Standard ..............................................................................................................102
4.1.1.8 Sample Preparation........................................................................................................... 102
4.1.1.9 Method Validation ............................................................................................................ 103
4.2 Physical Evaluation of In-situ Solution to Gel Formulations .................................................... 108

4.2.1 Measurement of the Sol-Gel Transition Temperatures (Tsol-gel) .............................................108
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4.2.2 Measurement of Rheological Parameters .................................................................................110
4.2.3 Clarity of the Formed Gel ...........................................................................................................112
4.2.4 Autoclaving ................................................................................................................................113
4.2.5 In-vitro Evaluation of In-situ Drug Formulations .......................................................................114
4.2.5.1 In-vitro Evaluation of In-situ Diclofenac Sodium Sol-Gel Formulations ............................114
4.2.5.2 In vitro Drug Release From DS In-situ Gels........................................................................117
4.2.5.3 Drug Release Kinetics from DS In-situ Gels .......................................................................121
4.2.5.4 In-vitro Evaluation of In-situ TM sol-gel Formulations......................................................131
4.2.5.5 Drug Release Kinetics from TM In-situ Gels ......................................................................137
4.2.5.6 In-vitro Evaluation of In-situ Insulin Sol-Gel Formulations ...............................................145
4.2.6 In-Vivo Evaluation of In-Situ Thermoreversible Gels .................................................................160
4.2.6.1 In-vivo Evaluation of DS In-situ gels ..................................................................................160
4.2.6.2 Subcutaneous Route .........................................................................................................160
4.2.6.3 Ocular Route .....................................................................................................................169
4.2.6.4 In-vivo Evaluation of Timolol Maleate In-situ Gels ...........................................................178
4.2.6.5 In-vivo Evaluation of Insulin In-situ Gels ...........................................................................187
5. CONCLUSION ................................................................................................................. 193
6. REFERENCES .................................................................................................................. 197
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DEDICATION
I would like to dedicate this thesis to my beloved mother who was
spring of spiritual, religious and moral support for me throughout
my life.
I also dedicate this thesis to my wife, my daughters Salwa, Meerab,
Aroosh and my son Muhammad for their sufferings and support.
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ACKNOWLEDGMENTS
All the praises to almighty Allah subhanatallah who gave me courage, knowledge,
patience and health to complete my Ph. D research work.
It is difficult to acknowledge everyone who has helped me over the last 5 ½ years in my
research work in earning my Ph.D. The work was difficult and I could not have done it
without their support and help.
First and foremost, I wish to thank my wife and family who have supported and prayed
for me throughout my research work. They have been steadfast and loving, made my
difficulties less difficult, and always been there when I needed them.
I am more than grateful to my supervisor Prof. Dr. Zafar Iqbal, B. Pharm., M. Pharm.
(Pak.), Ph.D, Post Doc. (UK). He is the reason that I decided to pursue a doctorate degree
and without his influence I would not be where I am now. From him I have learned the
value of continued excellence, education, and personal development throughout my
career.
I would like to thank the GSC and ASRB members; they have been invaluable mentors
in my doctorate progress. They have also been trusted advisors with me in my personal
endeavors and future career plans. I value their support and counsel and hope to be able
to continue our relationship for many years.
viii
I am also thankful to the HEC Pakistan for promoting the culture of higher education and
research. I am also grateful to the University of Peshawar for the financial support by
funding the research project and for providing all the facilities required. I am also
thankful for the support and prayers of the faculty members of the Department of
Pharmacy University of Peshawar.
I am thankful to whole of my research group and laboratory fellows Mr. Amirzada, Dr.
Abad Khan, Dr. Imran Khan, Mr. Yasir Shah, Mr. Latif Ahmed, Mr. Muhammad
Ismail, Mr. Amanullah, Ms. Naila, Ms. Shabnum Nazir, Ms. Saira, Mr. Abuzar
Khan, Mr. Muhammad Ismail, Mr. Fazli Khuda, Dr. Roohullah, Dr. Akhlaq
Ahmed, Mr. Abbas Khan, Mr. Khalid Javed and Mr. Hassan Qayum, for their
support and help. It was a pleasure to have you as colleagues and friends.
My thanks also go to Mr. Muzaffar Abbas, Mr Salar Ahmed and Mr. Gohar Ali who
helped me in carrying out the In-vivo studies. I have to say special thanks for their help
during my experimental work to Mr. Aman Gul, Mr. Ihtesham, Mr. Inamullah and
Mr. Saeed Shah. I am also thankful to the laboratory and clerical staff of the Department
of Pharmacy University of Peshawar for their co-operation.
In the last but not the least I wish to thank my parents who instilled in me perseverance
and faith in my abilities. I did not heed their advice until later in my life, but not too late
to make a difference. I am also grateful to my brothers, sisters and relatives who prayed
and supported me and shared the family responsibilities during my Ph. D studies.
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ABBREVIATIONS
AC Autoclaving
ACE Angiotensin converting enzyme
AH Aqueous Humour
API Active pharmaceutical ingredient
AUC Area under curve
AUMC Area under moving curve
CCD Charge coupled device
CL Clearance
Cmax Peak plasma concentration
CMC Critical micelle concentration
Cps Centipoise
CV Co-efficient of variation
DNA De-oxy ribonucleic acid
DS Diclofenac Sodium
DSC Differential scanning calorimetry
EDTA Ethylene diaminotetraamino acetic acid
G-CSF Glycosylated granulocyte colony stimulating factor
GI Gastro intestinal
GLUT Glutathione
HLB Hydrophilic lipophilic balance
HPLC High pressure liquid chromatography
HPMC Hydroxy propylmethyl cellulose
Hr Hour
IS Internal standard
IU International unit
LCST Lower critical solution temperature
LOD Limit of detection
LOQ Limit of quantification
x
MC Methyl cellulose
Min Minutes
ml Milliliter
MRT Mean residence time
MVL Multi vesicle liposomes
NCI Network chromatography interface
nm Nanometer
PCL poly(D,L-lactide) and poly(Ί-caprolactone)
PEG Polyethylene glycol
PEO Poly ethyleneoxide
pGH Porcine growth hormone
PK Pharmacokinetic
Pl Pluronic
PPO Poly propyleneoxide
PVC Polyvinyl chloride
RNA Ribonucleic acid
Rpm Revolution per minute
RSD Relative standard deviation
RST Rosuvastatin
S/N Signal to noise ratio
SD Standard deviation
TM Timolol Maleate
Tmax Time to achieved Cmax
UCST Uper critical solution temperature
UV Ultra violet
Vd Volume of distribution
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ABSTRACT
ABSTRACT
The aim and objective of the study was to develop and evaluate biodegradable polymer
drug delivery system that is capable of extended the drug release for prolong period of
time at predetermined rate and being a free flowing solution at room temperature and
converts to gel with increase in temperature.
In the current study four polymers (Poloxamer 407, Methyl cellulose, Hydroxypropyl
methylcellulose and polyethylene glycol) were used alone and in combination with each
other in different ratios. Pluronic PF-127 (Poloxamer 407) and methylcellulose (MC) gels
below 37oC at appropriate concentrations (P= 18% w/v, 20%w/v, MC=4%w/v,
PM=15/3%w/v, MPG3/2w/v and MPG1.5/10% w/v).
To evaluate the efficacy of the in-situ thermoreversible formulations in controlling the
drug release three drugs diclofenac sodium (hydrophobic), timolol maleate (hydrophilic)
and insulin (protein) were selected. The in-situ gels were evaluated for their physical
properties like Tsol-gel, viscosity, clarity of solution and gel. The drug delivery system
was also evaluated for drug content, in-vitro drug release and pharmacokinetic
parameters were also determine with in-vivo studies. The data obtained for drug content
after autoclaving the solutions indicates that autoclaving results in degradation of DS
while it has no significant effect on TM.
1
ABSTRACT
To predict the drug release kinetics and mechanism various mathematical models were
applied to the in-vitro dissolution data. To predict the pharmacokinetic parameters Pk-
Summit software was used.
Based upon the in-vitro and In-vivo data it was concluded that the system consisting of
the poloxamer 407 in concentration of 20% (DP20 and TP20) are the most capable
formulation for extending the drug release and maintaining therapeutic blood level of DS
and TM for longer duration. While the formulation IPM15/3 consisting of 15%w/v
poloxamer 406 and 3% MC w/v retarded the drug release and maintaining the plasma
insulin in steady state for longer duration of time.
The results obtained in current study suggests that the HPLC-UV method developed is
sensitive and rapid method for the determination of DS and TM in pharmaceutical
preparation and physiological fluids (plasma, AH). The data also suggests that the studied
polymers Poloxamer, MC and PG are good candidate to extend the drug release
possessing a unique thermoreversible property.
The physical data obtained during the experimental work indicates that the studied in-situ
thermorevesible sol-gel formulations were capable to retard the release of these drugs.
The drug delivery systems were smart enough to be used for the administration of the
studied drugs through subcutaneous and ophthalmic routes.
2
CHAPTER 1 INTRODUCTION
CHAPTER 1
INTRODUCTION
3
1. Introduction
“The delivery of a drug at predetermined rate and/or location according to the needs of
body and disease for definite time period is termed as controlled drug delivery”[1].
Various approaches have been adopted for controlling the release of the drug. The
common approaches are Drug modification and Dosage form modification[2].
Modification of dosage form is one of the most common approaches to retard the release
of the drug from the dosage form and thus extending the effective time period.
The applications of the polymers in pharmaceutical sciences particularly in drug delivery
systems and formulations are well known [3]. Some polymers separate from solution and
convert to gel upon solidification above a certain temperature. Natural polymers have
been used as food processing aids as well as in pharmacy. Thermo-reversible gelation has
been reported for gelatin and agarose [3-6].
Cellulose is water insoluble, but its hydrophilic derivatives are water soluble. When
cellulose derivatives have an optimum balance of hydrophilic and hydrophobic moieties,
they undergo a sol-to-gel transition in water. The sol–gel transition temperature depends
on the substitution of cellulose at the hydroxy group[7], Methyl cellulose and
Hydroxypropyl cellulose are typical examples[7]. With rise in temperature of solution of
these polymers the water becomes a poorer solvent and polymer–polymer interactions
become dominant resulting in a gel[8].
4
This transition temperature is known as the lower critical solution temperature (LCST).
Below this temperature, the polymers are water soluble. Above the LCST, these polymers
become hydrophobic and water insoluble that results in solidification [9]. Various
techniques such as spectroscopy [10], differential scanning calorimetry (DSC) and
rheology [9] can be employed to verify the sol–gel transition of such thermo-reversible
polymeric solutions [10].
Many polymers show a decrease in solubility as their hydrophobicity increases upon
temperature change. In such type of polymer solutions, three types of interactions are
observed, between the molecules of polymer, between water and polymer molecules and
between the molecules of water. For polymers exhibiting LCST, rise in temperature
results in a negative free energy of the system that decreases the interaction between
water and polymer molecules, facilitating rise in the other two types of associations. This
negative free energy is result of the increase in entropy. Due to water–water interactions
which are the governing associations in the system the entropy increases. This
phenomenon is called hydrophobic effect [10-13]. As a result of the increase in
hydrophobicity the polymer chains are linked by physical reversible linkage, and gels can
therefore return to solution after the temperature stimulus causing gelation is
removed[13].
The biodegradable thermo-reversible polymers have extensively been studied due to their
great compatibility, degradability within the biological system and temperature
sensitivity. Therefore, these polymers have been investigated as controlled in situ gel-
5
forming drug delivery system [14-17]. Different polymers like Poly (ethylene glycol)-
poly (propylene glycol)-poly(ethylene glycol) copolymer (PEG-PPG-PEG), known as
Poloxamer or Pluronic, has extensively been studied as a potential thermo-reversible drug
delivery system [9, 18].
Ketoprofen and spironolactone release kinetics were studied from Pluronics [19]. The
hydrophobicity of the drug markedly affected the drug release profile. The release of
more hydrophilic ketoprofen was maintained continuously over 2 weeks, and the release
rate was controlled by the initial polymer concentration. The more hydrophobic
spironolactone release was maintained over 2 months [19].
Block copolymers have been investigated intensively for the solubilization and
stabilization of water-insoluble drugs, such as cyclosporin A and paclitaxel, and various
protein pharmaceuticals, including Zn-insulin, porcine growth hormone (pGH), and
glycosylated granulocyte colony stimulating factor (G-CSF) [20].
This unique sol-to-gel transition has made the system attractive as an injectable drug
delivery system in an in situ gel-forming drug depot. Most applications are based on
Poloxamer PF-127 and include delivery of protein/peptide drugs, such as insulin,
epidermal growth factor, urease, interleukin-2, bone morphogenic protein, fibroblastic
growth factor and endothelial cell growth factor. Most release profiles show sustained
release kinetics over several hours [21-25]. Poloxamers have been suggested for use as an
ocular drug delivery carrier of pilocarpine, but an animal study of PF-127 showed marked
6
destruction of the retina [26]. The addition of poly(ethylene glycol) (PEG) or (poly vinyl
pyrrolidone) (PVP) accelerated pilocarpine release, while the addition of methylcellulose
slowed the release rate [27].
The solutions of these polymers are liquid at room temperature but when introduced into
the body will transform into gel, this phenomenon is known as sol-gel transition. It is
easy to manufacture, package and administer the drug quantitatively using liquid dosage
form compared with the gel/semisolid dosage form. Therefore, these polymers are useful
for the drug delivery through topical, subcutaneous, ocular routes. These formulations
may sustain the delivery of the drug for long period of time that will improve the
patient’s compliance.
7
CHAPTER 2 BACKGROUND
CHAPTER 2
BACKGROUND
8
2. Back ground
2.1 Drug Delivery System
Active Pharmaceutical Ingredient (API) is rarely administered solely as pure substance
but almost always given in the form of dosage form or delivery system. The drug delivery
system is formulated invariably using drug and excipient(s) in appropriate proportions.
Drug delivery is the technique or process of administering active pharmaceutical
ingredient to achieve a therapeutic response in humans or animals [28]. The objective of
any drug delivery system is to make available therapeutic amount of drug to proper site in
the physiological system. Principally a dosage form is formulated to achieve predictable
therapeutic response of the drug included in the formulation. The drug delivery system
can be classified as:
 Conventional drug delivery system
 Modified or controlled drug delivery system.
It has been recognized that conventional drug delivery system for instance simple tablets,
solution and injections may not be the best mode of drug administration. To improve the
compliance towards drug administration and reduce the dosage fluctuation, more efforts,
novelty and innovations have been entrusted to designing effective delivery systems,
specially controlled drug delivery system. Significant milestones have been achieved in
the field of controlled drug delivery as a result of collaborative research work between
chemists, polymer scientists, engineers, pharmacologists, engineers and medical
researchers [29].The objectives of drug delivery system includes;
9
 Spatial Placement: drug targeting to specific organ
 Temporal delivery: Controlling the rate of drug delivery to target site, thus
maintaining drug concentration.
The conventional delivery system has potential problems these are;
 Lack of specific targeted delivery (temporal delivery)
 Repeated dosage after specific interval. If the interval is not proper there will be
variable troughs and peaks.
 Patient non compliance
 Increased side effects
The above problems of conventional dosage form stimulate the formulator both in
industry and laboratory level to develop modified release dosage form. “The delivery of a
drug at predetermined rate and/or location according to the needs of body and disease for
definite time period is termed as modified/controlled drug delivery” [1]. Modified release
delivery system can be classified as [1]:
2.2 Delayed Release
These are type of dosage form that releases the drug at the time other than immediately
following the administration. The delayed release of the drug may be time dependant or
based on environmental conditions such gastric pH, enzymes or pressure etc. The
example includes enteric coated tablets, where a drug release is dependent on acid
resistant coating (CAP, Eudragit etc) repeated action tablets or spansules.
10
2.3 Extended Release
These include delivery system that maintains therapeutic plasma levels of administered
drug for prolong time. Such dosage form reduces the dosage frequency compared to the
immediate/conventional dosage form. The drug release is in continuous manner over
prolong period of time to maintain the plasma concentration, the drug release is mostly
independent of the environmental conditions.
2.4 Site Specific Targeting
In such system the drug delivery is targeted to the surrounding or in the pathologic tissue
or organ. This is a technique of drug delivery in a manner that increases the drug
concentration in the targeted area of the body relative to other tissues. The prime goal is
to extend, localize and have confined drug interaction with the targeted tissue or organ.
The targeted drug delivery reduces the total incorporated dose in the dosage form thus not
only provides better disease control but also reduces the systemic untoward effects.
Targeted drug delivery is of specific importance for oncology products.
Targeted drug delivery may be active (antibody delivery) or passive (enhanced
permeability and retention effect). Targeted drug delivery can be achieved by the use of
drug carriers like liposomes, niosomes, dendrimers, resealed erythrocytes, biodegradable
nanoparticles and micro-particles etc.
2.5 Receptor Targeting
In such system the target is a particular receptor with an organ or tissue. Folate receptor is
commonly used as marker for drugs effective in the cancer (Doxurubicin, antibodies,
11
interlukins, radio-pharmaceuticals). The beauty of the delivery system is that a specific
drug response is achieved with a minimal of side effects.
2.6 Fast Dissolve Drug Delivery System (Flash)
It is type of rapidly dissolving or disintegrating solid dosage form that delivers the drug
in the oral cavity and gets dissolve without the aid of water or chewing. Rapid dissolution
is achieved by forming porous slack network (Zydis, Eli Lilly), or by addition of some
effervescence producing ingredient (Oraslav, Cima) or with using a combination of
disintegrating agent and swelling phenomenon (Flash Tab, Prographarm)
Controlled drug delivery system offer various advantages over the conventional drug
delivery system, the advantages include; “avoid patient compliance problem, less total
drug incorporation, reduction, minimize or eradicate local side effects, diminishes or
curtail systemic side effects, reducing the drug potentiation with chronic use, minimize
accumulation of administered drug with chronic use, improve efficiency in treatment,
cure or control pathologic condition more promptly, minimizing drug plasma
concentration variation, improve bioavailability of some drugs, make use of special
effects (e.g. sustained release aspirin for morning relief of arthritis by dosing before
bedtime), economic savings”. [1, 2]
Various approaches have been adopted for controlling the release of the drug. The
common approaches are drug modification and dosage form modification [2].
12
2.7 Drug Modification
In this approach the physical or chemical structure of drug is altered to achieve better
control over the drug release or drug targeting. The common methods in this system
include; Drug complexes, Drug adsorbates and Pro-drug.
2.8 Dosage Form Modification
Modification of dosage form is one of the most common approaches to retard the release
of the drug from the dosage form and thus extending the effective time period. The
common approaches adopted for dosage form modification include [2].
2.8.1 Reservoir Devices
In such system the drug is wrapped in by a polymeric membrane from which the drug is
released either through diffusion, dissolution or erosion, these systems can also be
formulated using osmotic pressure. Such system make use of formation of either totally
or partially encapsulating drug with rate controlling drug surface by formation of coat
using polymer as coating material. The drug reservoir can be drug solid particles
(nanoparticles), a dispersion of drug solid particles, or a concentrated drug solution in a
liquid (micro emulsion) or solid-type dispersing medium.
2.8.2 Matrix Devices
This approach utilizes the dispersion of the drug in the polymer bed. The drug release
follows dissolution, erosion of the polymer or diffusion through polymer matrix alone or
combination of these mechanisms may take place.
13
2.8.3 Hybrid System
This system take advantages of both reservoir and matrix system and is term as the most
versatile system. This system enjoys the advantages of both systems. The drug is
wrapped in polymeric cover and then blended with polymer matrix, thus giving better
control and targeting of the drug.
14
2.9 Ophthalmic Drug Delivery:
2.9.1 Eye
The human eye is the organ which gives us the sense of sight. As anatomical part the eye
allows us to see and construe the dimensions, shapes and colors of objects. The light
emitted by the object is focused onto the retina and at the back of the eye. When the light
rays strike the lens, it passes through the vitreous humour, a jelly-like substance and
arrives at the retina. The retina works like a camera film, or a charge-coupled device
(CCD) of a digital camera[30].
Figure 2.1: Anatomy of the eye
The retina consists of three types of light sensitive photoreceptors; rods, cones and
ganglion cells. These receptors convert light rays through electric transduction to the
15
electrical signals. The impulses are transmitted through the optic nerve to the visual
cortex in the brain, where the image is processed and perceived.
Anatomically eye is divided into two segments i.e. the anterior and posterior segments
(Fig 2.1).
2.9.1.1 The Outer Layer
2.9.1.1.1 The Sclera and Cornea
Sclera is the white tough, fibrous outer layer that serves as protective cover. The front of
the sclera is covered by the conjunctiva, which is a thin, transparent membrane that
produces tear film that lubricates and protects the eye while it moves in its socket.
Cornea is a dome-shaped structure. It is transparent, and its function is to focus the
incoming light onto the retina.
2.9.1.1.2 Iris
Iris is coloured part that controls the size of the pupil. The iris sits between the anterior
chamber and the posterior chamber.
2.9.1.1.3 Pupil
Pupil is the black area in the centre of the iris. The size of pupil varies with the intensity
of the light.
16
2.9.1.1.4 Aqueous Humour
It is a transparent fluid, which is constantly being produced by the ciliary body. It has a
refractive index of 1.337. Aqueous humour is important for nourishing the lens and
cornea. It is drained through canal of Schlem.
2.9.1.1.5 Lens
It is a clear, flexible structure that changes shape so that to focus on objects at varying
distances.
2.9.1.1.6 Ciliary Body
When contracted accommodate the lens for close up vision and when relaxed fix the lens
for long-range vision.
2.9.1.1.7 Vitreous Humour
It is a jelly-like substance filling the back of the eye behind the lens. The vitreous humour
maintains shape and transmits light to the back of eye.
2.9.1.1.8 Choroid
It is a highly pigmented and separates sclera and the retina, choroid is rich in blood
supply thus also works as transporter of oxygen and nutrients to the retina.
17
2.9.1.2 The Inner Layer
2.9.1.2.1 Retina
Retina is the most sensitive part of the eye by virtue of containing millions of
photoreceptors cells that are linked to nerve fibers.
2.9.1.2.2 Blind spot
It is also known as the optic disc where all of nerve fibers converge to form the optic
nerve.
2.9.1.2.3 Fovea
It is the most sensitive area, providing the sharpest vision.
2.9.1.3 Drug Kinetics through Eye
Figure2.2: “Schematic presentation of the ocular routes of drug kinetics” [31].
1. “Trans-corneal permeation from the lachrymal fluid into the anterior segment
2. Non-corneal drug permeation across the conjunctiva and sclera into the anterior
segment
18
3. Drug distribution from the blood stream into the anterior segment
4. Elimination of drug from the anterior segment by the aqueous humour
5. Drug elimination from the aqueous humour into the systemic circulation
6. Drug distribution from the blood into the posterior eye segment
7. Intra-vitreal drug administration,
8. Drug elimination from the vitreous humour through posterior route
9. Drug elimination from the vitreous humour via anterior route to the posterior
segment”
2.10 Ocular Routes
Drug to the eye can be administered through several possible routes. The selection of the
route depends primarily on the target tissue. The anterior segmented is targeted with
topical and sub conjunctival administrations and intra-vitreal administration for posterior
segment[31].
2.10.1 Topical Ocular
Ocular drug administration is accomplished by eye drops, the disadvantage associated
with the eye drops is the short contact time on the eye surface. The non-conventional
formulation design (preformed gels, in-situ sol-gel, ointments and inserts) will increase
the contact and residence time, thus enhancing the duration of drug action [32]. On
administration of eye drop the peak concentration in the anterior chamber is reached after
20–30 minutes [33]. Drug is eliminated from the aqueous humour by two main
mechanisms: by aqueous turnover and by the venous blood flow [34] (Figure 2.2). The
aqueous turnover has a rate of about 3μl/min.
19
The other mechanism depends upon the drug permeability across the vessel walls.
Therefore the elimination of lipophilic drugs is faster as compared to hydrophilic drugs
from anterior chamber. The half-lives of drugs in the anterior chamber are typically short,
about an hour. Flow of aqueous humour from the posterior chamber to the anterior
chamber is another limiting factor.
2.10.2 Sub-conjunctival
Injection through this route is encouraged to deliver high drug concentration to the uvea.
The advancement and progress made by material scientists and pharmaceutical
formulator innovations have provided new stimulating possibilities to develop controlled
release drug delivery system to deliver drugs to the posterior segment of the eye [35].
After sub-Conjunctival injection drug is absorbed rapidly as it crosses the sclera which is
more permeable than the cornea. Interestingly the lipophilicity of drug has no pronounced
effect on drug permeability through sclera [36, 37]. Thus it seems easy to deliver drug to
sclera and choroid via this route.
2.10.3 Intra-vitreal
This route directly administers drug to the vitreous and retina (Figure 2.2). After intra-
vitreal injection the drug is eliminated by two main routes: anterior and posterior [34].
Drugs can be administered to the vitreous also in controlled release formulations
(liposomes, microspheres, implants).
20
2.11 Challenges in Ocular Drug Delivery
The anatomy, physiology and biochemistry of the eye render this organ impervious to
foreign substances. It is a challenge to the formulator to overcome and to provide the
drug in sufficient effective concentration to the target tissues. The challenges and
problems encountered by the formulator are rapid and extensive drug elimination.
Figure 2.3: Drug elimination pathways from the pre-corneal area [38].
After instillation, the flow of lachrymal fluid removes instilled solution from the eye by
naso-lachrymal duct rapidly in a couple of minutes [39]. The eye maintains its residence
volume at 7-10μl while the instilled eye drop is in the range of 20-50μl that results in
higher drainage rate to maintain the residence volume [38]. The systemic absorption via
blood capillaries in the conjunctival sac or from the nasal cavity also reduces the
21
availability of the drug from local absorption [40, 41]. This results in very low drug (1 -
6%) availability in the intra ocular tissues through cornea. The reason for this inefficient
drug delivery includes rapid tear turnover, lachrymal drainage and drug dilution by tears.
As most of the drug get systemically absorbed via the nose or gut after draining from eye.
This not only reduces the ocular bioavailability but also may lead to unwanted systemic
side effects and toxicity[38].
The following characteristics are required to optimize ocular drug delivery systems [38].
 “A good corneal penetration.
 A prolonged contact and residence time with corneal tissue.
 Ease of administration for the patient.
 A non-irritative, comfortable and biodegradable form”
Some common methods to prolong pre-corneal residence time include use of Hydrogels,
Liposomes, Inserts, micro and nano-carrier systems. In comparison with traditional
formulation, these systems have the following advantages [38].
 Increase contact time
 Prolonged drug release
 Reduced systemic side effects
 Reduced dosage frequency
 Better `patient compliance
22
2.11.1 Ophthalmic Dosage Forms
Ophthalmic preparations (eye preparations) are sterile, liquid, semi-solid, or solid
preparations that may contain one or more active pharmaceutical ingredient(s) intended
for application to the conjunctiva, the conjunctival sac or the eyelids.
Despite severe limitations and problems significant improvement in Ocular drug delivery
have been made. The improvements have been with objective of providing and
maintaining the drug concentration for an extended period.
2.11.1.1 Aqueous Solutions
Aqueous solution offers many advantages including the simplicity of large scale
manufacture, uniform dosage and no irritation. The major disadvantage associated is the
minimum contact time with the eye tissue, loss of active drug through drainage and
frequent administration. The contact time may be increased with addition of suitable
viscosity enhancer but this may offer instillation problem.
2.11.1.2 Suspensions
Suspensions are important ophthalmic delivery system that offers distinct advantages.
Most of the recently developed new drugs are water insoluble and are required to be
formulated as suspensions. From pharmaceutical formulation and physical stability view
point suspension is more difficult and challenging to formulate compared to conventional
ophthalmic solutions. The major problem encountered by formulator is to overcome non-
homogeneity of the dosage form, cake formation, aggregation of the suspended particles,
redispersability, effective preservation, and ease of manufacture [42].
23
2.11.1.3 Ointments
Ophthalmic ointments provide prolonged therapeutic actions. Major disadvantage of the
ophthalmic ointments is interference with the vision i.e. blurred vision due to refractive
index difference between the tears and the non-aqueous bases of the ointment [43].
2.11.1.4 Pre-Formed Gels and Muco-adhesive Polymer Systems
Muco-adhesive polymers have a property known as bio-adhesion meaning attachment of
a drug carrier to the epithelium and the mucosal surface. These polymers are able to
extend the contact time of the drug and thereby improve ocular bioavailability. There are
several bio-adhesive polymers now available with varying degree of muco-adhesive
performance [44].
2.11.1.5 Gel Forming Liquids
These are preparations which are clear free flowing liquid in packaging and can be
instilled in eye as drop but gel on contact with the eye by one of the following stimuli
[38];
 Physical stimuli like: Change in temperature, electric fields, light, pressure, sound
and magnetic fields.
 Chemical stimuli like: Change in pH and ion activation from biological fluid.
 Biological/ biochemical (bio-molecules) stimuli like: Change in Glucose level.
24
These “intelligent” or “smart” polymers play important role in drug delivery. It is widely
accepted that increasing the viscosity of a drug formulation in the pre-corneal region will
lead to increased bioavailability, due to slower drainage rate from the cornea [45].
2.11.1.6 Non-erodible Ocular Inserts
Alza Corporation marketed the first ocular insert (Ocusert) as controlled drug delivery in
1975. The Ocusert is soft and flexible membrane and designed to be placed in the cul-de-
sac between the sclera and the eyelid that continuously release pilocarpine at a steady rate
for one week. The major disadvantage associated with the system is the insertion and
removal on a weekly basis.
2.11.1.7 Prodrugs
Prodrugs are pharmacologically in active that is bio-transformed chemically or
enzymaticaly to pharmacologically active compound. Some key advantages of prodrugs
are
 Higher bioavailability
 Enhanced potency
 Lesser dose
 Extended duration of action
 Lesser side effects
 Higher chemical and physical stability
25
2.11.1.8 Microspheres and Nano-particles
Particulate polymeric delivery systems include microspheres and nanoparticles. Such
particles are commonly synthesized using biodegradable polymers such as polylactic co
polyglycolic acid. Drug delivery based on such system offer better control and
bioavailability of drug.
2.12 Subcutaneous Drug Delivery

The Parenteral administration route is the most effective and common for delivery of
active drug substances for which the bio-availability in limited by high first pass
metabolism.
Figure 2.4: skin tissue showing Hypodermis/Subcutaneous tissue
For the subcutaneous route, a needle is inserted into fatty tissue just beneath the skin. The
drug injected is absorbed in to the blood through the capillaries and thus introduced for
systemic effects. Macromolecules (Protein drugs), such as insulin, immunoglobulin that
are degraded by the oral route are administered via this route.
26
Fig. 2.5: Subcutaneous route: needle inserted beneath the skin
Subcutaneous injectable products are administered as aqueous solutions, suspensions or
emulsions. The injection volume via this route is usually 2 ml. The rate of absorption
from this route is slow and constant thus providing sustained and extended
pharmacological effects [46]. This route also provides better tolerance and steady state
concentration for hydromorphine in cancer patients.
Subcutaneous drug delivery systems can be used both for systemic or localized effect.
Drug release kinetics can be altered by changing the character of injectable biomaterial
template or depot, and the properties of the drugs like size, hydrophobicity, etc. The
depot injection will avoid patient discomfort by avoiding surgical procedure as involved
with implants. Thus a long term drug delivery system through subcutaneous injection got
multiple benefits [47].
The development of sustained-release formulations for intramuscular/subcutaneous
administration has become an increasingly important issue for the last two decades [48].
27
This will not only increase the patient compliance and comfort but also can minimize the
systemic side effects. Such type of parenteral drug delivery can be achieved by utilizing
drug delivery systems that will ensure continuous extended slow release of drug in
predictable manner.
28
2.13 Parenteral Depot System
The injection of a substance in a form that tends to keep the drug at injection site will
prolong the drug absorption. Long acting parenteral drug formulations are designed,
ideally to provide slow constant, sustained, prolonged action.
2.13.1 Reasons for development of PDS (Parenteral Depot System)
The PDS is biodegradable system which is converted to non toxicological by-product
with in the physiological system and no surgical removal of depleted system is required.
The drug release from this system can be controlled by following mechanisms;
 Drug diffusion through the polymer
 Polymer surface erosion with concomitant release of drug.
 Cleavage of covalent bond between the polymer bulks or at the surface followed
by diffusion drug release.
2.13.1.1 Advantages:
 Patient convenience
 Potential for controlled release
 Avoiding the peaks and troughs, thus minimizing the risk of toxicity and
ineffectiveness
 Decreasing the frequency of dosing
 Efficient drug delivery
 Flexibility
29
2.13.1.2 Disadvantages:
 Invasive
 Danger of device failure
 Commercial disadvantage
2.13.1.3 Types of Depot Formulations:
On the basis of different mechanism, depot formulation are categorized into four types
[49, 50]
 Dissolution controlled depot formulation
 Adsorption type depot formulation
 Encapsulation type depot formulation
 Esterification type depot formulation
2.13.2 Injectable Drug Delivery System
2.13.2.1 Emulsions
Emulsions are used extensively in parenteral products. Their use as depot type of
injection is limited because of physical instability. The possibility of coalescence and
dissolution in the surrounding body fluid has restricted the use of emulsion as choice for
long acting formulations.
2.13.2.2 Liposomes
Liposomes are bi-lipid vesicles which has internal aqueous core surrounded by
hydrophobic bi layer. Liposomes were employed as sustained release drug carriers,
30
therapeutic response of the drug entrapped within the liposomes is longer compared to
free drugs, but they are rapidly cleared off from the general circulation by macrophages
which make it poor choice for PDS. Other problems, such as physical and chemical
stability, sterilization and low drug entrapment, have limited the utility of liposomes.
In the area of injectable drug delivery systems, research into liposomes played a major
role in the past few decades. Significant efforts in basic and applied research institutions
led to the clinical development and ultimate approval by regulatory agencies for human
use of a lipid complex (Abelcet®, Amphotec®) and three liposome formulations,
AmbiSome®, DaunoXome® and a Stealth® liposome (DOXIL®) [51, 52]. The
liposomes can provide extended release of the active moiety by utilizing multivessicle
liposomes (MVL). It has been found that using long chain triglycerides as lipid bilayer
result in slower release rates from the MVL formulations than short chain triglycerides
[53].
2.13.2.3 Microspheres:
Microspheres are easy to deliver to the site of action but they have several inherent
disadvantages. The major issue is manufacturing processing as they are difficult to
sterilize, reproduce, also physical stability is a problem. The microspheres may also
migrate from the site of injection.
Depot injectable formulation with microsphere technology of leuprorelin was
achieved[54]. The drug release from these depot formulations was fairly constant for a
31
period of over one month or 3 months in animals and humans after subcutaneous or intra-
muscular routes.
2.13.2.4 Micelles
Micelles are amphiphilic in nature possessing both hydrophilic and hydrophobic ends,
being capable of delivering both water soluble and insoluble drugs. The formation of
micelles depends upon the concentration of polymer termed as critical micelle
concentration (CMC). The CMC has key role in the micelle stability, lower the CMC
more stable will be micelles. The micelles are also prone to migration as that of
microspheres. The dilution of micelles at the time of injection or within the body fluid
may results in burst release and dose dumping.
2.13.2.5 Thermoplastic Pastes
Thermoplastic polymers have low melting points i.e. between 25-65oC. These are
administered as solution at elevated temperature and solidify upon cooling to body
temperature. Bio-erodible thermoplastic pastes could be prepared from such monomers
as, caprolactone, trimethylene carbonate, dioxanone, D,L-lactide, glycolide, and ortho
esters [55-57].
As thermoplastic system is injected at temperature greater than 60 °C, the injection at
higher temperature can be very painful and increases the chance of necrosis and scar
formation at the site of injection is the major disadvantages associated with these delivery
system [58].
32
For the local delivery of taxol in the form of thermoplastic pastes was developed by
Zhang et al, the system was composed of triblock copolymer system of poly(D,L-
lactide)-block-poly(ethylene glycol)-block-poly(D,L-lactide) and blends of low
molecular weight poly(D,L-lactide) and poly(Ί-caprolactone) (PCL)[59].
2.13.2.6 Thermosets
Thermoset polymers can flow and can be molded in any shape. Upon heating, they
acquire their final shape. The process of molding into any shape is termed “curing” and
involves the establishment of covalent linkages between polymer chains to form a
macromolecular network [60].
The advantage of using this system is its facile syringe ability. The major disadvantage
associated with this system is burst drug release. This burst is due to the lag time for
solidification of the polymer.
33
2.13.3 Stimuli Sensitive Solution to Gel
These polymers also termed as smart or intelligent polymers changes from solution to gel
phase in response to external stimuli. Polymers possessing such ‘sensor’ properties can
undergo sol-gel phase transitions upon changes in the environmental condition [61].
These system deliver the drug in controlled manner for extended time period at targeted
tissues. The environmental sensitive polymeric drug delivery systems are also called
intelligent or smart hydrogels [62]. These polymers show a dramatic physical change
conversion from free flowing liquid to viscous gel in response to small environmental
changes. These smart polymeric systems can be classified into the following classes.
2.13.3.1 Temperature Sensitive Hydrogels
These polymers are capable to swell or de-swell as a result of temperature variation of the
surrounding fluid. These polymers may be positive or negative temperature sensitive and
thermally reversible. The most commonly used thermoreversible gels are these prepared
from poly ethylene oxide-poly propylene oxide (Pluronics®, Tetronics®, poloxamer)
[26, 63].
2.13.3.2 pH Sensitive Hydrogels
The pH-sensitive polymers contain acidic or basic functional groups and are known as
poly-electrolytes [63]. With increase in pH the polymers with acidic functional group
show increase in swelling, while the swelling decreases with in case of polymers with
basic functional group. The most of anionic pH-sensitive polymers are based on PAA
(Carbopol®, carbomer) or its derivatives, polyvinyl acetal diethylaminoacetate [61, 64].
34
2.13.3.3 In situ Solidifying Organogels
Organogels or oleaginous gels are amphiphilic lipids, which form different types of liquid
crystals upon contact with water. The most commonly employed lipids include glycerol
monooleate, glycerol monopalmitostearate, and glycerol monolinoleate. These
compounds form a cubic liquid crystal phase upon injection. This liquid crystalline
structure is gel-like and highly viscous. In vitro release of levonorgestrel from these
organogels was observed for up to 14 days.
Advantage of these systems is that they are biodegradable. Purity of waxes and stability
of oils are the major issues that need to be addressed [60, 65].
2.13.4 Polymers in Drug Delivery System
Polymers are macromolecules that are formed by covalent linkage of many small
molecules termed as monomers. The field of synthetic polymers is currently one of the
fastest growing materials industries. “The synthetic polymers provide good
biocompatibility and allow them to perform many biomimetic tasks that cannot be
performed by other synthetic materials, which include drug delivery, use as grafts for
arteries and veins and use in artificial tendons, ligaments and joints. The polymer used for
controlled release can be biodegradable, nonbiodegradable, non-biodegradable and
soluble [29]”. As drug-carriers, these polymers exist in the form of microspheres,
matrices and membranes.
35
2.13.4.1 Classification of Polymers
Polymers can be classified by number of ways for easy understanding.
2.13.4.1.1 Classification Based on Source of Availability
 Natural Polymers
The source of these polymers is natural (plants and animals) e.g. starch, protein,
cellulose, nucleic acid (DNA, RNA)
 Synthetic Polymers
The man made polymers which are synthesized in laboratories. For example polyethene,
PVC nylon, teflon, bakelite, terylene, synthetic rubber, pluronics, tetronics etc.
 Semi synthetic Polymers
These polymers are chemical derivatives of natural polymers synthesized in laboratories.
For example Ethyl cellulose, methyl cellulose, Hydroxy propyl cellulose,
hydroxylpropylmethyl cellulose, vulcanized rubber, Rayon etc.
2.13.4.1.2 Classification Based on Structure
 Linear polymers
These are polymers in which monomeric units are linked together to form linear chain.
Common examples are high density polyethylene, nylon, PVC etc.
36
A B
Figure 2.6: Chemical structure of Poly venyl chloride (A), and Polyethylene (B) and
Nylon (C)
 Branched Chain Polymers
These are polymers in which the monomers are joined to form long chains with side
chains or branches. Examples are low density polythene, glycogen, starch etc.
(Amylopectin).
 Cross Linked Polymers
These are polymers in which monomers unit are cross linked together to form three
dimensional network polymers. e.g. Bakelite, melamine, formaldehyde resin etc.
2.13.4.1.3 Classification Based on Type of Monomer
 Homopolymers
These are formed from identical monomers. e.g. Polyvenyl Chloride, polyethylene,
polymethylmethacrylic acid.
37
 Copolymers
These are formed from non-identical monomers. e.g. Nylon, terylene.
2.13.4.1.4 Classification Based on Monomer Arrangement
 Alternating Copolymers
A-B-A-B-A-B-A (polyester, polyamide)
 Random Copolymers:
A-A-B-A-A-A-B-B-A-B-B-A-A. (styrene, butadiene).
 Block Copolymers:
A-A-B-B-B-A-A-B-B-A-A-B-B
(Glycerol propoxylate-block-ethoxylate, Poly(styrene)-block-poly(acrylic acid)).
 Graft Copolymers:
A–A–A–A–A–A–A–A–A–A–A–A–
| | |
B B B
| | |
B B B
| | |
B B B
e.g. Polycarbonate-g-Polyacrylate
38
2.13.4.1.5 Classification Based on Basis of Mode of Synthesis
 Addition Polymers
This type of polymer is formed by addition of monomer without by product elimination.
Examples include, Polypropylene, polyethylene, polystyrene.
 Condensation Polymers
Such polymers are formed by the condensation of monomers with the elimination of
simple molecule like water, ammonia, HCl, alcohol etc. e.g. Nylon
2.13.4.1.6 Classification Based on Molecular Forces
 Elastomers
The monomers of the polymers are attached by weak forces and applying on stress these
polymers can easily be stretched and regains their original shape upon removal of the
stress. e.g. natural rubber
 Fibers
These polymers are held together by strong intermolecular forces. These polymers have
high tensile strength and less elasticity .e.g. Nylon 66, dacron, silk etc.
 Thermoplastics
These polymers get softened with application of heat and hardened on removal of heat
with no or little change in their characteristics. These polymers have intermediate type of
forces as compared to elastomers and fibres. e.g. Polythene, polystyrene, PVC, teflon etc.
39
 Thermosetting Polymers
These undergo permanent change on application of heat. Upon heating they get highly
cross linked to form hard infusible and insoluble products. Upon heating high cross
linking occurs in these polymers and they become permanently rigid. e.g. Bakelite,
melamine formaldehyde resin etc.
2.13.4.2 Properties of Polymers as Candidate for Controlled Release
There are several important Properties to be considered in selecting or developing a
polymer for controlled delivery [66] :
 Biocompatibility, safety and toxicology
 If the polymer is biodegradable then what is the degradation rate and byproducts
and their safety
 Cost
 Chemical, physical, and mechanical properties
 Solubility
 Processing requirements
 Compatibility with the active agent and other ingredients in the formulation.
 Required sterilization methods
 Thermal transition temperatures
40
2.14 Hydrogels
The polymeric network system that absorb huge amount of water without becoming
soluble in aqueous solution by cross linking of individual polymer chain [67]. The
hydrogel technologies have variety of applications including, food additives[68],
pharmaceuticals[69], biomedical implants [70] tissue engineering and regenerative
medicines [71], diagnostics [72], cellular immobility[73], separation of biomolecules or
cells[74] and barrier materials to regulate biological adhesions[75], Biosensor and drug
carriers[76].
Hydrogels can be classified on the basis of source as [77] Natural hydrogels and
Synthetic hydrogels. Hydrogels from natural source offer several advantages as they are
biocompatible, biodegradable and support cellular activity but there are disadvantages
associated with the hydrogels from natural origin that they may provoke immune
response because of the presence of pathogens and also may not provide sufficient
mechanical properties. Synthetic hydrogels, on the other hand are free from pathogenic
activity and with efficient fabrication biodegradable and physiologically friendly
hydrogels can be synthesized [67].
Hydrogels offer the following benefits which make them very good carriers for drugs and
tissue engineering [67];
 “Biocompatible
 Can be injected
 Easy to modify
41
 Timed release of growth factors and other nutrients to ensure proper tissue growth
 Entrapment of microbial cells to reduce the toxicity within polyurethane hydrogel
beads”
2.14.1 Environmental Sensitive Hydrogels
Environmentally sensitive hydrogels have the ability to sense changes of pH,
temperature, ions, light or the concentration of metabolite. Natural hydrogel materials are
being investigated for tissue engineering, which include agarose, methylcellulose and
hylaronan.
The polymeric solutions capable of changing their rheological behavior in response to
environmental stimuli are termed as smart or intelligent polymers. The environmental
stimuli may be physical or chemical and include;
2.14.1.1 Temperature sensitive hydrogels
2.14.1.2 pH Sensitive Hydrogels
2.14.1.3 Electric Signal Sensitive Hydrogels
2.14.1.4 Light Sensitive Hydrogels
2.14.1.5 Ion Sensitive Hydrogels
2.14.1.6 Pressure Sensitive Hydrogels
42
2.14.1.7 Temperature Sensitive Hydrogels
These polymers are sensitive to environmental temperature and change their solution
phase to gel phase. The common feature shared by almost all temperature sensitive
polymers that they contain hydrophobic group i.e. methyl, ethyl and propyl [63]. These
components of polymers must be insoluble above or below certain temperature, called the
upper or lower critical solution temperature (UCST and LCST). For drug delivery system
based on this property the polymers with LCST are more promising[26] . At LCST, the
interaction forces (hydrogen bonding) between water molecules and polymer weakens
compared to polymer-polymer and water-water interaction that results in phase separation
and the polymer forms gels[78]. As a result of the increase in hydrophobicity the polymer
chains are linked by physical reversible linkage, and gels can therefore return to solution
after the temperature stimulus causing gelation is removed[13]. These polymeric
solutions have display low viscosity (free flowing) at ambient temperature but form a
semi-solid gel at body temperature[79]. The most commonly used thermosensitive
polymers for controlled drug delivery include, polysaccharides (e.g., cellulose
derivatives, xyloglycan, chitosan), N-isopropylacrylamide and Pluronics (poloxamers,
(PEG-PLA-PEG)[20].
43
2.14.2 Pluronic (Poloxamers)
This is a group of nonionic surfactants consisting of polyethylene oxide (PEO) and
polypropylene oxide (PPO) copolymers. Commonly used Pluronics (Poloxamers) include
F-68 (188), F-87 (237), F-108 (338) and F-127 (407), all of them are freely water soluble.
Their chemical formula is
HO[CH2-CH2O]a [CH(CH3)-CH2O]b[CH2-CH2O]aOH,
“b” is higher than 14. Poloxamers are white, waxy, free-flowing granules practically
odorless and tasteless[80]. Different poloxamers and their physical properties are shown
in table 2.1.
PF-127 is the most commonly employed polymer among the group used as
thermorevesible vehicle as a carrier for most routes of administration including oral,
topical[81, 82], intranasal[83], vaginal, rectal[84, 85], ocular[86-92], and parenteral
routes[92]. The potential use of PF-127 as an artificial skin has also been reported[93].
Table 2.1: Types and physical properties of Pluronic/Poloxamers (USP 32)
Average
Pluronic Poloxamer A b Physical Form
molecular weight
L-44 124 12 20 Liquid 2090-2360
F-68 188 80 27 Solid 7680-9510
F-87 237 64 37 Solid 6840-8830
F-108 338 141 44 Solid 12700-17400
F-127 407 101 56 Solid 9840-14600
Where a= oxyethylene b= oxypropylene
44
Poloxamer 407 is available in the registered trademark of Pluronic F127 (BASF
laboratories), and Synperonic F127 (ICI laboratories), has a molecular weight of about
12,600 (9,840-14,600)[94] a and b are equal to 95-105 and 54 - 60, respectively. Its HLB
is 22 at 22oC [94-96].
Aqueous solutions of Poloxamer 407 show gelation on rise in temperature that is thermo
reversible. Thermo gelling property is completely reversible characterized by sol to gel
transition temperature. Below the transition temperature the solution remains free flowing
liquid, while above this temperature the solution form highly viscous semi solid gel. The
sol-gel transition is result of interaction between the segments of the block
copolymer[97], the molecules aggregate forming micelles at LCST . This micellization is
due to the dehydration of hydrophobic poly Propylene oxide blocks (Figure 2.7) and
hydration of poly Ethylene oxide chains[98].
Figure 2.7: Schematic representation of the association mechanism of Poloxamer 407 in

water
45
2.14.2.1.1 Solublization Property
Poloxamer 407 facilitates solubilisation of poorly water soluble molecules like
Indomethacin [99] or insulin[84, 100, 101]. Solubility of piroxicam in water was
increased by 11-fold by adding 22.5% w/w Poloxamer 407[102].
2.14.2.1.2 Stabilization Property
Pluronic F-127 enhances the stability of incorporated drugs in its matrix, particularly of
proteins [103-108]. The three dimensional structure of proteins is better preserved in the
formulations containing pluronic F-127 as ingredient [98, 103-106]. Poloxamer 407 is
also effectively been used with W/O/W multiple emulsion to stabilize the preparation
[109].
2.14.2.1.3 Gel Strength
Gel strength of pluronic solution increases with increase in temperature and
concentration. The gel strength may be altered in the presence of drugs or additives.
Additives like, diclofenac, ethanol and propylene glycol weaken Poloxamer 407 gel
while ingredients like sodium chloride, sodium mono hydrogen phosphate and glycerin
results in increasing the gel strength [110, 111].
2.14.2.1.4 Adhesive Properties
Adhesive characteristic is of great importance in topical formulations (e.g., rectal,
cutaneous or ophthalmic preparations) as prolonged residence-time is required.
Adhesiveness increases with gel strength.
46
2.14.2.1.5 Solution to Gel Transition Temperature
Sol-gel transition temperature increases with decrease in Poloxamer 407 concentration
[112]. As the transition temperature increases or decreases with the addition of drugs or
various additives the bio-adhesive forces decreases or increases respectively [27]. The
added substances interfere with the micellization of the pluronic and alter the dehydration
of hydrophobic PO blocks [97, 113, 114].
Molecules like diclofenac, ethanol, propylene glycol, and HCl reduce the gel strength and
bio-adhesive force and increase Tsol-gel while others like NaCl, Na2HPO4, NaH2PO4
do the opposite[111, 115]. Adding sodium alginate, polycarbophil or carbopol has been
reported to lower gelation temperature [85], use of benzalkonium chloride also decreased
Tsol-gel [116].
2.14.2.1.6 Drug Release from Pluronic Polymers
At low temperatures depending upon the polymer concentration, the polymeric solution
embedding the drug to be released is a free flowing liquid that can be injected into the
body via a syringe. At a higher temperature, the polymer molecules aggregate forming
micelles with the core hydrophobic poly (propylene oxide) (PPO) surrounded by the
hydrophilic poly- (ethylene oxide) (PEO) [117-119]. Upon sub-cutaneous or
intramuscular injection the temperature of this micellar solution increases to body
temperature, the micelles begin to entangle with other micelles forming gel within the
body. For pluronic F127 with 70% ethylene oxide ratio this concentration is about 20%
w/w at 37oC[120, 121]. The gel dissolves within the body by withdrawing water from its
47
surrounding, causing drug release that is dependent on the dissolution of the gel, which is
a zero-order process.
To predict the drug release from Pluronic gels for different drug and polymer
compositions, several aspects need to be investigated. The most common include, drug
solubility in pluronic solutions, diffusion of the drug through gel, and water penetration
rate into the gel [122]. Dissolution rate for concentrated gel is slower as compare to less
concentrated pluronic gels because the coefficient for water diffusion within the gel is
lower in the concentrated gels as than for the dilute polymer gels [122].
2.14.2.1.7 Applications of Pluronic F-127
The unique thermo reversible and controlled drug release characteristics of PF-127 make
it an attractive candidate as a pharmaceutical vehicle for drugs through different routes of
administration.
2.14.2.1.7.1 Topical and Dermal Applications
The pluronic gel is evaluated as topical drug delivery vehicle by several workers [123-
138]. Indomethacin was incorporated in 20% pluronic F-127 as a base for percutaneous
drug absorption[123]. Chi et al prepared and evaluated pluronic F-127 gel containing
ketotifen as drug, the preparation showed prolonged anti-inflammatory and analgesic
activities and less systemic side effects [126]. Fentanyl was successfully formulated in
PF-127 base to provide sustained release effect [129]. The effect of concentration of PF-
127 on the release rate of anticancer agents, 5-flurouracil and adriamycin was evaluated,
it was observed that with increase in polymer concentration the drug release decreases
48
[139]. The ceftiofur followed zero order kinetic being formulated in PF-127 and was
correlated well with the weight percentage of PF-127[134], formulation of benzoic acid
used as antiseptic in PF-127 formulation showed similar results [140].
Exploring the unique properties of PF-127, it has also been used in bone wound healing
in calvarial defects[135] (64), PF-127 gels also sufficiently mimic normal functions of
the skin epidermis acting as an “artificial skin”[141].
2.14.2.1.8 Buccal applications
Pluronic F-127 has been studied as vehicle for many drugs like triamcinolone [136],
Insulin[137] and human gingival fibroblast[142] administered through buccal cavity.
2.14.2.1.9 Rectal Application
Utilizing the muco-adhesive properties of pluronic F 127, the polymer is applied as
vehicle for rectal route within the concentration range of 17%-20%[143] and sustaining
the drug release for 10-15 hours[144].
2.14.2.1.10 Ophthalmic Applications
Pilocarpine HCl was incorporated in pluronic gels, the drug release and meiotic response
was significantly improved as compare with aqueous pilocarpine solution. The pluronic
in-situ gel showed increase tissue contact time and bioavailability[145]. 25%w/w solution
of Timolol showed 2.5 fold higher ocular concentration in aqueous humour as compared
to aqueous Timolol solution[146]. Poloxamer 407 (PF-127) are also evaluated for the
ocular delivery of the quinolones[147, 148] i.e. ciprofloxacin, moxifloxacin etc.
49
2.14.2.1.11 Other Routes
Number of workers has successfully investigated and evaluated poloxamers as vehicle for
the routes like, intramuscular[149-151] , intravenous [152], intraperitoneal [149]and
intranasal[153] routes.
2.14.3 Methyl Cellulose (MC)
Methyl cellulose also known as methyl cellulose ether is a white powder with yellow
cast. Methyl cellulose is a synthetic methoxy derivative of cellulose. The degree of
substitution is 1.6-1.9, on O-6, O-2 or O-3 position of glucose. This chemical
modification makes it soluble in cold water[154]. MC is available in number of viscosity
grades depending upon the molecular weight as given in Table 2.2.
Table 2.2: Methylcellulose viscosity grade with molecular weight (sigma-Aldrich)
Approximate Viscosity of Approximate

Product Number
2%, at 20 °C Molecular Weight
M 7140 15 cPs 14,000
M 6385 25 cPs 17,000
M 0262 400 cPs 41,000
M 0387 1500 cPs 63,000
M 0512 4000 cPs 88,000
50
Methylcellulose is used as a binder or thickener in pharmaceutical, cosmetic, food and
ceramic applications. Upon heating, methylcellulose converts to translucent gel in water,
this gelation is thermo-reversible in nature [155-157].
2.14.3.1 Biodegradation
Methyl cellulose and other cellulose derivatives are biodegradable as they are degraded
by several fungi and bacteria capable of producing cellulase enzyme [158]. Methyl
cellulose is biocompatible cellulose that has triggered its use in biomedical devices [159,
160]. As animal do not produce cellulose enzyme hence no resorption of cellulose in
animal and human tissues does occur.
2.14.3.2 Thermo-gelation
Methyl cellulose aqueous solution in concentration of 1-10% by weight forms a
thermorevesible gel [161]. In cellulose that is the raw material for methylcellulose, when
a certain number methoxyl groups are replaced for the hydroxyl groups, some hydrogen
bonds are broken and MC becomes water-soluble. At low temperatures, water molecules
surround the hydrophobic methoxyl groups forming “cagelike” structure, resulting in
making the MC gel [156, 162]. On increasing the temperature, these structures distort and
break to expose the hydrophobic regions, inducing the formation of aggregates and
resulting in gelation.
51
OR
H H H
OR H2C
RO O
O H O
H
O RO
H H
CH2 OR
H H H
OR n-2/2
Figure 2.8: chemical structure of methyl cellulose
The transition temperature depends on the degree of substitution, with increasing the
degree of substitution the hydrophobic character increases, thus lowering the transition
temperature. The degree of substitution can be properly adjusted (i.e. 1.9-2.6) to obtain
specific formulations gelling at 37 °C and thus potentially useful for biomedical
applications [163-165].
2.14.3.3 Applications of Methyl Cellulose
2.14.3.3.1 Oral Route
Methyl cellulose is commonly employed as tablet binder [166, 167], both in conventional
release and sustained release tablets depending upon its molecular weight and viscosity
grade [168]. Riboflavin was successfully incorporated in methyl cellulose matrix [169] to
provide sustained delivery of the drug. It is also used in oral pharmaceutical suspensions
as suspending agent [170, 171].
52
2.14.3.3.2 Ocular Route
Methyl cellulose is used in combination with thermorevesible polymers as mucoadhesive
agent or viscosity enhancer [146, 172, 173]. The addition of methylcellulose to the
aqueous solution of pluronic, carbopol, chitosan not only reduced the total concentration
of these polymers but also increase the ocular residence time thus enhancing the drug
bioavailability. Ciprofloxacin and timolol are successfully formulated in thermorevesible
ocular drug delivery vehicle in combination with methyl cellulose [146, 172].
2.14.3.3.3 Vaginal/Rectal Route
To enhance the residence time of the dosage form in the vagina and rectum use of
methylcellulose as mucoadhesive and thermo-gelation agent is successfully explored by
the pharmaceutical scientists [174, 175]. Addition of 1% MC as mucoadhesive agent in
poloxamer 18 % solution effectively enhanced the peak plasma concentration of
carbamezipine and reduced the gelation temperature [176]. In situ suppository system
was developed for mebaverine HCl and acetaminophen [175, 177].
2.14.4 Hydroxy propyl methyl cellulose (HPMC)
HPMC is a synthetic cellulose derivative it is propylene glycol ether of methylcellulose.
The chemical structure is shown as Fig. 1. The R substitution represents a –CH3 or a
CH2CH (CH3) OH group, or a hydrogen atom. The physicochemical properties of HPMC
are dependent upon: (i) the methoxy group content; (ii) the hydroxypropoxy group
content; and (iii) the molecular weight. The USP classifies the HPMC in four different
grades according to their relative –OCH3 and –OCH2CH (CH3) OH content. The four
grades are HPMC 1828, HPMC 2208, HPMC 2906 and HPMC 2910. The first two digits
53
in the nomenclature indicate the percentage of methoxy-groups while the last two
indicates percentage of hydroxypropoxy-groups [178].
The substitution comprises 19-30% of methoxy group and 3-12% hydroxypropoxy group.
HPMC is soluble in cold water and polyethylene glycols, but insoluble in alcohol. HPMC
is stable at wide range of pH i.e. 3-11. Aqeuous HPMC solution forms thermo-reversible
gel on heating [179] .
OR
H H H
OR H2C
RO O
O H O
H
O RO
H H
CH2 OR
H H H
OR n-2/2
Figure 2.9: Chemical structure of HPMC. The substituent R represents either a –CH3, or
a –CH2CH(CH3)OH group, or a hydrogen atom
2.14.4.1 Thermo-gelation
When aqueous solutions of HPMC are heated, they gel at higher temperatures. The
gelling temperature depends upon the HPMC grade and concentration. The solution to
gel transition is completely reversible i.e. upon cooling the gel liquefies to form free
flowing solution. The transition from sol-gel is primarily due to the hydrophobic
interaction between molecules [161]. At lower temperatures the molecules are
surrounded by water molecules and are in are hydrated form. There is little polymer-
polymer interaction in solution state. As the solution is heated and temperature raises, the
54
molecules gradually lose their water of hydration and polymer to polymer interaction
prevails resulting in sharp rise of viscosity and the solution tends to gel [161, 162].
2.14.4.2 Applications of Hydroxy propyl methylcellulose (HPMC)
2.14.4.2.1 Oral Route
HPMC is the most common excipient used in the manufacturing of oral tablets as binder
both in conventional and sustained release matrix tablets. It has good compressibility and
can be used equally well in wet granulation and direct compression. The HPMC begins to
swell forming a protective gel like layer around the tablet when it comes in contact with
physiological fluids leading to a sustained release of drugs [180, 181]. HPMC K15 and
K100 are studied to develop sustained release matrix tablets of diclofenac sodium,
ketoprofen, carbamezipine, naproxen sodium, ketrolac, losartan potassium, zidovudine,
isoniazid, resperidone and olanzepine etc[182-189]. HPMC is an alternate and
comparable polymer to replace gelatin as hard shell capsules [190-192]. The
hydroxypropyl methylcellulose is used as tablet coating film former as it has a very high
tensile strength and high force is needed to break the HPMC film.
2.14.4.2.2 Ophthalmic Route
To improve the ocular dug availability the thermo reversible gelation of HPMC is
successfully utilized to enhance the residence time and bioavailability of the drugs
including ciprofloxacin once a day, ofloxacin, gatifloxacin [146, 148, 172, 193-195].
55
2.14.5 Diclofenac Sodium
Diclofenac sodium a phenylacetic acid derivative is Nonsteroidal Anti-inflammatory
Agent [196-200].
2.14.5.1 Chemical Name
2-[(2,6-dichlorophenyl)amino] benzeneacetic acid.
2.14.5.2 Molecular Formula and Structure
C14H11C12NO2.NaC20H24Cl2N2O3
Figure 2.10: Chemical structure of Diclofenac Sodium
2.14.5.3 Mechanism of Action
Diclofenac inhibits cycloxygenase -1 and 2.
2.14.5.4 Uses
symptomatic treatment of osteoarthritis [201, 202], ankylosing spondylitis [203, 204], for
symptomatic treatment of infusion-related superficial thrombophlebitis [205], relief of
pain, including postoperative (e.g., orthopedic, gynecologic) pain, acute pain due to
minor strains, sprains, and contusions [206].
56
2.14.5.5 Dosage and Administration
2.14.5.5.1 Rheumatoid Arthritis
50 or 75 mg twice daily [207]
2.14.5.5.2 Ankylosing Spondylitis
100–125 mg daily, administer as 25 mg 4 times daily [203, 208].
2.14.5.5.3 Pain
50 mg 3 times daily [206]
2.14.5.5.4 Dysmenorrhea
50 mg 3 times daily [209]
2.14.5.6 Pharmacokinetics
2.14.5.6.1 Bioavailability
Diclofenac sodium is well absorbed following oral administration, it undergoes first pass
metabolism extensively and only 50-60% of drug is available unchanged systemically
[198, 210-214]. After administration of conventional oral tablets peak plasma
concentration is achieved within about 1 hour [210, 211], following topical application as
1% gel the absorption is low as compared to oral administration and peak plasma
concentration is reched in about 10-14 hours [215].
57
2.14.5.6.2 Distribution
Diclofenac is widely distributed [210, 211], after oral administration the concentration is
low in plasma as compared to synovial fluid [214, 216-219], more than 99% of drug is
protein bound [220, 221].
2.14.5.6.3 Metabolism
Diclofenac is metabolized in the liver, the major biotransformation reactions are
hydroxylation and conjugation reaction [210, 211, 219, 222].
2.14.5.6.4 Elimination Route
65% of drug is excreted in urine while the remaining 35% is eliminated in feces [210,
222, 223].
2.14.5.6.5 Half-life
The biological half life of diclofenac after oral administration is about 1-2 hours [212,
213, 224].
2.14.5.6.6 Contraindications
Known hypersensitivity to diclofenac [209], History of asthma, urticaria, or other
sensitivity reaction [225-227]
2.14.5.6.7 Adverse Effects
Abdominal pain or cramps, constipation, diarrhea, GI bleeding, peptic ulcer, vomiting,
dyspepsia, nausea, dizziness, liver function test abnormalities, renal function
abnormalities, anemia, prolonged bleeding time, pruritus, rash and tinnitus [228-230].
58
2.14.5.6.8 Drug Interactions
ACE inhibitors, Angiotensin II receptor antagonists, Antacids (magnesium- or aluminum-
containing), Anticoagulants (warfarin), Aspirin, Cyclosporine, Diuretics (furosemide,
thiazides), Lithium, Methotrexate, Quinolones (ciprofloxacin) [206, 231-241]
59
2.14.6 Timolol Maleate
Timolol (TM) (Fig. 2.11) 22 (S)-1-[(1-1-dimethyl)amino]-3-[[4-(4-morpholinyl9-1,2,5-
thiadiazol-3-yl]oxy]-2-propanol, is a nonspecific β-adrenergic blocker. TM was the first
β-blocker to be used as an antiglaucoma agent.
2.14.6.1 Chemical Formula and Structure
 C13H24N4O3S
Figure 2.11: Chemical structure of Timolol Maleate
2.14.6.2 Molecular Weight
 316.42
2.14.6.3 Description
White odorless powder
2.14.6.4 Melting Point
201.5-202.5oC [242, 243].
60
2.14.6.5 Solubility
Timolol maleate is soluble in 1 in 15 parts water, 1 in 21 of alcohol, 1 part in 40 parts of
chloroform and practically insoluble in ether [242, 243].
2.14.6.6 pH
2% aqueous solution has pH 3.8 to 4.3 [242, 243].
2.14.6.7 Uses
Hypertension, angina pectoris, cardiac arrhythmias, migraine and glaucoma
2.14.6.8 Dosage
2.14.6.8.1 Glaucoma
One drop of 0.25 % solution twice daily, the dose may be increased to 1 drop of 0.5%
solution twice daily.
2.14.6.8.2 Hypertension
15 to 60 mg daily
2.14.6.9 Pharmacokinetics
2.14.6.9.1 Absorption
After oral administration the absorption from the GI tract is almost complete i.e. 90%.
The peak plasma concentration is attained within 0.5 -3 hours [244, 245]. The first pass
effect for timolol is not significant [246]. The onset of action after ocular administration
is 10-20 minutes [247].
61
2.14.6.9.2 Distribution
After oral administration timolol bioavailability is 60% [248], the apparent volume of
distribution is 1.30 to 1.70 l/kg [248, 249]. Approximately 10% of the drug is bound to
plasma protein. After ocular administration timolol is distributed in cornea, iris,
conjunctiva, aqueous humour, liver, lung and kidney. Following the cutaneous route
timolol is 50-60 % absorbed systemically [250].
2.14.6.9.3 Biological Half life
2.5-5.0 hour [246, 249, 251].
2.14.6.9.4 Metabolism
Timolol is principally metabolized in liver through hydrolysis of the morpholino ring
with subsequent oxidation. 20% of the drug is eliminated as such via urine [246].
2.14.6.10 Contraindication
Timolol is contraindicated in patients with 2nd and 3rd degree AV block, cardiogenic
shock and asthma. Also contraindicated with other β-blockers [252].
2.14.6.11 Side Effects
Fatigue, confusion, depression, hallucinations, dizziness, vertigo, lethargy insomnia,
bradycardia, edema [252, 253].
2.14.6.12 Drug interactions
Clonidine, Epinephrine, NSAIDs, Prazosin, Insulin, Verapamil and theophylines.
62
2.14.7 Insulin
Insulin is a naturally occurring hormone, composed of two polypeptide chains with the
empirical formula of C254 H377 N65 O75 S6 and has a molecular weight of 5808 Da. The
two chains are connected to one another by three disulfide bonds as seen in Figure 2.12.
It is produced and secreted from the β-cells in the islets of Langerhans in the pancreas.
The primary role of insulin is to facilitate the transport of glucose from the bloodstream
to cells in the body.
Figure 2.12: Structure of human insulin; the amino acid diagram of human insulin,
showing the A and B chains and 3 disulfide bonds
2.14.7.1 Insulin Production and Secretion
In healthy humans, insulin is synthesized during the secretion from proinsulin containing
86 amino acid chain in the β-cells of the pancreas. In the β-cells as first step polypeptide
called pre-proinsulin containing 110 amino acid sequence is synthesized [254]. The pre-
proinsulin is cleaved at N-terminus in the endoplasmic reticulum upon translocation and
63
24-peptide sequence is removed and the polypeptide pro-insulin is produced. The three
disulfide bonds characteristic of insulin are formed upon folding of the pro-insulin as
tertiary protein [254]. Pro-insulin is then converted to insulin upon secretion, individually
four amino acids are removed and co secreted along insulin as C-peptide (connector
peptide)[254]. The mediator for insulin secretion is blood glucose level which is critically
controlled during fed and fasted state in healthy humans. Insulin release is also initiated
by glucagon-like peptide 1 (GLP-1), gastrin, vasoactive intestinal peptide, secretin,
gastrointestinal inhibitory peptide (GIP), cholecystokinin, gastrin-releasing peptide, and
enteroglucagon [255].
2.14.7.2 Effect of Insulin
Insulin concentrations in plasma are commonly measured in international units. One
international unit (IU) of insulin is equal to 6 nmol, that is equivalent to 34.8 μg [256].
The insulin concentrations for healthy adults ranges between 5-15 μIU/ml (30-90 pmol/l)
for basal levels and 60-90 μIU/ml (360-540 pmol/l) for postprandial levels [257].
Circulating insulin binds to the insulin receptor in liver, muscle, and adipose tissues. As a
result of insulin binding to the receptors signals are mediated to initiate the translocation
GLUT4 transporters intracellular vesicles that are exposed to extracellular surface as
result of fusion with plasma membrane of the cells [255], this results in increase glucose
diffusion into cytosol and will be utilized as source of energy by the cells via glycolysis
or stored as gylycogen or triglyceride [258]. In addition to glucose translocation from
plasma to cell, insulin has several other significant roles in metabolic homeostasis. It also
inhibits the breakdown of fat, glycogen and protein and stimulates the synthesis of
64
protein in muscle by increasing the amino acids uptake, regulates gene transcription,
increases glycogen and fatty acid synthesis, and inhibits the breakdown of glycogen, fat,
and protein for energy [255].
In fasting state plasma insulin level is low, in this state liver synthesizes glucose through
glycogenolysis and pyruvate pathway thus releasing the glucose [258]. Increased levels
of insulin after the meal in the hepatic portal vein, inhibits the glucose production via
these processes by the liver [259].
2.14.7.3 Insulin Regulation
Insulin has short plasma half -life of only 5- 6 minutes [255]. Insulin is degraded in liver,
kidney, and muscle tissues [260]. About 50 % of insulin secreted reaches general
circulation as it is extensively degraded by the liver. The Insulin is cleared off from blood
through renal clearance. Some insulin degradation occurs in peripheral tissues following
internalization into the cells, but this accounts for only a small amount of insulin
elimination. When injected subcutaneously, insulin first enters the general circulation,
thus having its initial effect on peripheral tissues. This does not mimic the natural process
of insulin secretion and the biological effect is different.
65
Figure 2.13:Diagram of the insulin and glucose regulation model
66
CHAPTER 3 MATERIALS AND METHOD
CHAPTER 3
MATERIALS AND METHODS
67
3. Materials and Methods
3.1 Materials
Timolol maleate (TM) purity 97.5% was provided by Schazo Pharma Pvt. Ltd. Lahore,
Rosuvastatin (RST), purity 98.4% was kind gift of Ferozsons Laboratories Pvt. Ltd.
Nowshera, Recombinant Human insulin was a kind gift from Zafa Pharmaceuticals
Karachi, Diclofenac sodium (DS) purity 99.30% (manufactured by Suzhou Ausun
chemical company limited), Methylcellulose (MC) 4000 cps, hydroxy propylylmethyl
cellulose (HPMC, Methocel E5) manufactured by Dow chemical and polyethylene glycol
(PEG 6000) manufactured by Clariant GMBH were the kind gifts of Medicraft
Pharmaceuticals. Pvt, Ltd Peshawar. Voren injection (DS) manufactured by Asian
Continental Pharma, Blotim 0.5% (TM) ophthalmic solution manufactured by Remington
Pharmaceuticals Lahore and Dofnac 0.1% (DS) ophthalmic eye drops manufactured by
Ethical Laboratories Karachi were purchased from local market. Ciprofloxacin,
levofloxacin, naproxen sodium, atenolol, propanolol, and atorvastatin were obtained from
Fluka (Sigma–Aldrich, Oslo, Norway). Methanol, acetonitrile, diethylether,
tetrahydrofuran, chloroform, dichloromethane, n-hexane, ethanol, phosphoric acid, and
triethylamine (HPLC grade), Pluronic F-127 (Poloxamer406), were purchased from
Sigma–Aldrich (Oslo, Norway). Purified water was prepared using a Millipore ultra-pure
water system (Milford, USA).
3.2 Instrumentation
Perkin-Elmer HPLC system (Norwalk, USA), consisted of a pump (series 200), on-line
vacuum degasser (series 200), auto-sampler (series 200), Peltier column oven (series
68
200), linked by a PE Nelson network chromatography interface (NCI) 900 with UV/VIS
(series 200). The whole HPLC system was controlled by Perkin-Elmer Total chrom
Workstation Software (version 6.3.1). UV/Visible spectrophotometer (Lambda 25, Perkin
Elmer), Centrifuge (Centurion. Scientific Ltd), Shaking water bath B.S.11 Lab
Companion (Jelo Tech Korea), pH meter (Hanna instruments 8417, USA) and Autoclave
HS-60 (Hansuin Medical Co. Ltd Korea).
3.3 Study Design
The present study is designed to develop and evaluate thermoreversible solution to gel
vehicle for sustained drug delivery through ocular and subcutaneous routes. The study
was carried out in two phases. In the first phase solutions of various concentrations of the
polymers including Poloxamer 407/Pluronic F-127 (P-127), Hydroxypropyl methyl
cellulose (HPMC), Methyl Cellulose (MC) and Polyethylene glycol were prepared alone
and in combination with each other. The formulations were evaluated for solution to gel
transition temperature. The selected formulations that gel below body temperature and
were free flowing at room temperature were further evaluated for rheological behavior
and gel clarity.
In second phase drugs (Diclofenac sodium, Timolol maleate and insulin) were
incorporated in the selected formulations, and were qualitatively evaluated both in-vitro
and in-vivo for sustained release profile. The in-vitro data was analyzed by Minitab® and
MS-Excel and fitted to various mathematical models, while the in vivo study was
processed by pk-summit® softwares.
69
3.3.1 Preparation of Polymer Solutions
The Polymer solutions of varying concentration (W/V%) alone and in different
combinations of PL-127, MC, HPMC and PEG 6000 were prepared by cold
method[261]. The formulations of the polymer alone and in combination are shown in
Table 3.1.Briefly, accurately weighed polymers were stirred in the calculated amount of
cold (4oC) distilled water. The dispersions were then stored in a refrigerator with
occasional stirring until clear solutions were obtained.
70
Table 3.1: Composition of polymer solutions for screening
PL F127 MC HPMC PEG Distilled

S.No Formulation
(gm) (gm) (gm) (gm) Water
1. P5 5.0 ----- ----- ----- qs to 100 ml
2. P10 10.0 ----- ----- ----- qs to 100 ml
3. P15 15.0 ----- ----- ----- qs to 100 ml
4. P18 18.0 ----- ----- ----- qs to 100 ml
5. P20 20.0 ----- ----- ----- qs to 100 ml
6. P23 23.0 ----- ----- ----- qs to 100 ml
7. P25 25.0 ----- ----- ----- qs to 100 ml
8. P30 30.0 ----- ----- ----- qs to 100 ml
9. H1 ----- ----- 1.0 ----- qs to 100 ml
10. H2 ----- ----- 2.0 ----- qs to 100 ml
11. H3 ----- ----- 3.0 ----- qs to 100 ml
12. H4 ----- ----- 4.0 ----- qs to 100 ml
13. H5 ----- ----- 5.0 ----- qs to 100 ml
14. H6 ----- ----- 6.0 ----- qs to 100 ml
15. H8 ----- ----- 8.0 ----- qs to 100 ml
16. H 10 ----- ----- 10.0 ----- qs to 100 ml
17. M1 ----- 1.0 ----- ----- qs to 100 ml
18. M2 ----- 2.0 ----- ----- qs to 100 ml
19. M3 ----- 3.0 ----- ----- qs to 100 ml
20. M4 ----- 4.0 ----- ----- qs to 100 ml
21. M5 ----- 5.0 ----- ----- qs to 100 ml
22. PM 10/3 10.0 3.0 ----- ----- qs to 100 ml
23. PM 15/3 15.0 3.0 ----- ----- qs to 100 ml
24. MPG 1.5/2 ----- 1.5 ----- 2.0 qs to 100 ml
25. MPG 1.5/5 ----- 1.5 ----- 5.0 qs to 100 ml
26. MPG 1.5/10 ----- 1.5 ----- 10.0 qs to 100 ml
27. MPG 3/2 ----- 3.0 ----- 2.0 qs to 100 ml
28. MPG 4/2 ----- 4.0 ----- 2.0 qs to 100 ml
29. HPG 1.5/2 ----- ----- 1.5 2.0 qs to 100 ml
30. HPG 1.5/5 ----- ----- 1.5 5.0 qs to 100 ml
31. HPG 1.5/10 ----- ----- 1.5 10.0 qs to 100 ml
32. HPG 3/2 ----- ----- 3.0 2.0 qs to 100 ml
33. HPG 6/2 ----- ----- 6.0 2.0 qs to 100 ml
34. HPG 10/2 ----- ----- 10.0 2.0 qs to 100 ml
P=Pluronic F127, M= Methyl cellulose 5cps, H= Hydroxy propyl methylcellulose 15 cps,

PG= polyethylene glycol 6000
71
3.3.2 Measurement of the Sol–Gel Transition Temperature (Tsol-gel)
The Tsol–gel of the preparations was determined by tube inversion method [27, 121]. 2
ml aliquot of each prepared solution was transferred to a 5 ml test tube in a digital
Shaking water bath B.S.11 Lab Companion (Jelo Tech Korea) maintained at 15oC and
sealed with a parafilm. The temperature of the bath was increased in increments of 3oC
(or 1oC in region of sol–gel transition temperature) upto 50oC. At each temperature point
the solution was allowed to equilibrate for 5 minutes. The samples were checked for
gelation by inverting the test tube at 90o, the solutions were said to be gel when no
fluidity was observed. Measurements were performed in triplicates and Student’s t-test at
P < 0.05 was performed for statistical significance.
3.3.3 Measurement of Steady Shear Viscosity
The rheological behavior before and after autoclaving of the solutions were studied using
Brookfield viscometer; type DVT-2. A 0.5 ml sample of the solution was placed at the
lower plate of the viscometer. The viscosity was determined at 35±0.1oC using spindle 52
at a shear rate ranging from 5 to 400 rpm. The shear rate (γ) in s−1 and the viscosity (η)
in centipoises (cps) were determined from the instrument readings and fitted to the power
law equation [262].
η = m γn−1 eq. 3.1
The consistency index (m) and the flow index (n) two dimensionless quantities for each
preparation were obtained. If the value for n=1 this indicates Newtonian behavior and if
the value of (n) is less than 1, this corresponds to shear thinning flow. The lower the
value of (n) the more shear thinning will be the preparation [113, 263, 264].
72
3.3.4 Clarity of the Formed Gel
The clarity of the solutions at various temperatures i.e. at 5oC, 25oC and gelling
temperature was observed visually.
3.3.5 Autoclaving
To study the effect of autoclaving on physical properties of the preparation, the selected
preparations were subjected to autoclaving sterilization conditions[178]. Briefly, screw
cap test tubes containing 10 ml of the preparation were placed in an autoclave HS-60
(Hansuin Medical Co. Ltd Korea). The autoclaving conditions were 121oC, 15 psi, and 20
minutes. The preparations were cool down to room temperature and were evaluated for
their physical properties i.e. Viscosity, clarity of solution and sol–gel transition
temperature, and compared to those before being autoclaved.
3.3.6 Preparation of Drug Polymer Solutions
Solutions of the drugs in selected polymer formulation (formulations that gels around
35oC) were prepared as below.
3.3.7 Preparation of Diclofenac Sodium In-situ Gel Formulations
The drug and polymers were accurately weighed according to the composition for the
formulations as shown in Table 3.2. Diclofenac sodium 5mg/ml solutions with different
polymers alone and in polymers combination were prepared using cold method [261,
265].
73
The polymer was dispersed in cold water with continuous stirring, the dispersion was
then stored in refrigerator for 24 hours to obtain clear polymer solution, to this solution
diclofenac sodium was added and dissolved with continuous stirring till clear solution is
obtained. The final solutions were sterilized by autoclaved and were evaluated for their
physicochemical properties i.e. Tsol-gel, viscosity, clarity, drug content, in-vitro drug
release and in-vivo pharmacokinetic parameters.
Table 3.2: Composition of Diclofenac Sodium thermoreversible gel
Diclofenac Sodium PL F127 MC PEG Distilled

S.No Formulation
(mg) (mg) (mg) (mg) Water
1. DP18 5.0 180 ----- ----- qs to 1 ml
2. DP20 5.0 200 ----- ----- qs to 1 ml
3. DPM 15/3 5.0 150 30.0 ----- qs to 1 ml
4. DMPG 1.5/10 5.0 ----- 15.0 100.0 qs to 1 ml
5. DMPG 3/2 5.0 ----- 30 20.0 qs to 1 ml
3.3.8 Preparation of Timolol Maleate In-situ Gel Formulations
formulations as shown in Tables 3.3. TM 5mg/ml solutions with different polymers alone
and in polymers combination were prepared using cold method [261, 265].
74
TM was added and dissolved with continuous stirring till clear solution is obtained. The
final solutions were sterilized by autoclaving and were evaluated for their
physicochemical properties i.e. Tsol-gel, flowability, clarity, drug content, in-vitro drug
release and in-vivo pharmacokinetic parameters.
Table 3.3: Composition of Timolol maleate thermoreversible gel
Timolol maleate PL F127 MC PEG Distilled

S.No Formulation
(mg) (mg) (mg) (mg) Water
1. TP18 5.0 180 ----- ----- qs to 1 ml
2. TP20 5.0 200 ----- ----- qs to 1 ml
3. TPM 15/3 5.0 150 30.0 ----- qs to 1 ml
4. TMPG 1.5/10 5.0 ----- 15.0 100.0 qs to 1 ml
5. TMPG 3/2 5.0 ----- 30 20.0 qs to 1 ml
3.3.9 Preparation of Human Insulin (recombinant) In-situ Gel Formulations
formulations as shown in Table 3.4. Insulin 1mg/ml (27.5 IU) solutions with different
polymers alone and in polymers combination were prepared using cold method [261,
265].
75
recombinant human insulin was added and dissolved with continuous stirring till clear
solution is obtained. The final solutions were sterilized by micro filtration and were
evaluated for their physicochemical properties i.e. Tsol-gel, viscosity, clarity, drug
content, in-vitro drug release and in-vivo pharmacokinetic parameters.
Table 3.4: Composition of Insulin thermoreversible gel
Insulin PL F127 MC Distilled

S.No Formulation
(mg) (mg) (mg) Water
1. IP18 1.0 180 ----- qs to 1 ml
2. IP20 1.0 200 ----- qs to 1 ml
3. IPM 15/3 1.0 150 30.0 qs to 1 ml
3.3.10 Measurement of Tsol- gel DS Formulations
The Tsol–gel of all thermorevesible solution was measured by tube inversion method [27,
121]. 2 ml of each solution was transferred to a 5 ml test tube in a digital Shaking water
bath B.S.11 Lab Companion (Jelo Tech Korea) maintained at gelation temperature and
sealed with a parafilm. The solution was allowed to equilibrate for 5 minutes to effect the
gelation. The samples were checked for gelation by inverting the test tube at 90o.
Measurements were performed in triplicates and Student’s t-test at P < 0.05 was
performed for statistical significance.
76
3.3.11 Measurement of Steady Shear Viscosity of Diclofenac Sodium Formulations
The steady shear viscosity of the DS in-situ gels was measured using cone and plate
viscometer. A 0.5 ml sample of the solution was applied to the lower plate of the
viscometer. The measurements were made at 37±0.1oC using spindle 52 at a shear rate
ranging from 5 to 400 rpm. The measurements were made before and after autoclaving.
All samples were analyzed in triplicate and for statistical significance student’s t-test at p
< 0.05 was performed.
3.3.12 Clarity of the Formed Gel of DS Formulations
The clarity of the DS solution and formed gels before and after autoclaving of the
formulations was observed visually at 5oC, 25oC and 37oC.
3.3.13 Drug Content of DS Formulations
All samples were analyzed in triplicate for the drug content using validated HPLC
method [266] before and after autoclaving. Only samples with drug content within 100 ±
10% of labeled amount were accepted.
3.3.14 Determination of In-vitro of Drug Release from DS Formulations
Membrane free models are recommended for in vitro evaluation of in-situ gels to be
employed as vehicle for ophthalmic and injectable formulations [100, 267, 268].
Membrane less model was used in the study[269] to evaluate the drug release from the
in-situ solution to gel formulations. The selected DS formulations were subjected to in
vitro drug release profile. From each preparation 2 ml solution containing DS was
transferred into 10 ml tubes. The test tubes containing the solutions were then
77
equilibrated for 5 minutes to effect the gelation in digital shaking water bath, maintained
at 37oC ± 1. Phosphate buffer pH 7.4 used as dissolution medium (2 ml) was poured
slowly on the surface of the gel not to disturb the surface layer. The water bath was
operated at 50 rpm. Whole of the dissolution medium was collected as sample after
predefined time intervals i.e.0.5, 1.0, 2.0, 4.0, 8.0, 12.0, 24, 36, 48, 72, 96 and 120 hours
depending upon formulation that remain intact for the specified time. As soon as samples
were collected fresh 2ml dissolution medium was added to the test tube.
The samples were suitably diluted and analyzed for diclofenac content using validated
HPLC method[266]. The dissolution samples were taken in triplicate for each
formulation.
3.3.15 Measurement of Tsol- gel of TM Formulations
The solution to gel transition temperature of the prepared formulation was recorded by
tube inversion method [27, 121]. For each formulation 2 ml of each solution was taken in
a 5 ml test tube sealed with parafilm in a digital Shaking water bath B.S.11 Lab
Companion (Jelo Tech Korea) maintained at gelation temperature. The solution was
allowed to equilibrate for 5 minutes to effect the gelation. The samples were checked for
gelation by inverting the test tube at 90o. Measurements were performed in triplicates and
Student’s t-test at P < 0.05 was performed for statistical significance.
3.3.16 Measurement of Steady Shear Viscosity of TM Formulations
The steady shear viscosity of the TM in-situ gels was measured using cone and plate
viscometer. From each formulation 0.5 ml of the solution was applied to the lower plate
78
of the viscometer. The measurements were made at 37 ± 0.1oC using spindle 52 at a shear
rate ranging from 5 to 400 rpm. The viscosity (η) in centipoises (cps) and the shear rate
(γ) in s−1 were noted and recorded from the instrument reading. The data was compared
with results obtained after sterilization by autoclaving. All samples were analyzed in
triplicate and for statistical significance student’s t-test at P< 0.05 was performed.
3.3.17 Clarity of the Formed Gel of TM Formulations
The clarity of the timolol solution and gel formulations before and after autoclaving was
determined visually at 5oC, 25oC and 37oC.
3.3.18 Drug Content of TM Formulations
All samples were analyzed in triplicate for the drug content using validated HPLC
method[266] before and after autoclaving. Only samples with drug content within 100 ±
10% of labeled amount were accepted.
3.3.19 Determination of In-vitro of Drug Release from TM Formulations
Timolol maleate release from the TM in-situ gel formulations was determined by
membrane less model [269]. The selected TM formulations were subjected to in vitro
drug release profile. 2ml of each preparation was taken in 10 ml test tubes. The test tubes
containing the solutions were equilibrated for 5 minutes to effect the gelation in digital
shaking water bath maintained at 37oC ± 1. Phosphate buffer pH 7.4 as dissolution
medium (2ml) was poured slowly on the surface of the gel not to disturb the surface
layer. The water bath was operated at 50 rpm. After predefined time interval i.e.0.5, 1.0,
2.0, 4.0, 8.0, 12.0, 24, 36, and 48 hours, whole of the dissolution medium was replaced
79
with fresh dissolution medium as sample. The samples were suitably diluted and
analyzed for TM content using validated HPLC method[266].
3.3.20 Measurement of Tsol- gel Insulin Preparations
The sol–gel transition temperature for the prepared formulations was measured by tube
inversion method [27, 121] before and after sterilization. 2 ml of each solution was
transferred to a 5 ml test tube in a digital Shaking water bath B.S.11 Lab Companion
(Jelo Tech Korea) maintained at gelation temperature and sealed with a parafilm. The
solution was allowed to equilibrate for 5 minutes to effect the gelation. The solutions
were checked for gelation by inverting the test tube at 90o. Measurements were
performed in triplicates and Student’s t-test at P < 0.05 was performed for statistical
significance.
3.3.21 Measurement of Steady Shear Viscosity of Insulin Preparations
The viscosity of the Insulin in-situ gels was measured using cone and plate viscometer
before and after sterilization. A 0.5 ml sample of the solution was applied to the lower
plate of the viscometer. The viscosity was determined at 37±0.1oC using spindle 52 at a
shear rate ranging from 5 to 400 rpm. The viscosity (η) in centipoises (cps) and the shear
rate (γ) in s−1 were recorded from the instrument reading.
All samples were analyzed in triplicate and for statistical significance student’s t-test at
P< 0.05 was performed.
80
3.3.22 Clarity of the Formed Gel of Insulin Preparations
The clarity of the formed gels before and after sterilization of the formulations was
observed visually.
3.3.23 Drug Content of Insulin Preparations
All samples were analyzed in triplicate for the drug content using HPLC method [270]at
a wavelength of 206 nm before and after sterilization. Only samples with drug content
within 100 ± 10% of labeled amount were accepted.
3.3.24 Determination of In-vitro Drug Release from Insulin Preparations
In vitro insulin release in dissolution medium was determined by membrane less model
[269]. The selected insulin formulations were subjected to in vitro drug release profile.
From each formulation 2ml solution was transferred to 10 ml test tubes. The test tubes
were equilibrated for 5 minutes to effect the gelation in digital shaking water bath
maintained at 37oC ± 1. Phosphate buffer pH 7.4 as dissolution medium was poured
slowly on the surface of the gel not to disturb the surface layer. The water bath was
operated at 50 rpm. Whole of the dissolution medium was collected as sample after
predefined time intervals i.e.0.5, 1.0, 2.0, 4.0, 8.0, 12.0, 24, 36, 48, 72, 96, 120, 144, 168,
192, 216, 240 and 264 hours depending upon formulation that remain intact for the
specified time. As soon as samples were collected fresh 2ml dissolution medium was
added to the test tube.
The samples were suitably diluted and analyzed by HPLC-UV method for insulin content
[270]. The dissolution samples were taken in triplicate for each formulation.
81
3.3.25 Drug Release Kinetics
The data obtained from in vitro experiments was fitted to various mathematical models
that are employed to assess the drug release kinetics. The results obtained were plotted as
drug released versus time.
3.3.25.1 Zero Order Kinetic Model
The dosage form that follows zero order release kinetics release same amount of drug by
unit of time, which is an ideal release profile independent of drug amount present in the
dosage form. The data obtained was analyzed by employing the equation 3.2. The data
was plotted as cumulative % drug release versus time.
(eq. 3.2)
Where Qt is the amount of drug dissolved in time t, Q0 is the initial amount of drug in the
solution and K0 is zero order release constant.
3.3.25.2 First Order Kinetic Model
This model relates decimal of logarithm cumulative % drug released versus time. The
drug release kinetics is said to be first order if the graphic is linear, indicating that the
drug release is dependent on the drug remaining in the dosage form. The equation for first
order kinetics is given as eq. 3.3.
(eq. 3.3)
.
Where Qt is the amount of drug dissolved in time t, Q0 is the initial amount of drug in the
solution and Kt is first order release constant.
82
3.3.25.3 Higuchi Model
The model was proposed by Higuchi (1961, 1963) that relates cumulative drug release
versus square root of time as shown in eq. 3.4.
√ (eq. 3.4)
3.3.25.4 Hixson –Crowell Model
The model was developed in 1931 by Hixson-Crowell, which relates cubic root of the
unreleased drug remaining in the dosage form versus time. As given by eq. 3.5.
/ /
(eq. 3.5)
3.3.25.5 Korsmeyer-Peppas Model
This model relates exponentially the drug release to the elapsed time. The equation is
given as eq. 3.6.
(eq. 3.6)
∞
Peppas in 1985 use the following values given in the table 3.5 to interpret the release
mechanism from the polymeric system.
Table 3.5:Interpretation of diffusion release mechanism from polymeric system
Release exponent (n) Drug transport mechanism Rate as a function of time
0.45 Fickian diffusion t-0.5
0.45<n<1.0 Anomalous transport tn-1
1.0 Case II transport Zero order release
Higher than 1.0 Super case II transport tn-1
83
3.3.26 In Vivo Evaluation of Formulations
3.3.26.1 Animal Handling
Male New Zealand white rabbits weighing 2.5 ± 0.15 kg were used both for subcutaneous
and ophthalmic route. Rabbits were kept individually in standard stainless steel cages, fed
with a commercial laboratory rabbit diet and were freely allowed to water. The animals
were fasted for 12 hours prior to study.
3.3.26.2 Diclofenac Sodium Administration and Sampling
3.3.26.2.1 Subcutaneous Route
Six animals were divided into two groups. Each group was treated with either the
representative DS in situ gel or the commercial diclofenac sodium injection by injecting
2ml of each test formulation subcutaneously to each rabbit. Blood sample of 500 µl from
each animal was collected from marginal ear vein five minutes before injecting the
formulation and at predetermined time intervals i.e. 0.5, 1, 2,4, 8,12, 24, 36, 48, 72, 96
and 120 hours for each formulation. Blood samples were collected in
ethylenediamintetraacetic acid (EDTA) glass tubes and centrifuged at 1600× g for 10 min
at 4oC.The plasma was collected and stored at -20oC until analysis. Diclofenac sodium
content in plasma was determined by using a validated High Performance Liquid
Chromatography (HPLC) technique[266].
Rabbits were divided into two groups. Each group was instilled with either the
representative DS in situ gel or the commercial diclofenac sodium eye drop (0.1%, w/v)
84
by instilling 20 µl of each test formulation into the right eye of each rabbit. After
instillation of drug solution three animals were then killed at the following time intervals:
0.5, 1, 2, 3, 4 and 6 hours for each formulation. 200 µl aqueous humour was withdrawn
with a 27-gauge, 1.3 cm needle attached to 1 ml disposable syringe. The aqueous humour
was stored at -20oC prior to HPLC analysis for Diclofenac sodium content.
3.3.26.3 Timolol Maleate Administration and Sampling
Rabbits were divided into two groups. Each group was instilled with either the
representative TM in situ gel or the commercial TM eye drop (0.5%, w/v) by instilling 20
µl of each test formulation into the right eye of each rabbit. After instillation of drug
solution three animals were then killed at predetermined time intervals: 0.5, 1, 2, 3, 4 and
6 hours for each formulation. 200 µl aqueous humour was withdrawn with a 27-gauge,
1.3 cm needle attached to 1 ml disposable syringe. The aqueous humour was stored at -
20oC prior to HPLC analysis for TM content.
3.3.26.4 Insulin Administration and Sampling

3.3.26.4.1 Subcutaneous route
New Zealand White rabbits with initial body weight of 2– 2.5 kg and maintained on
standard laboratory food were injected with10%w/v alloxan (125 mg/kg body weight) via
a marginal ear vein. The diabetic animals were divided into two groups. Each group was
treated with either the representative Insulin in situ gel or the commercial insulin by
injecting 2ml (2U) of each test formulation subcutaneously to each rabbit. Blood sample
of 500 µl from each animal was collected from marginal ear vein five minutes before
85
injecting the formulation and at predetermined time intervals i.e. 0.5, 1, 2,4, 8,12, 24, 36,
48, 72, 96, 120, 144, 168, 192, 216 and 240 hours for each formulation. Plasma insulin
was determined using insulin enzyme immunoassay kit (Dainabot Co., Ltd., Tokyo,
Japan).
3.3.27 Pharmacokinetic Parameters
To assess different pharmacokinetic parameters like tmax, Cmax, Half-life (t ½), Area under
cure (AUC), Area under moving curve (AUMC), Mean residence time (MRT) and
Volume of distribution (Vd), a non-compartmental model approach was applied[271] to
the data obtained for all the formulations.
3.3.28 HPLC analysis method
3.3.28.1 Materials and methods
3.3.28.1.1 Materials and reagents
Timolol maleate (TM) purity 97.5% (Schazo Pharma Pvt. Ltd. Lahore), rosuvastatin
(RST), purity 98.4% (Feroz Sons Laboratories Pvt. Ltd. Nowshera), diclofenac sodium
(DS) purity 99.0% (Medicraft Pharma. Pvt. Ltd. Peshawar), were the kind gift of local
pharmaceuticals. Ciprofloxacin, levofloxacin, naproxen sodium, atenolol, propanolol, and
atorvastatin were obtained from Fluka (Sigma–Aldrich, Oslo, Norway). Methanol,
acetonitrile, diethylether, tetrahydrofuran, chloroform, dichloromethane, n-hexane,
ethanol, phosphoric acid, and triethylamine (HPLC grade), were purchased from Sigma–
Aldrich (Oslo, Norway). Purified water was prepared using a Millipore ultra-pure water
system (Milford, USA).
86
3.3.28.1.2 Instrumentation
Perkin-Elmer HPLC system (Norwalk, USA), consisted of a pump (series 200), on-line
vacuum degasser (series 200), auto-sampler (series 200), Peltier column oven (series
200), linked by a PE Nelson network chromatography interface (NCI) 900 with UV/VIS
(series 200). The whole HPLC system was controlled by Perkin-Elmer Total chrom
Workstation Software (version 6.3.1). The data was acquired and quantified by this
software.
3.3.28.2 Preparation of Standard Stock Solutions
Stock solutions of analytes and IS (ciprofloxacin, levofloxacin, naproxen sodium,
atenolol, propanolol, and atorvastatin), each (1 mg/ml) were prepared in acetonitrile and
stored in amber glass vials at −20oC until analysis. Working standards solutions were
prepared in volumetric flasks (10 ml), using mobile phase in the concentration range of
0.05–2 µg/ml of each analyte, keeping IS concentration 1 µg/ml in each sample. The
calibration curves were constructed at seven concentrations levels for the standard
solutions of each analyte. Similarly, a 1:1 mixture containing 1 µg/ml of each analyte and
IS was also prepared.
3.3.28.3 Sample Preparation
3.3.28.3.1 Plasma sample
Blood samples were collected from human volunteers, at Department of Pharmacy,
University of Peshawar (Pakistan), in ethylenediamintetraacetic acid (EDTA) glass tubes
and centrifuged at 1600× g for 10 min at 4oC. The study was approved by the concerned
ethical committee. The plasma was collected and stored at −20oC until analysis. For
87
sample preparation the plasma was first thawed at room temperature and a volume (200
µl) was spiked with the respective volume in the range of 10–400 µl of standard stock
solution (50 µg/ml), of TM, RST and DS each to prepare their respective dilutions in the
range of 0.05–2 µg/ml at 0.05, 0.100, 0.250, 0.500, 1.00, 1.50, and 2 µg/ml for each
analyte. The equal volume (10 µl) of IS (1 µg/ml), was added to each sample to make its
concentration 1 µg/ml in each sample and vortexed for 3 min. The liquid–liquid
extraction procedure was applied as given in section 3.3.28.4.
3.3.28.3.2 Aqueous Humour Sample
Aqueous humour was collected in borosilicate glass tubes from the bovine eyes. The
collected sample was stored in screw capped air tight glass vials at -20oC till analysis. At
the time of analysis aqueous humour was thawed and a volume (200 µl), was spiked with
the respective volume in the range of (10–400 µl), of the standard solutions of TM, and
DS each to prepare their respective dilutions in the range of 0.05–2 µg/ml at 0.05, 0.100,
0.250, 0.500, 1.00, 1.50, and 2 µg/ml for each analyte. An equal volume (10 µl) of IS (1
µg/ml) was added to each sample to make its concentration 1 µg/ml in each sample and
vortexed for 3 min. The extraction was carried out as given in Section 3.3.28.4.
3.3.28.4 Liquid-Liquid Extraction
Sample (200 µl) was transferred to plastic eppendorf tube (ca≈2 ml), and spiked with
each analyte in its respective concentration range and a constant amount of IS (1 µg/ml)
was added to each sample. The samples were vortex-mixed for 3 min and methanol (600
µl) was added for de-proteination. Extraction was carried out with 1 ml of mobile phase.
The samples were then centrifuged for 5 min at 2000×g and 4oC. After centrifugation the
88
clear supernatant was transferred to eppendorf tube and the volume was made to 1 ml
with mobile phase. Sample (50 µl) of the supernatant was injected into HPLC for
analysis.
Calibration curves were constructed for all the analytes in the range of 0.05–2 µg/ml at
0.05, 0.100, 0.250, 0.500, 1.00, 1.50, and 2 µg/ml for each analyte using naproxen
sodium (1 µg/ml) as IS in mobile phase, spiked plasma and spiked aqueous humour and
different columns like Hypersil BDS C18 column (250 mm ×4.6 mm,5 µm); Symmetry
C8 (250 mm ×4.6 mm, 5 µm); Discovery HS C18 column (150 mm ×4.6 mm, 5 µm),
Symmetry C8 columns (150 mm ×3.9 mm, 5 µm) were evaluated for separation of drugs
in the mixture.
3.3.28.5 Chromatographic Conditions
The HPLC analysis of the studied compounds was performed using ACN: 0.2% TEA
(60:40, v/v), pH 2.75 adjusted with 85% phosphoric acid, as mobile phase pumped at
flow rate of 1 ml/min, in isocratic mode on Hypersil BDS C18 column (250 mm×4.6
mm, 5 µm). The column oven temperature was kept at 45 oC and the peak response was
monitored at a wavelength of 284 nm. The sample (50 µl) was injected into HPLC
system and the data was acquired using Perkin-Elmer Total chrom Workstation Software
(version 6.3.1).
89
3.3.28.6 Chromatographic Conditions and Experimental Parameters Optimization
3.3.28.6.1 Selection of Stationary Phase (column)
Different particulate reversed-phase chromatographic columns (stationary phases) such
as; Hypersil BDS C18 column (250 mm×4.6 mm, 5 µm); Symmetry C8 (250 mm×4.6
mm,5 µm); Discovery HS C18 column (150 mm×4.6 mm, 5 µm), Symmetry C8 column
(150 mm×3.9 mm, 5 µm), protected by a Perkin Elmer C18 (30 mm×4.6 mm, 10 µm;
Norwalk, USA), pre-column guard cartridge were tried for the analysis of TM, RST and
DS.
3.3.28.6.2 Mobile Phase Composition
The mobile phase composition was optimized using various organic solvents including
methanol, acetonitrile, tetrahydrofuran, and water in different composition in isocratic
mode for the analysis of the above mentioned compounds. The mobile phase composition
that resulted in a better resolution and shorter analysis time of the studied compounds was
selected as mobile phase for the simultaneous analysis.
3.3.28.6.3 Mobile Phase Flow Rate
The mobile phase flow rate was adjusted in isocratic mode for the analysis of studied
analytes after applying various flow rats in the range of 0.9–2 ml/min.
3.3.28.6.4 Column Oven Temperature
The column oven temperature significantly affects the elution and resolution of different
compounds. The column oven temperature was therefore evaluated in the range of 25–
50oC to show its effects on the analysis of above mentioned compounds.
90
3.3.28.6.5 Internal Standard
Different compounds including ciprofloxacin, levofloxacin, naproxen sodium, atenolol,
propanolol, and atorvastatin were tried to be used as IS. The compound that showed
better compatibility, best recovery and shorter analysis time was selected as IS for the
suggested method.
3.3.28.6.6 Sample Size
The sample loop size was evaluated in the range of 20–50 µl to adjust the sample size and
minimize the problems like column loading and lack of sensitivity of the mentioned
compounds.
3.3.28.6.7 Detector Wavelength
For simultaneous determination of TM, RST, and DS using naproxen sodium as IS the
detector’s wavelength was evaluated in the range of 280–300 nm. The wavelength that
resulted in the optimal sensitivity and better resolution was chosen as the wavelength for
simultaneous analysis of studied compounds.
3.3.28.7 Validation of the Method
The proposed method was validated according to standard guidelines [272]. The
precision, specificity, sensitivity, linearity, recovery, robustness, stability of solutions and
system suitability parameters were evaluated. The proposed method was validated with
respect to the following parameters.
91
3.3.28.7.1 Specificity
The specificity of the suggested method was tested through separation of studied
compounds in the mobile phase, 1:1 mixture (containing 1 µg/ml of each analyte and IS),
plasma and aqueous humour samples spiked with appropriate concentration of each
analyte.
3.3.28.7.2 Percent Recovery
Percent recovery was tested to measure the accuracy of the suggested method. The %
recovery was determined from spiked plasma and aqueous humour samples at two
selected concentration levels of each analyte keeping IS concentration same in each
sample. Recovery was calculated according to the following equation:
(eq. 3.7)
Where [A] is the peak area response ratios of the analytes with reference to IS in the
mobile phase; C is the peak area response ratios of the analytes with reference to IS in
spiked plasma/aqueous humour.
92
Fig. 3.1.Overlay of different representative chromatograms. A = spiked aqueous humour,

B = spiked plasma sample and C = standard solution. Peaks: 1, timolol; 2, rosuvastatin; 3,
naproxen sodium; 4, diclofenac sodium. The chromatograms were obtained at column
oven temperature of 45 oC using mobile phase ACN:0.2% TEA in the ratio of 45:55 v/v
and flow rate of 1.0 ml/min.
3.3.28.7.3 Linearity
The linearity of the proposed method was determined from the calibration curves
constructed at seven concentration levels. Calibration curves were constructed for all the
analytes in the mobile phase, spiked plasma and spiked aqueous humour samples by
plotting their peaks response ratios (ratios of peak areas of analytes to IS) with respect to
their respective spiked concentrations using a linear least squares regression analysis. The
slope (m), intercept (b), and correlation coefficient (r) were calculated from their
respective regression equations.
93
3.3.28.7.4 Precision
Precision of the method was determined through injection repeatability and analysis
repeatability of spiked plasma and aqueous humour samples. Injection repeatability was
assessed through injecting 10 times the same plasma and aqueous humour spiked with 1
µg/ml of each analyte into the HPLC system. The retention time and peak area of each
analyte were expressed as mean, standard deviation (SD), and covariance (%RSD) as
precision of the suggested method. Analysis repeatability was evaluated from the analysis
of five spiked samples prepared from the same plasma/aqueous humour spiked with 1
µg/ml of each analyte, and the results were expressed as mean, standard deviation (SD),
and covariance (%RSD) of the recovered amount. Intra-day and inter-day studies were
carried out on spiked plasma/aqueous humour samples at 8:00, 16:00,and 24:00 h, for 1
week at alternate days to assess the intermediate precision. The results were expressed as
mean, standard deviation (SD), and covariance (%RSD) of the recovered amount.
3.3.28.7.5 Drug Concentration
The recovered amount was calculated in the form of concentration by the following
equation:
X A
C= × ×CS ×FD (eq. 3.8)
Y B
Where
X = Area of peak of analyte in sample i.e. Plasma/Aqueous humour/invite sample.
Y = Area of peak of internal standard added to the sample.
A = Area of peak of internal standard in 1:1 mixture.
B = Area of peak of the analyte standard in 1:1 mixture.
94
C = Amount of analyte in1:1 mixture.
FD=Dilution factor
3.3.28.7.6 Limit of Detection and Quantification
The limit of detection (LOD) and limit of quantification (LOQ) for all the analytes were
quantified at a concentration whose signal-to noise ratio (S/N) was three and ten,
respectively. For LOD and LOQ evaluation dilutions of the analytes were prepared in the
ranges of 0.5–5 ng/ml and 5–20 ng/ml for all the analytes. The LOD and LOQ were then
determined from the peaks by the software at signal-to noise ratio (S/N) of three and ten,
respectively.
3.3.28.7.7 Robustness
The robustness of the proposed method was tested through small deliberate changes in
the various chromatographic conditions, like mobile phase composition (±2%), column
oven temperature (±5oC), detector wave length (±2 nm) and flow rate of mobile phase
(0.2 ml/min).Stability studies of standard solutions and spiked plasma/aqueous humour
samples stored at 25oC, 4oC and −20oC were carried out for 1 month.
3.3.28.7.8 Percent Stability
The % stability was calculated by the following equation.
% 100 (eq. 3.9)
Where St is stability of analyte at time t, and S0 is stability at initial time.
95
CHAPTER 4 RESULTS AND DISCUSSION
CHAPTER 4
RESULTS AND DISCUSSION
96
4. Results and Discussion
The objective of the current study was to develop sustained release in-situ solution to gel
polymeric delivery system for the drugs to be administered through subcutaneous or
ocular route. Four polymers (Pluronic F-127, Methyl cellulose, hydroxypropyl
methylcellulose and polyethylene glycol 6000) were used in different concentrations
alone and in combination with each other in different ratios. Total of thirty four (34)
formulations were prepared and evaluated for physical and chemical attributes. All the
formulations were subjected to determine the Tsol-gel, out of which only seven
formulations were evaluated for rheological properties and clarity of the formed gels.
Five In-situ thermorevesible solution-gel formulations were selected for in-vitro drug
release by incorporating the selected drugs. On the basis of in-vitro evaluation selected
drug formulations were subjected to in-vivo evaluation. To quantify the amount of drug
(DS,TM) in the in-vitro and in-vivo samples HPLC –UV method was developed and
validated as per standard guidelines[272].
4.1 HPLC –UV Method for Analysis of Diclofenac Sodium and Timolol Maleate
The method developed was novel in the sense that simultaneous determination of TM,
RST, and DS was carried out for the first time using naproxen sodium as an IS. All the
analytes were separated applying the proposed method in standard mixtures, plasma
samples, and aqueous humour samples as presented in Fig. 3.1. The suggested method
was found accurate and quite specific for the simultaneous analysis of these compounds
in plasma and aqueous humour samples. Complete separation of the target compounds
was achieved in 7 min using the proposed method.
97
4.1.1 Optimization of HPLC Experimental Parameters
Different experimental parameters were optimized in the specified ranges to choose the
optimum mobile phase, stationary phase, mobile phase flow rate, detector’s wavelength,
column oven temperature and pH.
4.1.1.1 Mobile Phase
The mobile phase comprised of ACN: 0.2% TEA in the ratio of 60:40 (v/v), was selected
for the analysis of the above mentioned compounds. The retention times of the studied
compounds decreased with increasing the ratio of acetonitrile (ACN) in the mobile phase.
The overall analysis time decreased significantly with increasing the ACN content.
Figure 4.1:Influence of ACN ratios in the mobile phase on the elution of different analytes. A = having
45% ACN, B = having 50% ACN, C = having 55% ACN, D = having 60% ACN and E = having 70%
ACN. Peaks: 1, timolol; 2, rosuvastatin; 3, naproxen sodium; 4, diclofenac sodium. The chromatograms
were obtained at column oven temperature of 45 oC at a flow rate of 1.0 ml/min.
The effect of ACN on the retention time of DS was greater in comparison with TM and
RST. Increasing the ratio of ACN above 60% resulted in the co-elution of TM peak with
the solvent front although the analysis time decreased appreciably as presented in Fig 4.1.
98
4.1.1.2 Stationary Phase
The optimal stationary phase that resulted in better separation and resolution of the
studied compounds among the various tested stationary phases was selected for the
simultaneous determination of above mentioned analytes. The better separation was
achieved on Hypersil BDS C18 column among the tested columns. The poor resolution of
TM was obtained as TM was co-eluted with the peak of solvent front in case of other
tested columns.
4.1.1.3 Flow Rate
The flow rate of mobile phase greatly affected the analysis of the studied analytes.
Although run time decreased significantly at higher flow rates along with better
resolution of the peaks, however; sensitivities of the analytes decreased as shown in Fig.
4.2.
Figure 4.2:Influence of flow rate of the mobile phase on the elution of different analytes. A = at 0.8
ml/min, B = at 1.0 ml/min, C = at 1.25 ml/min, D = at 1.5 ml/min and E = at 2 ml/min. Peaks: 1, timolol; 2,
99
rosuvastatin; 3, naproxen sodium; 4, diclofenac sodium. The chromatograms were obtained at column oven
temperature of 45 oC using mobile phase ACN:0.2% TEA in the ratio of (60:40, v/v).
The flow rate greatly affected the retention of DS in comparison with other analytes. Co-
elution of TM with solvent front resulted at higher flow rates. The flow rate 1 ml/min was
chosen as optimal flow rate for the simultaneous analysis of these compounds.
4.1.1.4 Column Oven Temperature
Peak sensitivities were not affected significantly by column oven temperature, however,
run time was decreased and resolution of the peaks was increased at higher temperature.
The optimal temperature for the simultaneous analysis chosen was 45oC as shown in Fig.
4.3.
Figure 4.3:Influence of column oven temperature on the elution of different analytes. A = at 50oC, B = at
45oC, C = at 40oC, D = at 35oC, E = at 30oC and F = 25oC. Peaks: 1, timolol; 2, rosuvastatin; 3, naproxen
sodium; 4, diclofenac sodium. The chromatograms were obtained at a flow rate of 1.0 ml/min using mobile
phase ACN:0.2% TEA in the ratio of (60:40, v/v).
4.1.1.5 pH
Higher sensitivities and better resolution of the analytes were achieved at pH 2.75 of the
mobile phase among the different tested pH of the mobile phase as shown in Fig. 4.4. The
100
sensitivity of DS was greatly affected by pH of the mobile phase as compared with other
analytes. Variation in the retention times of the analytes was observed at different pH as
shown in Fig. 4.4.
Figure 4.4:Influence of various pH of the mobile phase on the elution of different analytes. A = at pH
2.5, B = at pH 2.75, C = at pH 3.0 and D = at pH 3.5. Peaks: 1, timolol; 2, rosuvastatin; 3, naproxen
sodium; 4, diclofenac sodium. The chromatograms were obtained at column oven temperature of 45 oC
using mobile phase ACN:0.2% TEA in the ratio of (60:40, v/v), and flow rate of 1.0 ml/min.
4.1.1.6 Wave Length
Detector’s wavelength has been selected after recording the sensitivities of the analytes at
various wavelengths. The greater sensitivities of TM, RST and DS were recorded at 284
nm. Small changes in the retention times of the analytes have been observed as evident in
Fig 4.5.
101
Figure 4.5:Influence of detector wavelengths on the elution of different analytes. A = at 280 nm, B = at
284 nm, C = at 290 nm, D = at 295 nm and E = at 300 nm. Peaks: 1, timolol; 2, rosuvastatin; 3, naproxen
sodium; 4, diclofenac sodium. The chromatograms were obtained at column oven temperature of 45 oC
using mobile phase ACN:0.2% TEA in the ratio of 60:40 (v/v), and flow rate of 1.0 ml/min.
4.1.1.7 Internal Standard
Internal standard (IS) that has shown better sensitivity, recovery, stability and
compatibility with other analytes was chosen among the various tested compounds.
Naproxen sodium was used as IS in these studies as it resulted in better recovery and
good compatibility in comparison with other tested compounds.
4.1.1.8 Sample Preparation
Standard stock solutions of TM, RST, DS and naproxen sodium were prepared in
acetonitrile and working dilutions were prepared in the mobile phase on daily basis.
Extraction from plasma and aqueous humour samples was carried out using different
organic solvents. De-proteination was carried out using methanol and the analytes were
then extracted applying various organic solvents.
102
The mobile phase resulted in better recoveries of the analytes as compared with other
solvents used for extraction. The supernatant separated after centrifugation was directly
injected into HPLC system and complete separation of all the target analytes was
achieved.
4.1.1.9 Method Validation
The developed method was validated in terms of linearity, selectivity, sensitivity,
recovery, precision, and robustness according to standard guidelines. The target
compounds were separated in standard mixtures, plasma and aqueous humour samples
using the proposed method with no extraneous peak. The method was found quite
suitable for the analysis of the above studied analytes.
103
Table 4.1: Validation parameters of HPLC-UV Method
Parameters Analytes
Timolol Maleate Rosuvastatin Diclofenac Sodium
Linearity
0.05-2 µg/ml 0.05-2 µg/ml 0.05-2 µg/ml
Calibration range (µg/ml)
Standard solution
Regression equation y=1.523x+0.023 y=1.592+0.038 y=3.955x+0.010
Correlation coefficient (R2) 0.999 0.999 0.999
Spiked plasma
Regression equation y=1.483x+0.063 y=1.592+0.035 y=3.286x+0.040
Correlation coefficient (R2) 0.999 0.999 0.999
Spiked aqueous humour
Regression equation y=1.443x+0.054 y=3.274x+0.057
Correlation coefficient (R2) 0.999 0.998
Accuracy (%recovery) mean±SD; %RSD mean±SD; %RSD mean±SD; %RSD
Spiked sample (0.05µg/ml) 95.17±1.24; 1.30 97.15±1.03; 1.06 94.77±0.96; 1.01
Accuracy (amount recovered) mean±SD; %RSD mean±SD; %RSD mean±SD; %RSD
Spiked sample (0.05µg/ml) 0.049±0.0010;2.04 0.48±0.0008; 1.67 0.047±0.0009; 1.91
Percent recovery (relative)
mean±SD; %RSD mean±SD; %RSD mean±SD; %RSD
(spiked human plasma) spiked sample
98.72±1.19; 1.20 96.04±3.09; 3.22 95.14±1.19; 1.25
(1.0µg/ml)
Percent recovery (absolute) mean±SD; %RSD mean±SD; %RSD mean±SD; %RSD
Spiked Plasma 92.65±2.32; 2.50 94.43±1.58;1.67 93.54±1.38; 1.47
Percent recovery (relative)
mean±SD; %RSD mean±SD; %RSD
(spiked aqueous humor) spiked sample
94.99±0.98; 1.03 98.23±1.13; 1.15
(1.0µg/ml)
Percent recovery (absolute) mean±SD; %RSD mean±SD; %RSD
spiked aqueous humor 92.56±1.72; 1.86 95.85±1.52; 1.59
Repeatability mean±SD; %RSD mean±SD; %RSD mean±SD; %RSD
Injection repeatability
Spiked sample (2.0µg/ml) 2.9±0.02;0.69a 3.8±0.01;0.26a 6.2±0.02; 0.32a
Spiked sample (2.0µg/ml) 27437.33±109.20;0.40b 28328±110.49; 0.30b 77533.33±136.55;0.18b
Analysis repeatability
1.91±0.02; 1.04 1.92±0.02; 0.76
Spiked sample (2.0µg/ml) 1.90±0.01; 0.53
Sensitivity
Limit of detection (ng/ml) 0.800 0.500 0.250
Limit of quantification (ng/ml) 2.000 1.500 1.000
a; retention time (minutes of the analyte), b; peak area of the analyte
104
The linearity of the method was evaluated from the calibration curves of standard
mixtures, spiked plasma and aqueous humour samples constructed at seven concentration
levels of all the analytes in the range of 0.05–2 µg/ml. Calibration curves of standard
mixtures of TM, RST, and DS and their spiked plasma samples and aqueous humour
samples are shown in Fig 4.6a and b, respectively.
Figure 4.6:(a) Calibration curves of timolol, rosuvastatin and diclofenac sodium standard solutions and
spiked plasma samples. (b) Calibration curves of timolol and diclofenac sodium standard solutions and
spiked aqueous humour Note: Each point is a mean of triplicate injections.
The regression equation and their respective correlation co-efficient (r) are given in Table
4.1. Accuracy of the proposed method was determined on the basis of percent recovery at
three concentration levels (0.05, 1 and 2 µg/ml), for TM, RST, and DS. The results are
shown in Table 4.1.
105
Table 4.2: Intra-day and Inter-day studies
Spiked concentration
Concentration recovered (µg/ml)
(µg/ml)
Intra-Day(mean±SD) %RSD Inter day (mean±SD) %RSD
Timolol maleate
1.0 0.9386±0.0095 0.6357 0.9166±0.0368 4.0104
1.5 1.5098±0.0063 0.4170 1.4527±0.0543 3.7410
2.0 1.8933±0.0035 0.1834 1.8552±0.0405 2.1821
Rosuvastatin
1.0 0.9499±0.0111 1.1729 0.9071±0.0334 3.6859
1.5 1.4332±0.0054 0.3779 1.4171±0.0205 1.4473
2.0 1.8675±0.0028 0.1492 1.8421±0.0197 1.0708
Diclofenac sodium
1.0 0.9404±0.0100 1.0586 0.9093±0.0196 2.1580
1.5 1.4800±0.0104 0.7052 1.4395±0.0284 1.9763
2.0 1.9166±0.0146 0.7622 1.8767±0.0197 1.0516
The precision of the method was evaluated through injection repeatability, analysis
repeatability, and intra-day, inter-day studies as shown in Table 4.2. The intra-day co-
efficient of variation (% RSD) was in the ranges of 0.183–0.635, 0.949–1.867, and
0.940–1.910 for TM, RST, and DS, respectively. Similarly, their respective values for
inter-day studies were in the range of2.182–4.010, 1.070–3.685, and 1.052–2.158 for TM,
RST, and DS, respectively.
Figure 4.7:Chromatograms representing LOD and LOQ values of TM, RST and DS.
106
LOD and LOQ values were determined at precision and accuracy of 20% variations for
the evaluation of sensitivity of the suggested method. The respective chromatograms of
LOD and LOQ are given in Fig 4.7. The respective values are given in Table 4.1.
Stability studies were conducted at room temperature (25oC), and at freezer temperature
(−20oC), for the standard mixtures and spiked plasma samples of TM, RST, and DS and
aqueous humour samples spiked with TM, and DS for 1 week, respectively. The results
obtained have shown that these compounds are stable at freezer temperature. The TM and
DS standard solutions as well as spiked samples were degraded at room temperature.
Ruggedness of the proposed method was determined through small deliberate changes in
various experimental parameters and the observed changes in the peak area and retention
times were found non-significant.
107
4.2 Physical Evaluation of In-situ Solution to Gel Formulations
4.2.1 Measurement of the Sol-Gel Transition Temperatures (Tsol-gel)
The Tsol–gel for all the formulations (34) was determined by tube inversion method [27,
121]. The data is shown in table 4.3. Out of 34 formulations seven formulation converts
to gel from free flowing solution (at 25oC) between 30 to 35oC. The transition
temperature obtained were 30oC, 31oC, 32oC, 33oC, 34oC, 34oC and 35oC for MPG 4/2,
P20, M4, P18, PM 15/3, MPG 3/2 and MPG 1.5/10 respectively. The lowest transition
temperature (30oC) was observed for MPG 4/2 and the highest (35oC) for MPG 1.5/10.
The selected formulations were evaluated in triplicate for precise Tsol-gel at observed
transition temperature ± 0.5oC; data is given in table 4.3. The results confirmed the
transition temperature is dependent on the concentrations for the selected polymers, the
transition temperature increases as the polymer concentration decreases [112].
The combination of the polymers results in the reduction of the polymer concentration
maintaining the transition temperature around the body temperature. Pluronic F-127 in
15% W/V concentration has the gelation point above 50oC, while addition of 3% MC
(PM15/3) decreases the Tsol-gel to 34oC. This might be due to increase in the viscosity as
MC is viscosity enhancer and also due to combined micellization phenomenon of the
polymers[273] thus reducing the critical micellization concentration. All the selected
formulation showed thermoreversible gelation i.e. reducing the temperature below the
transition temperature converts the gel formulations to free flowing liquid.
108
Table 4.3: Tsol-gel of various Polymer Formulations
Temperature (oC)
S. No Formulation 15 20 25 28 30 31 32 33 34 35 36 37 40 45 50
1. P5 × × × × × × × × × × × × × × ×
2. P10 × × × × × × × × × × × × × × ×
3. P15 × × × × × × × × × × × × × × ×
4. P18 × × × × × × × ✔ - - - - - - -
5. P20 × × × × × ✔ × × × × × × × × ×
6. P23 × × ✔ × × × × × × × × × × × ×
7. P25 × ✔ - - - - - - - - - - - - -
8. P30 × ✔ - - - - - - - - - - - - -
9. H1 × × × × × × × × × × × × × × ×
10. H2 × × × × × × × × × × × × × × ×
11. H3 × × × × × × × × × × × × × × ×
12. H4 × × × × × × × × × × × × × × ×
13. H5 × × × × × × × × × × × × × × ×
14. H6 × × × × × × × × × × × × × × ×
15. H8 × × × × × × × × × × × × × × ×
16. H 10 × × × × × × × × × × × × × ✔ -
17. M1 × × × × × × × × × × × × × × ×
18. M2 15 20 25 28 30 31 32 33 34 35 36 37 40 45 50
19. M3 × × × × × × × × × × × × ✔ × -
20. M4 × × × × × × ✔ - - - - - - - -
21. M5 × ✔ - - - - - - - - - - - - -
22. PM 10/3 × × × × × × × × × × × × × × ×
23. PM 15/3 × × × × × × × × ✔ - - - - - -
24. MPG 1.5/2 × × × × × × × × × × × × × × ×
25. MPG 1.5/5 × × × × × × × × × × × × × × ×
26. MPG 1.5/10 × × × × × × × × × ✔ - - - - -
27. MPG 3/2 × × × × × × × × ✔ - - - - - -
28. MPG 4/2 × × × × ✔ - - - - - - - - - -
29. HPG 1.5/2 × × × × × × × × × × × × × × ×
30. HPG 1.5/5 × × × × × × × × × × × × × × ×
31. HPG 1.5/10 × × × × × × × × × × × × × × ×
32. HPG 3/2 × × × × × × × × × × × × × × ×
33. HPG 6/2 × × × × × × × × × × × × × × ×
34. HPG 10/2 × × × × × × × × × × × ✔ × × ×
P=Pluronic F127, M= Methyl cellulose 5cps, H= Hydroxy propyl methylcellulose 15 cps, PG=
polyethylene glycol 6000
109
4.2.2 Measurement of Rheological Parameters
The rheological behavior at 25oC and 37oC of the solutions was studied using cone and
plate viscometer (Brookfield viscometer; type DVT-2) results are depicted in table 4.4.
The highest viscosity (238 cps) was observed for the formulation MPG4/2 containing
methylcellulose 4% and 2% polyethylene glycol. The viscosity order was 238 ± 2.9
(MPG4/2), 222.7 ± 3.1 (M4), 130.3 ± 0.5 (PM15/3), 128.3 ± 2.5 (P20), 109.3 ± 0.5 (P18),
100 ± 0.8 (MPG3/) and 97.7 ± 1.2 (MPG1.5/10). The addition of viscosity enhancer 3%
MC to Pluronic F-127 produce solution of comparable viscosity while reducing the
Pluronic concentration from 20% to 15%. The increase in viscosity was linear with rise in
the temperature; all the formulations were in gel form at 37oC. The recorded viscosities
are 533 ± 2.1 (MPG4/2), 334.7 ± 1.2 (M4), 517.3 ± 1.9 (PM15/3), 310.7 ± 1.2 (P20),
341.7 ± 1.7 (P18), 292.7 ± 2.5 (MPG3/) and 283.7 ± 1.2 (MPG1.5/10).
The flow index (n) of all the formulations at 25oCwas almost one with very low
consistency index (m) values (Table 4.4), indicating Newtonian flow of the solutions.
Increase in the values of m and decrease in the values of n was observed at high
temperature i.e. at 37oC indicating shear thinning of the formulation. The lower the value
of (n) the more shear thinning will be the preparation [113, 263, 264]. The lowest n value
(0.1324±0.003) was obtained for the P18 formulation (Figure 4.8).
110
Table 4.4: Rheological Data of Selected Formulations
25oC 37oC
Consistency Consistency
Viscosity Flow Index Viscosity Flow Index
S. No Formulation Tso-gel (oC) Index Index
Cps (n) Cps (n)
(m) (m)
1. MPG 4/2 29.8±0.05 238.0±2.9 0.8800±0.007 413.3±1.25 553.3±2.1 0.2859±0.003 200326±136
2. P20 31.0±0.08 128.3±2.5 0.9079±0.004 378.7±2.05 334.7±1.2 0.2567±0.004 170063±65
3. M4 32.1± 0.05 222.7±3.1 0.9040±0.006 388.7±1.25 517.3±1.9 0.2377±0.004 181026±48
4. P18 32.8±0.05 109.3±0.5 0.9278±0.003 322.3±1.89 310.7±1.2 0.1324±0.003 138948±47
5. PM15/3 33.9±0.05 130.3±0.5 0.9470±0.005 348.3±2.05 341.7±1.7 0.1542±0.003 122764±93
6. MPG3/2 33.7±0.05 100.0±0.8 0.9682±0.002 302.7±2.87 292.7±2.5 0.1945±0.004 118419±82
7. MPG1.5/10 35.0±0.09 97.7±1.2 0.9904±0.002 354.7±3.30 283.7±1.2 0.2690±0.002 149776±227
n=3
The addition of MC to pluronic solution and polyethylene glycol to MC supplemented
the hydrogen bonding thus comparable Tsol-gel and gel strength was obtained with
decreased concentration of pluronic (15%) and MC (3%) [268]. The formulation M4 and
MPG 4/2 were excluded from further studies as the solutions were highly viscous and
have poor syringe-ability.
111
25oC
37oC
1.0
Flow Index (n)

0.8
0.5
0.3
0.0
Figure 4.8: Flow index (n) of selected formulations
4.2.3 Clarity of the Formed Gel
Solutions were checked for clarity at various temperatures i.e. at 5ºC, 25ºC and 37ºC. All
the solutions were clear and transparent at 5ºC and 25ºC, the gel formed for pluronic
formulation was transparent and clear while the MC forms turbid gel, the gel formed by
combination of pluronic and MC (PM15/3) was semitransparent (Fig. 4.9)
1 2
Figure 4.9: Appearance of solution at 25oC=1 and 37oC=2. A=P18, B=PM15/3,

C=MPG1.5/10, D=MPG3/2 and E=P20.
112
4.2.4 Autoclaving
Viscosity, clarity and Tsol-gel of the preparations at 25ºC before and after autoclaving
were determined, data given in table 4.5.
Table 4.5: Physical Evaluation of Selected Formulation before and after autoclaving
Before Autoclaving After Autoclaving
Sol-gel Sol-gel
Viscosity Clarity of Viscosity Clarity of
S.No Formulation Temp Temp
At 25oC gel At 25oC gel
(oC) (oC)
1. P20 128.3±2.5 +++++ 31.0±0.08 129.1±1.5 +++++ 31.2±0.04
2. P18 109.3±0.5 +++++ 32.8±0.05 108.9±2.1 +++++ 32.6±0.07
3. PM 15/3 130.3±0.5 +++ 33.9±0.05 130.8±1.8 +++ 34.1±0.08
4. MPG3/2 100.0±0.8 ---- 33.7±0.05 101.3±0.8 ---- 33.8±0.07
5. MPG1.5/10 97.7±1.2 ---- 35.0±0.09 97.9±1.6 ---- 35.2±0.06
+++++= good Transparency, +++ =Fair Transparency, ----- Turbid
Autoclaving did not show any significant effect on the viscosity; clarity and Tsol-gel of
the solutions. The results are consistent with the earlier studies [99, 274, 275].The
preparations were free flowing at 25ºC before and after autoclaving.
113
4.2.5 In-vitro Evaluation of In-situ Drug Formulations
4.2.5.1 In-vitro Evaluation of In-situ Diclofenac Sodium Sol-Gel Formulations
4.2.5.1.1 Tsol- gel of DS Formulations
Transition temperature data from sol-gel of the prepared in-situ thermoreversible gels is
depicted in Table 4.6. The data indicates that addition of diclofenac sodium in the
polymer solutions results in slight increase of the transition temperature. This might be
due to the interference in the micellization of these polymers by altering the dehydration
of the hydrophobic polyoxyethylene (PO) block [97, 113, 114]. The data in table 4.6
shows the effect of autoclaving on the Tsol-gel. The P-Values (n=3)obtained using
student’s t-test were 0.423, 0.225, 0.425, 0.478 and 1.00 for DPM18, DPM20, DPM15/3,
DMPG 3/2 and DMPG1.5/10 respectively, suggesting that autoclaving has no significant
effect on the transition temperature of the in-situ gels [274].
4.2.5.1.2 Viscosity of DS Formulations
Viscosity of DS in-situ gel (n=3) was measured using cone and plate viscometer
(Brookfield viscometer; type DVT-2), results are shown in Table 4.6.The addition of DS
to the polymer solutions has negligible effect on the viscosity of the solutions. The effect
of autoclaving was also determined which was not significant and is consistent with
previous studies [274], ensuring that autoclaving has no significant effect on the
rheological properties of the gelling polymers.
114
Table 4.6: Physicochemical evaluation of Diclofenac sodium thermoreversible in-situ gels
Drug Drug Viscosity Clarity Clarity

P Viscosity P Tsol-gel Tsol-gel P
S. No Formulation content content Before Before After
value After AC value Before AC After AC value
Before AC After AC AC AC AC
1. DP18 100.63±0.49 95.17±0.59 0.003 119.0±0.8 117.67±1.5 0.057 33.33±0.24 33.67±0.24 0.423 +++++ +++++
2. DP20 100.49±0.47 94.32±0.10 0.003 132.0±0.8 130.7±0.5 0.270 31.0±0.41 31.50±041 0.225 +++++ +++++
3. DPM 15/3 100.63±0.21 94.23±0.35 0.001 145.7±0.9 145.0±0.8 0.529 34.85±0.24 35.17±0.24 0.425 +++ +++
4. DMPG 1.5/10 100.40±0.26 94.53±0.25 0.004 101.7±0.5 102.7±1.2 0.478 36.2±0.24 36.33±0.24 0.478 ---- ----
5. DMPG 3/2 100.30±0.40 94.00±0.70 0.005 113.3±1.2 112.7±0.9 0.270 35.3±0.24 35.33±0.24 1.00 ---- ----
(n=3, mean ±SD), Clarity = +++++ (good), +++ (Fair), ---- (poor)
115
4.2.5.1.3 Clarity of the DS Solutions and Formed Gels
Solutions of all the formulations were checked for clarity at 5ºC, 25ºC and gelling
temperature, all the solutions were clear indicating the solubility of the added ingredients
at all the temperature excepts for the solutions containing MC (DMPG 3/2 and DMPG
1.5/10) that formed turbid gels at the gelling temperature. The turbidity may be due to the
hydrophobic interaction of the methoxyl group of the polymer chain [161]. The gel
formed by the combination of pluronic and MC i.e. DMP 15/3 were semitransparent.
Autoclaving sterilization did not affect the clarity of these formulations. The clarity of the
pluronic solutions indicates the surfactant activity of these polymers which help in the
salvation [274], while the addition of PEG (co-solvent) in the MC formulations helps in
the solubility of the drug.
4.2.5.1.4 Drug Content of DS In-situ Gels
The diclofenac sodium contents for the formulations were between100.30±0.40 to
100.63±0.49 and 94.00±0.70 to 94.23±0.35 before and after autoclaving respectively. The
data in Table 4.6 suggests that the diclofenac content decreases significantly after the
sterilization through autoclaving as indicated by the P-Values which were between
0.001to 0.005 for the tested samples. The drug content decreases upto 6% for all the
formulation that is within the official limits [178]. The decrease in % labeled amount of
DS might be due to its degradation by exposure to high temperature during autoclaving.
The results are consistent with the previous studies [276]. Sterility is one of the prime
requirements for both ophthalmic and parenteral delivery system. It is important to
116
employ appropriate technique for sterilization to avoid the degradation of the DS, hence
filtration at room temperature will be an ideal alternate technique for the sterilization.
4.2.5.2 In vitro Drug Release From DS In-situ Gels
Drug release from the DS in-situ solution to gel formulations was determined using
membrane free models [100, 267, 268]. Phosphate buffer pH 7.4 was used as dissolution
medium. Samples were collected at the predefined time intervals and were analyzed for
drug content using validated HPLC method [266].
120.0
100.0
%Cummulative DS release
80.0
DP20
DP18
60.0
DPM15/3
40.0 DMPG1.5/10
DMPG3/2
20.0
0.0
0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180
Time (Hr)
Fig. 4.10: In-vitro release profile of Diclofenac sodium form various in-situ sol-gel
The release profiles of DS in- situ gels (Fig. 4.10) show that all the formulation retarded
the drug release above twelve (12) hours except for the formulation DMPG 1.5/10. The
t100% (time when 100% drug was released) was 4, 24, 72, 96 and 144 hours for DMPG
1.5/10, DMPG 3/2, DPM 15/3, DP18 and DP 20, respectively. Table 4.7 tabulates the in-
vitro release data of DS from different preparations at various time intervals. The slowest
drug release was observed for the formulation DP 20, that contains 20% pluronic F-127
117
as in-situ thermoreversible gelling polymer. The addition of 3% w/v MC to pluronic in
DPM 15/3 extended the drug release upto 72 hours.
118
Table 4.7: Cumulative % In-vitro Diclofenac sodium release from diclofenac sodium in-
situ gels
Formulations DP18 DP20 DPM15/3 DMPG1.5/10 DMPG3/2
Time (Hr) %drug release %drug release %drug release %drug release %drug release
1 4.56±0.11 3.92±0.17 15.90±0.15 23.66±0.32 15.58±0.36
2 10.68±0.10 9.56±0.31 34.23±0.25 74.60±0.36 36.92±0.18
4 21.75±0.20 13.85±0.22 55.85±0.24 101.44±0.49 62.34±0.56
8 30.52±0.14 18.91±0.20 66.94±0.37 83.75±0.15
12 41.78±0.21 25.66±0.17 74.37±0.31 95.68±0.14
24 52.68±0.16 33.81±0.28 83.94±0.15 103.45±0.28
36 64.48±0.28 42.72±0.22 90.97±0.14
48 75.65±0.13 52.95±0.25 97.04±0.08
72 85.74±0.21 64.56±0.30 100.22±0.25
96 99.76±0.28 76.79±0.24
120 100.50±0.45 89.76±0.23
144 99.36±0.31
168 100.3±0.56
n=3
119
Formulation DMPG 1.5/10 containing 1.5% MC and 10% PEG as thermoreversible
gelling polymer could only extended the drug release for 4.0 hours.
Fig 4.11: Representative chromatogram of in-vitro DS release from DP 20 in-situ gel at

different time interval, A=1 hr, B=12 hrs, C=24 hrs, D=36 hrs, E=72 hrs, F=144 hrs,
G=168 hrs.
Initially no burst release of the drug was observed from the formulation DP 20 and DP18
after the first hour as only 3.92% ± 0.17 and 4.56% ± 0.11of the drug was released,
respectively. DP 20 released only 25% of the loaded drug by 12th hour, 50 % of the drug
was released after 48 hours and then 75% at 96 and 100% drug at 144 hours. The drug
release from DP 18 was 21.75 % (4 hr), 52.68% after 24 hours, 75 % of the drug was
released at 48 hours and t100 was 120 hours. A burst release at first hour for the
formulations DPM 15/3, DMPG 3/2 and DMPG 1.5/10 was observed the drug release
was 15.90%, 15.58% and 23.66%, respectively. The hydrophobic nature [277] of the DS
120
also played the role in retarding the release of drugs from the formulations, DS being less
water soluble will diffuse slowly from the hydrogel formed.
The results obtained showed that increase in the pluronic concentration have positive
effect on sustaining the drug release from the in-situ gels i.e. with increase in polymer
concentration the release profile reduces[278]. The higher the concentration of the
polymer longer the path length for diffusion, also the mesh size of the polymer network is
smaller with higher polymer concentration. With increasing the polymer concentration
the water penetration is slow within the hydrogel which causes reduction in the polymer
erosion and degradation. All these factors accounts for slow drug release from the
concentrated polymer system as compared to less polymer concentration [278].
4.2.5.3 Drug Release Kinetics from DS In-situ Gels
The drug release from the thermoreversible gels are governed by various parameters.
Solubility of the drug in the polymer and water, water diffusion rate into the polymer gel,
the drug diffusion rate from the polymer gel and dissolution of the polymer within the
experimental conditions are all important factors that affect the kinetics of drug release.
Initially the drug is released through Fickian diffusion followed by dissolution [122].
The data obtained from the in-vitro experiments were fitted to different mathematical
model i.e. Zero order, First Order, Higuchi, Hixson-Crowell, Korsmeyer Pappas, to
predict the kinetics and release mechanism of the drug.
121
4.2.5.3.1 DP 20 In-situ Gel
The in-vitro dissolution results obtained from membrane less technique for the
formulation DP20 was fitted to the above mathematic models. The release constant and
regression coefficient (R2) values obtained from the mathematical models Zero Order (eq.
3.2), First Order (eq. 3.3), Higuchi model (eq. 3.4), Hixson Crowell model (eq. 3.5) and
Korsmeyer Pappas model (eq.3.68) are shown in Table 4.8.
Table 4.8: Release Parameters of Diclofenac Sodium from DP20, DP18, DPM15/3,
DMPG 3/2 and DMPG 1.5/10 in-situ sol-gel formulations.
Formulation Zero Order First Order Higuchi Hixson Crowell Korsmeyer
R2 Koh-1 R2 K1h-1 R2 KH(h-0.5) R2 KHC(h-1/3) n R2
DP20 0.968 0.640 0.814 0.011 0.995 8.428 0.954 0.022 0.421 0.881
DP18 0.899 0.939 0.815 0.021 0.988 10.55 0.958 0.036 0.470 0.960
DPM15/3 0.717 1.373 0.966 0.027 0.839 10.13 0.961 0.060 0.455 0.978
DMPG3/2 0.711 3.389 0.992 0.113 0.870 22.39 0.997 0.249 0.670 0.978
DMPG1.5/10 0.871 24.13 1.000 0.477 0.925 75.65 0.990 1.825 1.291 0.969
It was found that the in-vitro DS release from the DP20 best fits to the Higuchi model
(Fig. 4.14) as the R2 value obtained was the highest (0.995) indicating the drug release
followed Fickian diffusion. Fig. 4.12 indicates that good linearity (R2=0.968) was
122
obtained for Zero Order indicating the drug release is independent of drug concentration.
Hixson – Crowell plot for the data was also constructed (Fig. 4.15), the R2 value was
0.954 indicating polymer erosion and dissolution.
120.0
y = 24.139x + 10.242
R² = 0.8712
100.0 y = 6.8251x + 21.999

R² = 0.8927
y = 1.3733x + 41.731 y = 0.9395x + 20.326

% cumulative DS release
y = 0.6408x + 14.042
R² = 0.7173 R² = 0.8992 R² = 0.9687
80.0
60.0
DP20
40.0 DP18
DMPG3/2
20.0 DMPG1.5/10
DPM15/3
0.0
0 20 40 60 80 100 120 140 160
Time (Hr)
Fig. 4.12: % Cumulative Diclofenac Sodium Release Vs Time (Zero Order Model) from
Diclofenac sodium in-situ thermoreversible sol-gel.
The value of release exponent “n” was 0.421 (Fig. 4.16) by plotting the fraction
diclofenac sodium released vs. log time (Korsmeyer-Pappas), indicating Fickian diffusion
for the release of DS from the in-situ gels.
In current study the in-vitro dissolution study was performed using membrane free
models, hence the drug mechanism is combined effect of Fickian diffusion and the
dissolution of the polymer. Water uptake through diffusion results in the subsequent
dilution of pluronic F-127 micelles governs the drug release. The drug diffusion and
123
polymer dissolution (90%) followed zero order kinetics [267]. Same results were
obtained for pilocarpine, metronidazole and cephelexin [177, 267] using Pluronic F-127
as thermorevesible polymer for sustaining of these drugs.
4.2.5.3.2 DP 18 In-situ Gel
The in-vitro dissolution drug release obtained for the formulation DP18 was fitted to
different mathematical models to explore the drug kinetics and release mechanism. The
values showed best linearity for the Higuchi model (R2= 0.988). Values for regression
coefficient for the various equations are summarized in Table 4.8. The highest value for
R2 (Fig. 4.14) was obtained for Higuchi Model (0.988) followed by Hixson-Crowell
(0.958) Fig. 4.15.
3.00
2.50
Log% DS remaining
Log(100) -Logt
y = 0.0214x - 0.1065
2.00 R² = 0.8151
y = 0.0271x + 0.1746 y = 0.0113x - 0.0858

R² = 0.9661 R² = 0.8148
y = 0.1137x - 0.0437
1.50 R² = 0.9924
DP20
1.00
y = 0.4778x - 0.3606 DP18
R² = 1
DMPG3/2
0.50 DMPG1.5/10
DPM15/3
0.00
0 20 40 60 80 100 120 140 160
-0.50
Time (Hr)
Fig. 4.13: % Log cumulative Diclofenac Sodium Remaining Log(100)-Logt Vs Time

(First Order Model) from Diclofenac sodium in-situ thermoreversible sol-gel.
The release data also showed reasonable linearity for zero order (R2= 0.899), indicating
the drug release is independent of the drug concentration within the gel. The value
124
(0.470) of release exponent “n” obtained with Korsmeyer-Pappas equation, suggests that
drug release from DP18 formulation followed anomalous transport. Hixson – Crowell
plot (Fig. 4.15) also confirms polymer erosion with the (R2= 0.958).
The “K” values for Zero order (0.939), First order (0.021), Higuchi equation (10.55) and
Hixson-Crowell equation (0.036) were higher compared with the DP 20 which were
0.640, 0.011, 8.428, 0.022 respectively. These values indicate that lower the
concentration of the pluronic F-127 the higher will be the diffusion of the drug from gel
matrix and rapid will be the degradation rate of the polymer.
140.0
y = 22.395x + 8.039
R² = 0.8704
120.0 y = 75.657x - 44.755
y = 10.136x + 27.139
R² = 0.8391
R² = 0.9259 y = 10.553x - 0.6132
R² = 0.988
y = 8.4289x - 4.6873
R² = 0.9959
100.0
% cumulative DS release
80.0
60.0 DP20
DP18
40.0 DPM15/3
DMPG3/2
20.0 DMPG1.5/10
0.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Time (Sq.Rt)
Fig. 4.14: % cumulative Diclofenac Sodium release Vs Sq. Rt. Time (Higuchi Model)
from Diclofenac sodium in-situ thermoreversible sol-gel.
Reduction in pluronic F-127 concentration in case of DP 18 results in weak gel
compared with the DP 20, hence increase in the rate of water diffusion and rapid water
125
uptake results in rapid dissolution of the drug and faster diffusion as the path length is
reduced [279]. The decrease in concentration of the pluronic results in the increase in the
number and size of the water channels, hence results in high drug release [280, 281].
4.2.5.3.3 DPM 15/3 In-situ Gel
Formulation DPM 15/3 contains 15 % Pluronic P-F127 and 3% methyl cellulose. Methyl
cellulose was added to reduce the Tsol-gel below the physiological temperature. The
formulation showed reasonable gel strength and sustained the drug release upto 72 hours.
The mathematical models zero order, first order, Higuchi model, Hixson-Crowell model
and Korsmeyer-Pappas model were applied to the dissolution data obtained from in-vitro
dissolution testing, data is shown in Table 4.8. The results inferred that the drug release
follow first order (Figure 4.13) as the value (0.966) of the regression co-efficient was
highest, indicating the drug release was dependent on the drug concentration remaining
within the system. The data also showed good linearity (R2=0.961) for Hixson-Crowell
(Figure 4.15), indicating surface erosion of the in-situ gel. The data was also fitted to
Korsmeyer-Pappas equation by plotting log% drug release as a function of log time (Fig.
4.16), the value of release exponent was 0.455 indicating the drug followed almost
Fickian diffusion from the gel.
Methyl Cellulose is a high molecular weight water soluble polymer, when dissolved in
water the MC solutions are highly viscous which is concentration dependent, the
viscosity of MC is higher compared with the Pluronic solutions. The addition of 3% MC
to 15% Pluronic yield high viscosity solutions (Table 4.5 and 4.6). The resultant high
viscosity is comparable to that of 20% pluronic solutions might be the reason for
126
retarding the drug release from the formulation DPM 15/3. MC forms micelles in the
aqueous solution upon heating thus it has junction of the micelle zone with the pluronic
F-127 [282].
7.0
y = 0.2499x + 0.1223
y = 1.8253x - 1.6349 R² = 0.9976
R² = 0.9902
6.0
y = 0.0607x + 0.6292
R² = 0.9613
5.0
Cube Root % DS remaining
4.0
Wo-Wt
y = 0.036x + 0.1371
R² = 0.9588
y = 0.022x + 0.0441
R² = 0.9547
3.0
DP20
2.0
DP18
DPM15/3
1.0 DMPG3/2
DMPG1.5/10
0.0
0 20 40 60 80 100 120 140 160
Time (Hr)
Fig. 4.15: Cube root% Diclofenac Sodium remaining (Wo-Wt) Vs Time (Hixson-Crowell
Model) from Diclofenac sodium in-situ thermoreversible sol-gel.
The data suggests that the drug diffusion rate was high that may be due to rapid water
uptake that enhance the drug release rate [277] from the formulation compared with the
DPM20 and DPM18.
4.2.5.3.4 DMPG 3/2 In-situ gel
Solutions of 3% MC has Tsol-gel at 40ºC, the addition of 2% polyethylene glycol to the
solution reduces the transition temperature to 34ºC with good viscosity of 110 cps. The
formulation extended the in-vitro release of diclofenac sodium for 24 hours. The
127
regression coefficient “R2” values obtained for various models indicates highest values
for Hixson-Crowell equation (0.997) showing that the drug release follows diffusion as
well as erosion. Good linearity with R2 value of 0.992 (Figure 4.13) for first order was
obtained inferring the concentration dependent drug release from the in-situ
thermorevesible gel.
The value for release exponent (n=0.670) reveals that the drug release follows anomalous
diffusion. The values of release constant “K” for all the equations applied were high
indicating rapid drug release from the combined polymers formulation.
4.2.5.3.5 DMPG 1.5/10 In-situ Gel
This formulation could only hold its integrity in the in-vitro dissolution medium for 4
hours. The viscosity of the solution was upto 100 cps but forms a soft delicate gel, this
might be due to reduction in the MC content. The Tsol-gel (36ºC) was also high.
The R2 value for the Hixson-Crowell was 0.990 suggesting the drug release through
polymer erosion in the dissolution medium. The “N” values obtained by applying the
Korsmeyer-Pappas equation were 1.291 which is above 1 indicating a super case II
release. The value of release exponent suggests that the polymer degrades rapidly as a
result of its dissolution and erosion. The formulation was not suitable for subcutaneous
route but can be used for ocular route.
128
1.20
y = 0.4456x + 0.2249
y = 0.6706x + 0.1861 R² = 0.978 y = 0.4705x - 0.0457
y = 1.2918x + 0.2768 R² = 0.9789 R² = 0.9607
R² = 0.969
1.00
Fraction (Mt/M) DS released

0.80
y = 0.4211x - 0.1036
R² = 0.8813
0.60
DP 20
DP 18
0.40
DMPG 3/2
0.20 DMPG 1.5/10

DPM 15/3
0.00
0.0 0.5 1.0 1.5 2.0 2.5
Log Time
Fig. 4.16: Fraction (Mt/M) Diclofenac Sodium released Vs Log Time (Korsmeyer-
Pappas Model) from Diclofenac sodium in-situ thermoreversible sol-gel.
4.2.5.3.6 Conclusion
In-vitro evaluations of the formulations divulge that autoclaving has no significant effect
on the Tsol-gel, viscosity and clarity of the solutions. The sterilization through
autoclaving significantly decreased the drug content of all the formulations. The in-vitro
drug release data of the prepared formulation indicates that t100 (144 hrs) was obtained for
DP20 while the lowest (4.0 Hrs) was obtained for DMPG1.5/10. The application of
mathematical release models suggest that the formulation DP 20, DP18 follow the
Higuchi model for the drug release suggesting that major portion of the drug is released
through Fickian diffusion. Formulation DPM 15/3, DMPG 3/2 and DMPG 1.5/10 showed
high linearity for Hixson-Crowell model suggesting that the drug release from these in-
situ gels followed diffusion of the drug along with drug dissolution as a result of polymer
erosion.
129
The drug release from the DP 20 and DP 18 was independent of drug concentration as
highest R2 value was obtained for zero order equation. The formulations can deliver the
drug at constant rate for the predetermined time period. While the drug release was
dependant on the drug concentration present in the gel for the formulations DPM 15/3,
DMPG 3/2and DMPG 1.5/10.
The data obtained from the Korsmeyer-Pappas equation suggests that the drug release
from DP20 and DPM15/3 follow diffusion, formulations DP18 & DMPG 3/2 followed
anomalous transport, while drug release from DMPG 1.5/10 followed super case II
mechanism.
130
4.2.5.4 In-vitro Evaluation of In-situ TM sol-gel Formulations
4.2.5.4.1 Tsol- gel of TM Formulations
The results obtained for the transition temperature of the TM in-situ gel formulations are
shown in Table 4.9. It can be seen from the data obtained that addition of TM slightly
decreases gelling temperature of the solutions that may be due to hydrophilic nature of
TM, TM might cause modification of the micellization process of poloxamer gels thereby
increase their Tsol–gel [27, 175]. This may be due to the dehydration of polyoxyethylene
(PO) block thus reducing the micellization process and increase polymer entanglement
with each other [97, 113, 114]. Autoclaving has no significant effect on the Tsol-gel of
the TM in-situ thermoreversible gel formulations (p< 0.4-1.0).
4.2.5.4.2 Viscosity of TM Formulations
Addition of TM to the polymer solution has no significant effect on the viscosity of the
solutions as indicated from the data in Table 4.9. P-Values obtained for the effect of
terminal sterilization were 0.300, 0.250, 1.00, 0.472 and 0.86 for the formulations TP18,
TP 20, TPM15/3, TMPG3/2 and TMPG1.5/10, respectively. All the P-Values are non-
significant showing that autoclaving has no significant effect on viscosity of the
formulations. The results obtained are in accordance with previous studies [274].
131
Table 4.9: Physicochemical evaluation of Timolol Maleate thermoreversible in-situ gels
Drug Content Drug content P Viscosity Viscosity P Tsol-gel Tsol-gel P Clarity Clarity
S. No Formulation
Before AC After AC value Before AC After AC value Before AC After AC value Before AC After AC
1. TP18 98.18±0.306 97.22±0.293 0.002 114.0±1.0 112.3±1.5 0.300 33.2±0.3 33.0±0.3 0.423 +++++ +++++
2. TP20 98.63±0.740 98.10±0.830 0.153 127.0±2.0 124.7±0.6 0.250 31.0±0.5 30.7±0.3 0.184 +++++ +++++
3. TPM 15/3 98.88±0.530 98.47±0.470 0.016 135.7±1.2 135.7±0.6 1.000 34.8±0.3 34.8±0.3 1.000 +++ +++
4. TMPG 1.5/10 98.50±0.514 97.90±0.312 0.086 100.0±1.0 99.7±0.6 0.742 35.5±0.5 35.8±0.3 0.529 ---- ----
5. TMPG 3/2 98.67±0.108 98.21±0.226 0.040 109.3±2.1 109.0±1.0 0.860 35.2±0.3 35.2±0.3 1.000 ---- ----
n=3
132
4.2.5.4.3 Clarity of TM Solutions and Formed gels
Solutions of all the formulations were checked for clarity at 5ºC, 25ºC and gelling
temperature, Timolol maleate is water soluble drug the solutions obtained for all the
formulations were clear. The gels obtained at the gelling temperature were clear [274]
except for the formulations TMPG3/2 and TMPG 1.5/10 that showed some turbidity.
Terminal sterilization by autoclaving has no effect on the clarity of these formulations.
The results obtained indicate that the addition of TM in the polymer solution has no
effect on the physical appearance of these solutions.
4.2.5.4.4 Drug Content of TM Formulations
The formulations prepared were analyzed with validated HPLC [266] method in triplicate
for the drug content. The assay of the formulations showed that the drug contents were in
the range of 98.18%±0.306 to 98.88%±0.530. Autoclaving has negligible effect on the
TM content of the formulations. The drug content after sterilization by autoclaving were
well above 97%. The data presages that TM is stable at autoclaving conditions.
4.2.5.4.5 In vitro Drug Release from TM Formulations
Timolol maleate release from the prepared formulations was determined by membrane
less model [269]. The TM content was determined using validated HPLC-UV method
[266].
The in-vitro drug release profiles of TM in- situ gels (Fig. 4.17) indicates that formed
gels were strong enough to extend the release of hydrophilic timolol maleate. The
formulation TPM 15/3 extended the release over the longer period of time, while TMPG
133
1.5/10 retarded for about 8 hours. The t100% was 8, 8, 12, 12 and 24 hours for TMPG
1.5/10, TP 18, TMPG 3/2, TP20 and TPM 15/3, respectively.
120.0
% cummulative Timolol Release
100.0
80.0 TP 20
TP18
60.0
TPM 15/3
TMPG 3/2
40.0
TMPG 1.5/10
20.0
0.0
0 4 8 12 16 20 24 28
Time (Hr)
Fig. 4.17: In-vitro release profile of Timolol Maleate form various in-situ
thermorevesible sol-gel (n=3)
The drug release from the in-situ thermoreversible gels after the first hour was in the
order of TMPG 1.5/1>TP18>TP20>TMPG3/2>TPM15/3. All the formulation released
less than 25% (Fig.4.17) of the drug after first hour except for the formulation TMPG
1.5/10 for which a burst release at first hour was 44.90%. The in-vitro release data at
different time intervals depicted in Table 4.10; indicates that the slowest release was
observed for the formulation (TPM 15/3) containing 15% pluronic and 3%
methylcellulose. This might be due to the bonding between the TM and MC polymer and
swelling of the MC in aqueous medium, thus increasing the path length for the diffusion
of both penetrating water and eluting drug from the hydrogel network [283].
The results obtained for the formulation reveals that increase in the pluronic
concentration has positive [278] effect on sustaining the drug release from the in-situ gels
134
i.e. with increase in poloxamer concentration the release profile reduces as the number
and size of the pores decreases within the network.
135
Table 4.10: % Cumulative In-vitro drug release from Timolol Maleate in-situ gels
Formulation TP18 TP20 TPM15/3 TMPG1.5/10 TMPG3/2
%drug release %drug release %drug release %drug release %drug release
Time (Hr)
Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD
1 24.82±0.22 23.19±0.25 21.65±0.34 44.90±0.21 22.07±0.25
2 56.02±0.22 49.39±0.38 47.53±0.32 73.55±0.46 50.66±0.44
4 85.88±0.17 73.87±0.34 67.74±0.54 93.20±0.79 73.19±0.57
8 100.45±0.56 88.11±0.65 78.47±0.47 102.27±0.38 89.92±0.81
12 99.36±0.42 86.07±0.46 ---- 100.71±0.89
24 98.00±0.65 ---- ----
(n=3)
136
4.2.5.5 Drug Release Kinetics from TM In-situ Gels
The release kinetics and mechanism of drug release from the in-situ gels was evaluated
by fitting the dissolution data shown in Table 4.10 obtained from the in-vitro experiments
to different mathematical model i.e. zero order, first order, Higuchi, Hixson-Crowell,
Korsmeyer Pappas.
4.2.5.5.1 TP 20 in-situ Gel
The release constant (k) and regression coefficient (R2) values obtained from zero order
(eq. 3.2), first order (eq. 3.3), Higuchi model (eq. 3.4), and Hixson-Crowell model (eq.
3.5) and (n) values from Korsmeyer Pappas model (eq. 3.6) are shown in Table 4.11.
Table 4.11: Release Parameters of Timolol Maleate from TP20, TP18, TPM15/3, TMPG
3/2 and TMPG 1.5/10 in situ sol-gel formulations.
Formulation Zero Order First Order Higuchi Hixson Crowell Korsmeyer
R2 Koh-1 R2 K1h-1 R2 KH(h-0.5) R2 KHC(h-1/3) n R2
TP20 0.834 6.147 0.933 0.175 0.924 29.25 0.978 0.286 0.695 0.986
TP18 0.761 7.156 0.999 0.242 0.911 40.38 0.994 0.711 0.852 0.976
TPM15/3 0.698 2.704 0.986 0.065 0.850 17.82 0.933 0.118 0.537 0.967
TMPG3/2 0.836 6.330 0.987 0.123 0.926 30.10 0.932 0.424 0.715 0.987
TMPG1.5/10 0.820 9.811 0.999 0.301 0.864 29.78 0.994 0.723 0.637 0.950
137
Highest linearity with R2 value of 0.978 for the formulation was obtained for Hixson-
Crowell model (Figure 4.22), indicating the drug release follows both erosion and
diffusion. The Korsmeyer-Pappas power law also supplements the release mechanism
being anomalous transport as the value for release exponent is 0.695. Fig. 4.18 and 4.20
represent zero order and first order kinetics fit, respectively for the dissolution data
obtained, indicating good linearity for first order as compared with the zero order, the
value for regression co-efficient “R2” is 0.933 and 0.834, respectively.
120.0
y = 7.1567x + 51.643 y = 6.33x + 33.134
R² = 0.7612 R² = 0.8366
y = 6.1479x + 33.59 y = 2.7048x + 43.586

y = 9.8118x + 30 R² = 0.6985
100.0 R² = 0.8202
R² = 0.8343
%cumulative TM release
80.0
60.0
TP 20
40.0 TP 18
TPM15/3
20.0 TMPG3/2
TMPG1.5/10
0.0
0 5 10 15 20 25 30
Time (Hr)
Fig. 4.18: % Cumulative Timolol maleate Release Vs Time (Zero Order Model) from
TM in-situ thermoreversible sol-gel.
The dissolution data was also plotted for Higuchi model Fig. 4.21, which also showed
good linearity with the R2 value of 0.924 indicating diffusion of the drug from the
formulation.
138
Fig 4.19: Representative chromatogram of in-vitro TM release from TP 20 in-situ gel at

different time interval, A=1 hr, B=4 hrs, C=8 hrs, D=12.
4.2.5.5.2 TP 18 In-situ Gel
The R2 values obtained from the mathematical models indicates that the drug release
from the formulation depends on the concentration of TM remaining in the system, as
best linearity (R2=0.999) was observed for first order equation (Fig. 4.20). The R2value
(0.994) for the Hixson-Crowell (Fig. 4.22) suggests the drug release occurs due to erosion
of the polymeric system. The data was plotted for Korsmeyer-Pappas power law (Fig.
4.23) to better understand the release mechanism. The value of release exponent “n”
obtained was 0.852 suggesting anomalous transport i.e. the drug release from the system
follows diffusion of the drug from the in-situ gels and erosion of the polymer. The
R2value of 0.976 also suggest good linearity for the power law equation.
139
2.5
y = 0.1754x - 0.1236
R² = 0.9337
2.0
% log drug remaining

(Log100-logt)
y = 0.3017x - 0.036
1.5 R² = 0.9996
y = 0.2427x - 0.1227
R² = 0.9998
y = 0.1233x + 0.0337 y = 0.065x + 0.1303
R² = 0.9873 R² = 0.9863 TP20
1.0
TP 18
TPM15/3
0.5 TMPG3/2
TMPG1.5/10
0.0
0 5 10 15 20 25 30
Time (Hr)
Fig. 4.20: % Log cumulative Timolol Maleate Remaining Log(100)-Logt Vs Time (First
Order Model) from TM in-situ thermoreversible sol-gel.
4.2.5.5.3 TPM 15/3 In-situ Gel
Formulation TPM 15/3 contains 15 % Pluronic P-F127 and 3% methyl cellulose. The
formulation extended the drug release upto 24 hours, which is the most among the TM in-
situ gel formulations prepared. The dissolution data compiled in Table 4.10 was fitted to
the mathematical models zero order, first order, Higuchi, Hixson-Crowell and
Korsmeyer-Pappas. Highest linearity (R2=0.986) was obtained for first order equation,
suggesting concentration dependant drug release. The data also showed good linearity
(R2=0.933) for Hixson-Crowell (Fig. 4.22), indicating surface erosion of the in-situ gel.
The value of release exponent (n=0.537) obtained by plotting the fraction of drug
released vs. log time indicates the drug followed anomalous transport from the in-situ gel.
140
120.0
y = 40.383x - 6.3257y = 30.105x + 2.8499
R² = 0.9114 R² = 0.9264
y = 29.252x + 4.1488
100.0 y = 29.785x + 24.551 R² = 0.9246
% cumulative TM Released
R² = 0.8649
y = 17.825x + 20.213
R² = 0.8501
80.0
60.0
TP20
TP18
40.0
TPM15/3
TMPG3/2
20.0
TMPG1.5/10
0.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0
Time (Sq.Rt)
Fig. 4.21: % cumulative Timolol maleate release Vs Sq. Rt. Time (Higuchi Model) from
TM in-situ thermoreversible sol-gel.
The addition of Methyl Cellulose to the pluronic solution increases the viscosity. MC
forms micelles in the aqueous solution when heated thus it has junction of the micelle
zone with the pluronic F-127 [282]. The MC establishes hydrogen bonding with pluronic
[146] and the drug, thus retarding the diffusion and the erosion of the polymer surface.
7.0
%Cube root TM remaining (Wo-Wt)
6.0 y = 0.7232x + 0.0885 y = 0.7111x - 0.3748

R² = 0.9948 R² = 0.9945
5.0 y = 0.424x - 0.0598

R² = 0.9322
4.0
y = 0.2865x + 0.2832
R² = 0.9785 TP20
3.0
y = 0.1189x + 0.6882 TP18
R² = 0.9336
2.0 TPM15/3
1.0 TMPG3/2
TMPG1.5/10
0.0
0 5 10 15 20 25 30
Time (hr)
Fig. 4.22: Cube root% Timolol maleate remaining (Wo-Wt) Vs Time (Hixson-Crowell
Model) from TM in-situ thermoreversible sol-gel.
141
4.2.5.5.4 TMPG 3/2 In-situ Gel
This formulation extended the TM release for twelve (12) hours. Table 4.11 shows
regression coefficient “R2” values obtained for various models. The graph obtained by
plotting the log % drug remaining vs. time (Fig 4.20) shows the R2 value (0.987), which
is the highest among the various release models, indicating the drug release is dependent
on drug remaining within the polymer system.
The plot for Higuchi and Hixson-Crowell equations also show good linearity with the R2
values of 0.926 and 0.932, respectively. It can be inferred from the values of regression
co-efficient of Higuchi and Hixson-Crowell that the drug release follows diffusion and
surface erosion of the polymers.
The value for release exponent (0.715) suggests anomalous transport. The values of
release constant “K” for all the equations applied were high indicating rapid drug release
from the combined polymers formulation.
4.2.5.5.5 TMPG 1.5/10 In-situ Gel
The formulation could only hold its integrity in the in-vitro dissolution medium for eight
(8.0) hours. The data presented in table 4.11 obtained from the application of
mathematical models suggests the drug release follow First order kinetics (R2= 0.999).
The R2value for the Hixson-Crowell was 0.994 suggesting the drug release through
surface erosion of the polymer in the dissolution medium. The “n” value obtained by
applying the Korsmeyer-Pappas equation was 0.637. The value of release exponent is
below one (1) indicating anomalous transport from formulation to dissolution media.
142
1.2 y = 0.6957x + 0.2664

R² = 0.9864 y = 0.7158x + 0.2601
R² = 0.9877
y = 0.8529x + 0.2828
R² = 0.9767
1
fraction Mt/M TM released

y = 0.637x + 0.4972 y = 0.537x + 0.284
R² = 0.9504 R² = 0.9675
0.8
0.6
TP 20
TP 18
0.4
TMPG 3/2
TMPG 1.5/10
0.2
TPM 15/3
0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Log Time
Fig. 4.23: Fraction Mt/M Timolol Maleate release Vs Log Time (Korsmeyer-Pappas
Model) from TM in-situ thermoreversible sol-gel.
In-vitro evaluations of the TM in-situ thermorevesible gel formulations demonstrate that
autoclaving have no significant effect on the Tsol-gel, viscosity, drug content and clarity
of the solutions. The in-vitro drug release data of the prepared formulation indicates that
formulation TPM 15/3 extended the drug release for the longer period of time with t100 of
24 hours. While the formulation TP18 and TMPG 1.5/10 could only retard the drug
release for 8 hours. The application of mathematical release models suggests that all the
formulations followed first order release kinetics. The formulations also showed good
linearity for Hixson-Crowell model indicating the drug release from these in-situ gels
followed diffusion of the drug along with polymer dissolution.
The release exponent “n” values (Fig. 4.23) for all the formulations obtained by applying
Korsmeyer-Pappas equation were higher than 0.5 and lower than 1.0, indicating that the
drug release for all the formulation follows anomalous transport.
143
It can also be determined from the in-vitro release data that all the formulations extended
the drug release between 8-24 hours, suggesting the formulation can be good candidates
for the ocular drug release.
144
4.2.5.6 In-vitro Evaluation of In-situ Insulin Sol-Gel Formulations
4.2.5.6.1 Tsol- gel of Insulin Formulations
The sol–gel transition temperature for the prepared formulations was measured by tube
inversion method [27, 121] before and after filtration. Tsol-gel of insulin in-situ gel
formulation IP20, IP18 and IP15/3 is shown in Table 4.12. It can be observed from the
data that the addition of insulin to the polymer system significantly increased the Tsol-gel
[27, 175]. The transition temperature of Pluronic 18%(P18), pluronic 20% (P20) and PM
15/3, MPG 3/2 and MPG 1.5/10 was increased from 32.1ºC, 31ºC, 33.9ºC, 33.7ºC and
35ºC to 34.20ºC, 31.8ºC, 35.5ºC, 40.7ºC and 43.7ºC, respectively.
The Tsol-gel for the formulation IMPG 3/2 and IMPG 1.5/10 was above 37ºC. These
formulations were excluded from further evaluation and study. The increase in transition
temperature is due to the dehydration of polyoxyethylene (PO) block thus reducing the
micellization process and increase polymer entanglement with each other [97, 113, 114].
The results are in accordance with the early studies [115], which suggests that addition of
hydrophobic ingredients to the aqueous solution of polymers forming thermorevesible in-
situ gels increases the Tsol-gel. The filtration has no significant effect on the Tsol-gel of
the insulin in-situ thermoreversible gel formulations.
4.2.5.6.2 Viscosity of Insulin Formulations
The viscosity shows no significant difference on the addition of insulin to the polymer
solutions. The viscosities of the formulations IP18, IP20 and IPM15/3 were in the range
of 115.3, 127.7 and 143.3 before filtration and 115, 127.7 and 144.0 cps after filtration,
145
respectively. The results illustrates that filtration has no significant effect on the viscosity
of the in-situ thermorevesible gels. The P-Values obtained for the viscosity before and
after the filtration were insignificant as shown in Table 4.12. The results obtained are in
accordance with previous studies [274].
4.2.5.6.3 Clarity of Insulin Solutions and Formed Gels
Solutions of all the formulations were checked for clarity at 5ºC, 25ºC and at gelling
temperature, Human Insulin is water insoluble soluble drug the solutions obtained for all
the formulations were clear, confirming the surfactant effect of the pluronic which
favours solubility of insulin [84, 100, 101].The gels obtained at the gelling temperature
were clear [274] except for the formulation IPM 15/3 which formed turbid gel on
transition from solution phase. Filtration sterilization has no effect on the clarity of these
formulations.
146
Table 4.12: Physicochemical evaluation of Recombinant Human Insulin thermoreversible in-situ gels
P P P
Assay Viscosity Tsol-gel Clarity
Value Value Value
Before After Before After Before After Before After

Formulation
Filtration Filtration Filtration Filtration Filtration Filtration Filtration Filtration
IP18 98.72 ±0.42 98.6±0.40 0.192 115.3±1.15 115.0±1.00 0.423 34.2± 0.29 34.2±0.29 1 +++ +++
1
IP20 98.35±0.62 98.3±0.57 0.423 127.7±0.58 127.7±0.58 31.8±0.29 32.0±0.00 0.423 +++ +++
(NS)
IPM 15/3 98.96±0.19 98.8±0.28 0.314 143.3±2.08 144.0±1.73 0.184 35.5±0.00 35.3±0.29 0.423 + +
IMPG 1.5/10 43.7±0.58 43.7±0.58 1
IMPG 3/2 40.7±0.59 40.8±0.29 0.423
(n=3)
147
4.2.5.6.4 Drug Content of Human Insulin Preparations
The formulations prepared were analyzed with HPLC-UV method [270] in triplicate for
the drug content at a wavelength of 206 nm. The assay of the formulations IP18, IP20
and IPM15/3 was 98.72 ± 0.42, 98.35 ± 0.62 and 98.96 ± 0.19, respectively. The assay
was also performed after the filtration of the formulation that did not showed any
significant effect on the insulin content of formulations. The P-Values were insignificant
for all formulations.
4.2.5.6.5 Determination of In-vitro Drug Release from Insulin Formulations
In vitro insulin release in dissolution medium was determined by membrane less model
[269]. The selected insulin formulations were subjected to in vitro drug release profile
using phosphate buffer pH 7.4 as dissolution medium. Samples were collected after
predefined time intervals i.e.0.5, 1.0, 2.0, 4.0, 8.0, 12.0, 24, 36, 48, 72, 96, 120, 144, 192,
216, 240 and 264 hours depending upon formulation that remain intact for the specified
time.
The samples were suitably diluted and analyzed for insulin content by HPLC technique
.The dissolution experiment was repeated in triplicate for each formulation. The
formulations with insulin content 100 ± 10% were included in the in-vitro dissolution
studies.
The in-vitro drug release/time profiles of insulin thermoreversible in-situ gels (Fig. 4.24)
indicate that the formulation IPM 15/3 is the most efficient formulation in terms of
148
sustaining the drug release. The formulation IPM 15/3 extended the insulin release for
264 hours (11 days). While the formulations IP18 and IP20 extended the drug release for
36 and 72 hours only.
The drug release after second hour was 3.56%, 10.50% and 32.12% from IPM15/3, IP20
and IP18, respectively. The formulation IP 18 showed a burst release of insulin after first
hour. This burst release is due to the higher transition temperature and week gel strength
as insulin is hydrophobic drug that increases the Tsol-gel and also reduces the gel
strength.
The slowest drug release was attained for the formulation IPM15/3(Figure 4.24), which is
composed of combination of pluronic F-127 15% and methylcellulose 3%. This might be
due to the bonding between the insulin and MC polymer and swelling of the MC in
aqueous medium, thus increasing the path length for the diffusion of both penetrating
water and eluting drug from the hydrogel network [283]. Insulin is also hydrophobic and
large molecular weight drug as compared to diclofenac and timolol, and it might be
entangled in the polymeric matrix.
4.2.5.6.6 Drug Release Kinetics from Insulin In-situ Gels
The release kinetics and mechanism of insulin from thermoreversible sol-gel formulation
was predicted by fitting the dissolution data obtained (Table 4.13) to various
mathematical models. The models applied were zero order, first order, Higuchi time
square root equation, Hixson-Crowell cube root model, and Korsmeyer Pappas power
law.
149
The drug release from the thermoreversible gels are governed by various parameters.
Solubility of the drug within the polymer and water, water diffusion rate into the polymer
gel, the drug diffusion rate from the polymer gel and dissolution of the polymer within
the experimental conditions are all important factors that will affect the kinetics of drug
release.
Pluronic F-127 attains core-shell structure in an aqueous medium. The hydrophilic PEO
block occupies the outer shell region and the hydrophobic PEO block is embedded in the
shell as core.
120.0
% Cummulative Insulin Released
100.0
80.0
IP20
60.0
IP18
40.0 IPM15/3
20.0
0.0
0 24 48 72 96 120 144 168 192 216 240 264 288
Time (hr)
Fig. 4.24: In-vitro release profile of Recombinant Human Insulin form various in-situ
thermorevesible sol-gel (n=3)
Insulin is a hydrophobic drug and mostly resides inside the hydrophobic hydrogel
network. The drug release from the PEO hydrophilic block is through diffusion, while the
150
rest of the drug released is governed by the erosion and degradation of the hydrophobic
PPO domain of hydrogel.
Insulin in aqueous solution exists in various association states such as monomer, dimer,
hexamer and aggregate [284]. It is thought that insulin without zinc exists in aggregate
form inside the in-situ gel. Thus this form of insulin may not diffuse fast from in-situ
gels.
151
Table 4.13: % Cumulative In-vitro drug release from Insulin in-situ gels
Formulation IP18 IP20 IPM15/3
%drug release %drug release %drug release

Time (Hr) Mean ± SD Mean ± SD Mean ± SD
1 32.12±0.12 10.50±0.21 3.56±0.22
2 48.3±0.25 18.24±0.23 6.07±0.34
4 66.6±0.15 25.41±0.28 11.68±0.42
8 83.4±0.31 32.51±0.17 17.42±0.18
12 89.0±0.25 43.09±0.18 23.49±0.15
24 94.1±0.14 52.47±0.21 29.24±0.18
36 100.2±0.17 66.97±0.28 35.03±0.28
48 83.23±0.41 40.81±0.51
72 99.48±0.22 46.11±0.29
96 51.40±0.22
120 56.76±0.24
144 62.33±0.27
168 67.49±0.18
192 74.42±0.09
216 82.43±0.26
240 91.47±0.24
264 99.24±0.18
(n=3)
152
4.2.5.6.7 IP 18 insulin In-situ Gel
The drug release time profile of insulin in-situ gel formulation in Fig 4.24, illustrates that
the formulation IP18 extended the drug release for 36.The regression coefficient “R2” and
release exponent “n” values obtained for the formulations are depicted in Table 4.14. As
evident from the data best linearity (R2=0.930) for drug release from IP 18 was obtained
for Hixson-Crowell model, suggesting that the drug release is the result of degradation of
the polymer. Figure 4.26 indicates that good linearity with “R2” value of 0.900 was
obtained for first order, suggesting drug release dependence on drug remaining within the
hydrogel network.
Table 4.14: Release Parameters of Human Insulin from IP18, IP20 and IPM15/3 in situ
sol-gel formulations.
Model Zero Order First Order Higuchi Hixson-Crowell Korsmeyer
Formulation R2 KoH-1 R2 K1H-1 R2 KH(h-0.5) R2 KHC(h-1/3) n R2
IP 18 0.689 1.22 0.900 0.040 0.826 10.09 0.930 0.109 0.239 0.902
IP 20 0.970 1.142 0.839 0.027 0.991 11.41 0.955 0.046 0.467 0.992
DPM15/3 0.966 0.309 0.723 0.004 0.982 5.580 0.898 0.010 0.503 0.982
Plots for zero order and Higuchi equations are shown in Figure 4.25 and 4.27
respectively. The value of regression co-efficient “R2” is 0.689 and 0.826, respectively.
The value of release exponent (n=0.239) obtained from Korsmeyer-Pappas equation
153
indicates that the drug release follows Fickian diffusion that is in agreement with the first
order.
120.0 y = 1.222x + 62.732

y = 1.1421x + 23.268 y = 0.309x + 17.929
% Cummulative Insulin Released R² = 0.6895 R² = 0.9663
R² = 0.9706
100.0
80.0
IP20
60.0
IP18
40.0
IPM15/3
20.0
0.0
0 24 48 72 96 120 144 168 192 216 240 264 288
Time (hr)
Fig. 4.25: % Cumulative Recombinant Human Insulin Release Vs Time (Zero Order
Model) from different insulin in-situ thermoreversible sol-gel.
4.2.5.6.8 IP 20 insulin In-situ Gel
The in-vitro insulin release at 37ºC from the formulation is compiled in Table 4.13. The
cumulative % released of insulin after 2 and 12 hours from the formulation was 10.50%
and 43.09%, respectively. The slope and regression co-efficient obtained by plotting the
release data to different release models is shown in Table 4.14.The insulin release from
formulation showed best linearity for Higuchi model with R2 value of 0.991, the
formulation also showed good linearity (R2= 0.970) for zero order equation, suggesting
that the drug release is independent of the drug within the polymer network. The value of
release exponent obtained from Korsmeyer-Pappas model is 0.467, indicating the drug
release follow anomalous transport and also supports the drug release is independent of
drug concentration. The R2 (0.955) obtained from Hixson-Crowell suggests that portion
154
of the drug is released through drug diffusion and drug dissolution due to polymer
degradation.
2.5
2.0
y = 0.0408x + 0.3372
%loginsulin remaining
R² = 0.9006
y = 0.0271x - 0.1356 y = 0.0049x - 0.0533
1.5 R² = 0.8396 R² = 0.723
(log100-logt)
IP20
1.0
IP18
IPM15/3
0.5
0.0
0 50 100 150 200 250 300
-0.5
Time (hr)
Fig. 4.26: % Log cumulative Human Insulin Remaining Log (100)-Logt Vs Time (First
Order Model) from Insulin in-situ thermoreversible sol-gel.
4.2.5.6.9 IPM 15/3 insulin In-situ Gel
The formulation consists of 15%w/v Pluronic F-127 and 3%w/v methylcellulose. The
formulation was the most promising among the prepared insulin thermoreversible in-situ
solution to gel formulations. Figure 4.24 indicates that the formulation extended the drug
release for 264 hours (11 days). The values of regression coefficient “R2”, slope and
release exponent “n” are tabulated in Table 4.14. The Figure 4.27 indicates that the
release data best fits to Higuchi model as the R2 value obtained was 0.982 which is the
highest among the released model applied to the data. The R2 value suggests the drug is
released through diffusion. The Zero order release model also showed good linearity
155
indicating the drug release is independent of insulin concentration within the delivery
system.
120.0 y = 10.095x + 45.577

R² = 0.8265
%Cummulative Insulin Released
100.0 y = 11.412x + 1.2957

R² = 0.9915
y = 5.5802x + 0.1792
R² = 0.9822
80.0
60.0
40.0
IP20
20.0 IP18
IPM15/3
0.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0
Sq.Rt Time
Fig. 4.27: % cumulative Human Insulin release Vs Sq. Rt. Time (Higuchi Model) from
Insulin in-situ thermoreversible sol-gel.
The value obtained for the release exponent “n” by application of Korsmeyer-Pappas
equation was higher than 0.45 indicating the anomalous transport of the drug from
hydrogel system.
156
Fig 4.28: Representative chromatogram of in-vitro Insulin release from IPM 15/3 in-situ
gel at different time interval, violet=1 hr, black=12 hrs, blue=96 hrs, orange=264 hrs.
In general, the PF127 gel formation occurs due to the progressive dehydration of the
polymer micelles as temperature increases, leading to increased chain entanglement. This
entanglement was increased by the addition of methylcellulose to the polymer system,
resulting in increase in gel strength and thus reducing the drug release [285].
The in-vitro release of insulin from the in-situ gel formulation at 37ºC shown in Fig. 4.24,
indicates that the formulation IPM15/3 retarded the drug release for the greater time
period.
As shown in Fig. 4.24 and Table 4.13, IP18 produced the fastest release of insulin among
the formulations studied. The alteration of transition temperatures may alter the diffusion
of drugs in the gels, hence influencing release rates.
157
6.0
y = 0.109x + 0.910
R² = 0.930
5.0
CRT Insulin Remaining

4.0
y = 0.0465x + 0.112
y = 0.0102x + 0.1172
(Wo-Wt)
R² = 0.9557
R² = 0.8984
3.0
2.0
IP20
IP18
1.0
IPM15/3
0.0
0 50 100 150 200 250 300
Time (hr)
Fig. 4.29: Cube root% Human Insulin remaining (Wo-Wt) Vs Time (Hixson-Crowell
Model) from Insulin in-situ thermoreversible sol-gel.
The physicochemical properties of drugs also affect the drug release characteristics from
formulations [283]. As insulin is hydrophobic large molecular protein drug it is slowly
soluble in the penetrating water and thus released slowly and gradually from the in-situ
thermoreversible gel.
The inter-micellar gap decreases with increasing the concentration of PF127, the degree
of swelling also increases with increase in polymer concentration. This results in the
reduction of Tsol-gel and therefore altering the viscosity and the consistency
characteristics of the formulation. This is probably the most important factor influencing
the release characteristics of the drug [261, 286, 287].
158
2.50
y = 0.2391x + 1.66 y = 0.4679x + 1.1121
Log % cumulative Insulin Released

R² = 0.902 R² = 0.9926
2.00
y = 0.5035x + 0.7422
R² = 0.9827
1.50
1.00
IP20
0.50 IP18
IPM15/3
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Log Time
Fig. 4.30: log %cumulative Human Insulin release Vs Log Time (Korsmeyer-Pappas
Model) from Insulin in-situ thermoreversible sol-gel.
The IPM15/3 formulation was selected for the in-vivo study based on the results obtained
from the in-vitro study.
159
4.2.6 In-Vivo Evaluation of In-Situ Thermoreversible Gels
4.2.6.1 In-vivo Evaluation of DS In-situ gels
The in-situ thermorevesible gels preparations containing diclofenac sodium was
evaluated in-vivo using rabbits as experimental animals to determine various
pharmacokinetic (PK) parameters. The PK parameters were evaluated following
administration through ocular and subcutaneous route.
4.2.6.2 Subcutaneous Route
Formulations DP20, DP18, DPM 15/3, DMPG3/2, DMPG1.5/10 and conventional
commercial diclofenac sodium solutions were injected (5mg/kg) through subcutaneous
route. The plasma drug concentration time profile is shown in Figure 4.23.The data
obtained for the preparations was analyzed using non-compartmental model by PK-
summit® software. The pharmacokinetic parameters determined include; Peak plasma
concentration (Cmax),Time (Tmax) to attain Cmax, area under plasma drug concentration –
Time curve time zero to last sample drawn (AUC0-t), area under curve plasma drug
concentration –Time curve time zero to infinity(AUC0-∞), area under the movement curve
(AUMC0-∞), mean residence time (MRT), volume of distribution (Vd), clearance and
half-life (t1/2).The formulations were compared statistically with diclofenac sodium
conventional injection using student’s t-test at95% significance level.
4.2.6.2.1 DP18 and DP20 DS In-situ gel
The plasma drug concentration at different time intervals after the subcutaneous injection
of different in-situ gel preparations and the conventional parenteral diclofenac solution is
160
shown in Figure 4.31. The pharmacokinetic parameters of DS of these preparations are
shown in Table 4.15.
3
DPL20
DPL18
2.5
DMPG3/2
Plasma Drug Conc. µg/ml
DPM 15/3
2
Ref
1.5
0.5
0
0 20 40 60 80 100 120
Time (h)
Fig 4.31: Plasma Concentration (µg/ml) of Diclofenac sodium at various time intervals
after subcutaneous injection of commercial diclofenac sodium injection and DS in-situ
gels.
The Cmax for DP18, DP20 and conventional parenteral solution was 2.3798 ± 0.16, 2.4017
± 0.109 and 0.671 ± 0.061, respectively. The Cmax values obtained for the formulation
DP18 and DP20 were significantly high. The P-Values of DP 18 and DP 20 with respect
to reference formulation were 0.003 and 0.004, respectively. The Cmax of the in-situ gels
was 3.5 fold higher than the conventional parenteral solution. The higher Cmax may be
due to the gel that remains intact and remains for the longer duration at the site of
absorption releasing the drug continuously at constant rate.
The Tmax was 48 hours for both DP18 and DP20 in-situ gels preparations compared with
the conventional parenteral solution which was 2 hours. The plasma drug concentration
161
vs. time of different in-situ gels and commercial parenteral solution is shown in Figure
4.30. The plasma drug concentration-Time plot of formulations DP18 and DP 20 give
two peak concentrations, the first peak was observed at first hour indicating the burst
release of drug [60], which is due to transition lag time from solution to gel of the in-situ
gels. The second high concentration peak is observed at 48 hours that is the Tmax for
both formulations. This second high concentration peak is due to the dissolution [122]
and degradation of the polymer hydrogel. The in-vivo drug absorption is in accordance
with the in-vitro drug release and confirms that the drug release is anomalous transports
and follows both diffusion and erosion of the polymeric system.
Fig 4.32: Representative chromatogram of in-vivo plasma DS release from DP 20 in-situ

gel at different time interval, A=1 hr, B=24 hrs, C=48 hrs, D=72 hrs, E=96 hrs.
162
The P-Values are also significant for the pharmacokinetic parameters AUC0-t, AUC0-∞
and Cl (Clearance). The AUC, Cl, MRT and biological half-life values clearly suggests
that the in-situ gels have higher bioavailability as compared to reference solution.
The AUC0-t values indicate that bioavailability of diclofenac from DP 18 and DP 20 was
21.5 and 34 fold, respectively, higher than the conventional solution that indicates the
longer systemic release and absorption of the DS from the in-situ-gel formulations. The
relative bioavailability of DS from DP 18 and DP 20 was 227% and 367% higher
compared with reference formulation.
The elimination half-life obtained was in the order of 23.109 ± 9.70, 18.363 ±1.03 and
4.038 ± 0.19 for DP20, DP18 and reference formulation, respectively indicating the
prolong release of the drug from these formulations. Mean residence time (MRT) is the
ratio of AUMC/AUC, the value of MRT was 70.901 ± 13.19 and 50.77 ± 1.36 for DP20
and DP18,respectively, which is higher compared to the conventional preparation (6.953
± 0.31), indicating that the drug is slowly eliminated from the body after administration
of the in-situ gel compared to conventional preparation. The higher MRT value suggests
that the steady state plasma drug concentration was maintained during the studied time
period.
The plasma drug concentration-Time plots of the formulation DP 18 and DP 20 showed
that the plasma drug concentration was in the range of 1.985 ± 0.16 - 2.379 ± 0.17 and
2.045 ± 0.055 - 2.401 ± 0.11 µg/ml through-out the maintenance period i.e. 0-72 hours
163
and 0-96 hours, respectively, which is well above MEC of diclofenac sodium [288]. On
the other hand the conventional commercial parenteral solution could only maintain the
plasma drug concentration for 12 hours.
The rapid decline in the drug plasma concentration of the formulation DP 18 depicted in
the Figure 4.31 as compared to DP20 may be due to rapid degradation rate of the polymer
as the polymer concentration is less in DP18 compared to DP20.
4.2.6.2.2 DPM 15/3 DS In-situ gel
The plasma drug concentration at different time intervals after the subcutaneous injection
of 2 ml (10mg) DPM15/3 in-situ gel preparations and the conventional parenteral
diclofenac solution is shown in Figure 4.31. The formulation DP 15/3 maintained the
steady state drug plasma concentration for 72 hours. It is evident from the Figure 4.31
that the drug concentration at all points is higher in case of in-situ gel compared to the
reference solution. The Figure 4.31 also indicates that there is initial burst release in the
first hour, which is characteristic to in-situ gels because of the solution to gel transition
lag time[60] and the second burst release observed is due to the degradation of the
polymer in the physiological system.
The pharmacokinetic parameters calculated using PK-Summit is given in Table 4.15.
High Cmax (2.724 ± 0.06) was achieved by the formulation compared to the reference
formulation (0.671 ± 0.061).
164
The Tmax observed for the formulation was 48 hours compared to 2 hours of the
commercial conventional formulation. As illustrated in Figure 4.31 there is initial burst
release followed by constant plasma drug concentration. The Cmax attained at 48 hours is
due to the second high release of the drug from the in-situ gels that may be due to the in-
vivo degradation of the polymers.
Moreover higher values for AUC0-t, AUC 0-∞, AUMC0-∞ and MRT were attained for the
in-situ gel compared to reference parenteral solution (Table 4.15). The P-Values were
0.001, 0.001, 0.001, 0.001, 0.001, 0.001 and 0.013 for AUC0-t, AUC0-∞, AUMC0-∞, MRT,
Vd, CL, and Half-life, respectively.
The higher values of these parameters suggest higher bioavailability of the drug from the
thermorevesible gels. The bioavailability from the formulation is 18.6 fold higher than
the reference conventional parenteral solution. The relative bioavailability was 161%
higher for the formulation DPM 15/3 in comparison with reference conventional
formulation.
The volume of distribution (Vd) is significantly high for the reference formulation
compared with the DPM 15/3 in-situ gel formulation; the high Vd value indicates that
extensive distribution of the drug into the body tissues as it was absorbed rapidly due to
availability of high drug concentration at the site of absorption from the conventional
parenteral solution.
165
Table 4.15: Pharmacokinetic parameters of diclofenac sodium commercial formulation and in-situ gel after subcutaneous
administration
Formulation DPL 20 DPL 18 DMPG3/2 DPM15/3 Reference
P P P P
Pk Parmeter Unit Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD
Value Value Value Value
E Half-life Hr 23.109±9.70 0.008 18.363±1.03 0.003 9.071±0.76 0.003 6.785±0.07 0.013 4.038±0.19
Cmax (obs) µg/ml 2.4017±0.109 0.004 2.3798±0.16 0.003 2.640±0.01 0.001 2.724±0.06 0.001 0.671±0.061
Tmax (obs) Hr 48±0.0 ---- 48±0.0 ---- 1±0.0 ---- 48±0.0 ---- 2±0.0
AUC(0-t) (obs area) µg-hr/ml 156.61±11.71 0.002 99.197±6.11 0.001 32.50±0.77 0.001 85.757±0.21 0.001 4.608±0.15
AUC (0-∞) µg-hr/ml 202.22±44.68 0.017 124.65±6.95 0.001 35.78±0.44 0.001 88.058±0.12 0.001 5.493±0.09
AUMC (0-∞) µg-hr*hr/ml 14731 ±6097.78 0.005 6329.5±389.96 0.001 815.65±35.86 0.001 3370.8±19.18 0.001 38.19±12.43
MRT (area) Hr 70.901±13.19 0.015 50.775±1.36 0.001 22.793±1.001 0.001 38.279±0.27 0.001 6.953±0.31
Vd (area) / kg ml/kg 802.39±152.59 0.002 1065.3±87.52 0.001 1829.5±161.7 0.000 555.94±6.42 0.001 5304.60±4.56
CL (area) / kg ml/hr/kg 25.486±5.19 0.001 40.19±2.27 0.001 139.735±1.72 0.001 56.780±0.08 0.001 910.190±0.10
Half-life from Vd and CL Hr 23.109±9.70 0.008 18.36±1.03 0.002 9.071±0.76 0.003 6.7851±0.07 0.013 4.038±0.19
n=3
166
The Vd was small in case of DPM 15/3 in-situ gel formulation as the drug concentration
was maintained at steady state this may be due to the equilibrium between the drug
absorption and elimination. DS was slowly released from the gel formulation compared
with the conventional reference formulation that provides 100% of the drug immediately
at the site of absorption.
The elimination rate of the drug from the in-situ gel was significantly low compared to
the reference conventional solution. The MRT was statistically significant; the MRT
value was 38.279 ± 0.27 that was 5.5 times greater than the conventional reference
preparation. The gel remains intact for 48 hours maintaining the steady state
concentration and then degraded rapidly resulting in depletion of the drug and rapid
decline in the plasma concentration.
4.2.6.2.3 DMPG 3/2 DS In-situ Gel
Formulation DMPG 3/2 maintained the plasma drug concentration for 48 hours (Figure
4.31). The drug plasma – time profile shows characteristic initial burst release [60].
Plasma drug concentration as a function of time plot shows (Figure 4.31) that after 12
hours there is rise in the plasma concentration suggesting the dissolution of the polymer
within the physiological system.
The Cmax 2.640±0.01was observed after one hour. The Cmax obtained for the formulation
was significantly high (p< 0.001) compared with commercial parenteral solution. The in-
situ gel formulation shows greater values of AUC0-t, AUC 0-∞, AUMC0-∞, MRT and t1/2
that were statistically significant.
167
The relative bioavailability of the DMPG 3/2 was 65% greater than that of the reference
formulation. This indicates that the bioavailability of the drug from the formulation
DMPG3/2 was 6.5 fold higher compared with the commercial conventional solution. The
half-life was 9.071 ± 0.76, and 4.038 ± 0.19 for DMPG3/2 and reference formulation,
respectively. The half-life of the gel formulation was statistically significant, indicating
availability of the drug for prolong period of time.
The formulation DMPG 3/2 has lower Cmax, AUC, AUMC and MRT values compared
with the other prepared in-situ gel formulations.
The bioavailability of all in-situ gels was significantly high compared with the reference
conventional parenteral solution. The elimination half-life of the drug obtained for all in-
situ gel formulations was significantly greater than that of conventional solution. The
volume of distribution (Vd) for all the prepared in-situ gels were lower than the Vd of the
conventional reference parenteral solution.
Consequently, the diclofenac sodium content from the administration of conventional
parenteral preparation could not be detected in the plasma after twelve (12) hours
indicating that it was eliminated from the body or below the limit of detection.
The plasma drug–time profile (Fig: 4.31) values suggested that the in-situ
thermorevesible gels prepared with Pluronic, MC and their combination increased the
168
absorption and prolong the elimination half-life of diclofenac sodium resulting in better
bioavailability as compared with the conventional parenteral solution. Among various in-
situ gels formulations DP20 was the most robust to extended the in-vitro drug release for
120 hours that was fully supported by the in-vivo data obtained. The study also revealed
that the combination of pluronic and methylcellulose (DPM15/3) show comparable drug
control and sustainability as that of the pluronic formulations.
4.2.6.3 Ocular Route
The formulations DP20, DP18, DPM 15/3, DMPG3/2 and DMPG1.5/10 were evaluated
for different pharmacokinetic parameters after instillation 50 µl (25µg DS) of each
formulation into the rabbit’s eye. The pharmacokinetic parameters determined include;
peak aqueous humour drug concentration (Cmax),time to attain Cmax (Tmax), area under
curve aqueous humour drug concentration-Time Curve (AUC0-t), area under curve
aqueous humour drug concentration-time curve from time (T = 0) to infinity (AUC0-∞),
area under the movement curve (AUMC0-∞), mean residence time (MRT), volume of
distribution (Vd), clearance and half-life (t1/2).
The concentration of DS determined in rabbits’ aqueous humour samples is shown in
Figure 4.34. The data obtained was analyzed using non-compartmental analysis by PK-
summit® software to calculate the different pharmacokinetic parameters.
The in-situ thermorevesible formulations were compared statistically with diclofenac
sodium 0.1% conventional eye drops using student’s t-test with 95% significance level.
169
4.2.6.3.1 DP18 and DP20 DS In-situ Gel
Aqueous humour drug concentration-Time profile of the DP18 and DP20 following
instillation of eye drops is shown in Figure 4.34.The pharmacokinetic parameters of DS
in aqueous humour of these preparations are depicted in Table 4.16. The data was
statistically evaluated using Minitab 14 and the difference between in-situ gel
preparations and reference formulation was conducted using student t-test at 95%
confidence interval.
Fig 4.33: Representative chromatogram of in-vivo DS release in AH from DP 20 in-situ

gel at different time interval, A=0.5 hr, B=1 hrs, C=2 hrs, D=3 hrs, E=4 hrs, F=6 hrs.
The Cmax obtained was 2.95 ± 0.08, 4.07 ± 0.04 and 0.94 ± 0.07 for DP18, DP20 and
conventional eye drops, respectively. The P-Values were 0.003 and 0.004 for DP18 and
DP20, respectively that shows highly significant difference of Cmax between the in-situ
gel formulations and Reference drug preparation. The Cmax for the in-situ gels were 3.12
and 4.2 folds greater than the conventional eye drops for DP18 and DP20, respectively.
170
The Time to attain Cmax was one hour for DP18 and two hours for DP20 in-situ gels
compared with the conventional eye drop which was half (0.5) hour. The AUC0-t, AUC0-∞
and AUMC0-∞ values for the formulation DP18 was 6.84 ± 0.11, 8.88 ± 0.43 and 36.10 ±
4.65, respectively. The relative bioavailability was 40% and 56% higher for the
formulation DP 18 and DP 20 respectively compared with reference eye drops.
The values of these parameters obtained for the formulation DP20 were not only higher
than the commercial eye drops but also than the DP18 in-situ gel, indicating that higher
the concentration of the polymer more stronger and adhesive the gel will be, thus
increasing the residence and contact time of the formulation within the eye [289].
The aqueous humour drug concentration-Time profile shown in Figure 4.34, indicates the
absorption, distribution and extended elimination phase for the in-situ gels formulations.
The rapid decline of the DS concentration in AH confirms the in-vitro data obtained,
which suggests the release through drug diffusion and polymer erosion [290].
The diclofenac concentration (µg/ml) in aqueous humour for DP18 and DP20 was 0.450
and 0.524 µg/ml, respectively, after 6 hours indicating that the gels are able to maintain
the steady state concentration within the aqueous humour for the period.
171
4.5
4
Aq-Homour Drug Conc. (µg/ml)

3.5
3 Refernce
DMPG3/2
2.5
DP18
2
DP20
1.5
DPM15/3
1
0.5
0
0 1 2 3 4 5 6 7
Time (h)
Fig 4.34: Concentration (µg/ml) of diclofenac sodium in rabbits’ aqueous humour at

various times after instillation of a commercial diclofenac sodium eye drops and DS in-
situ gel formulations
4.2.6.3.2 DPM 15/3 DS In-situ Gel
The formulation consist of 15% Pluronic F-127 and 3% methyl cellulose. The DS
concentration at all points was higher compared with reference eye drops. The data
obtained various Pharmacokinetic parameters calculated with PK-summit software is
shown in Table 4.16.
Aqueous humour drug concentration-Time profile of the DPM 15/3 formulation
following instillation of eye drops is shown in Figure 4.34. The pharmacokinetic
parameters of DS in aqueous humour of the preparation are given in Table 4.16. The data
was statistically evaluated using Minitab 14 and the difference between in-situ gel
172
The Cmax (2.95 ± 0.08) attained at one hour (Tmax) is higher in comparison to the
reference conventional eye drops. The values for AUC0-t, AUC0-∞, AUMC0-∞ and MRT
were significantly high for the in-situ gel as compared with the conventional eye drops.
The relative bioavailability was 32% higher compared with the reference formulation.
This higher bioavailability might be due to the bio-adhesive character of the gel imparted
by MC[291].
The value of MRT was 3.33 ± 0.34 which is higher than the reference formulation (1.83
± 0.11). The greater MRT value also confirms the increased contact time of the gel
formulation compared to the reference formulation. The elimination rate of the drug from
the in-situ gel was significantly low indicating low drainage and high residence of the gel
in the ocular tissue. The elimination half-life were 2.14 ± 0.69 and 0.82 ± 0.14for the
formulation and reference, respectively. The half-life of the in-situ gel formulation was
significantly high with the p-value of 0.013.The formulation DPM15/3 has low relative
bioavailability compared with DP 18 and DP 20 i.e. 0.80 and 0.57 respectively.
4.2.6.3.3 DMPG 3/2 DS In-situ Gel
Aqueous humour drug concentration-Time profile of the DMPG 3/2 following instillation
of eye drops is shown in Figure 4.34.The pharmacokinetic parameters of DS in aqueous
humour of the preparation are given in Table 4.16. The data was statistically evaluated
using Minitab 14 and the difference between in-situ gel preparations and reference
formulation was conducted using student t-test at 95% confidence interval.
173
The drug content-time profile Figure 4.34 shows that the formulation is capable of
maintaining the drug concentration above the MEC of diclofenac for six (6) hours. The
pharmacokinetic data obtained for the in-situ formulation is shown in Table 4.16.The
Figure 4.34 suggests that there is initial burst release resulting in peak plasma
concentration (Cmax) at half (0.5) hour. It can be seen from the in-vitro evaluation of the
formulation that the Tsol-gel was above 35oC, thus the lag time for conversion of solution
to gel was high which may be the cause of the burst release.
The Cmax of the formulation was 1.84 ± 0.008 µg/ml compared with 0.94 ± 0.07 µg/ml
reference formulation. The Cmax value was statistically high, with p-value of 0.001.
The values of AUC0-t, AUC0-∞, AUMC0-∞, MRT and t1/2 were statistically significant
compared with the reference formulation (Table 4.16) that indicates the bioavailability of
the drug from the in-situ gel formulation was higher in comparison to the commercial eye
drops. The relative bioavailability was 22% more for the in-situ gel formulation
compared with the reference formulation.
The MRT was higher for the formulation indicating the high retention time of the
formulation in the eye as compared to the conventional eye drops, providing longer
absorption duration. The clearance rate of diclofenac sodium from the ocular tissue with
the administration of the gel formulation was low compared with the commercial eye
drops, the values are 111.91 and 50.97 for commercial eye drops and DMPG3/2. The bio-
adhesive characteristic of the methyl cellulose increases the residence and contact time of
174
the formulation with in the eye resulting in greater bioavailability from the formulation
compared with the conventional eye drops which are rapidly drained off from the ocular
tissues.
175
Table 4.16: Pharmacokinetic parameters of Diclofenac sodium commercial formulation and in-situ gel formulation after ocular
administration
Formulation DP 18 DP 20 DPM 15/3 DMPG3/2 Reference
Pk Parmeter Unit Mean ± SD P-Value Mean ± SD P-Value Mean ± SD P-Value Mean ± SD P-Value Mean ± SD
E Half-life Hr 3.13±0.36 0.003 2.45±0.40 0.008 2.14±0.69 0.013 1.55±0.07 0.003 0.82±0.14
Cmax µg/ml 2.95±0.08 0.003 4.07±0.040 0.004 2.95±0.08 0.001 1.84±0.08 0.001 0.94±0.07
Tmax Hr 1.00±0.00 ---- 2.00±0.00 ---- 1.00±0.00 ---- 0.50±0.00 ---- 0.5±0.00
AUC(0-t) µg-hr/ml 6.84±0.11 0.001 10.66±0.35 0.002 6.10±0.39 0.001 4.48±0.33 0.001 1.89±0.01
AUC(0-∞) µg-hr/ml 8.88±0.43 0.001 12.51±0.11 0.017 7.19±0.62 0.001 4.94±0.39 0.001 2.24±0.08
AUMC(0-∞) µg-hr*hr/ml 36.10±4.65 0.001 42.00 ±2.41 0.005 24.03±4.33 0.001 13.44±1.26 0.001 4.11±0.39
MRT Hr 4.05±0.33 0.001 3.36±0.22 0.015 3.33±0.34 0.001 2.72±0.04 0.001 1.83±0.11
Vd ml/kg 126.75±8.60 0.001 70.67±12.14 0.002 106.44±27.17 0.001 112.96±3.67 0.001 132.25±17.21
CL ml/hr/kg 28.19±1.35 0.001 19.99±0.18 0.001 34.95±2.93 0.001 50.79±3.98 0.001 111.91±3.89
Half-life from Vd and CL Hr 3.13±0.36 0.002 2.45±0.40 0.008 2.14±0.69 0.015 1.55±0.07 0.003 0.82±0.14
n=3
176
The bioavailability of all in-situ gels was significantly high compared with the reference
conventional ophthalmic solution. The elimination rate of the drug from all the in-situ gel
formulations was significantly low than that of conventional eye drops.
The diclofenac sodium content from the instillation of conventional commercial eye drop
could not be detected in the aqueous humour after three (3) hours indicating that it was
below the limit of detection or eliminated from the aqueous humour, while the DS
content for the gels was detected for six (6) hours after the instillation. The concentration
of the DS determined with HPLC method for DP20, DP18, DPM15/3 and DMPG3/2
after 6 hours was0.450 ± 0.02, 0.524 ± 0.015, 0.348 ± 0.02 and 0.206 ± 0.02 (µg/ml),
respectively. The concentration of diclofenac achieved at sixth hour were above the MEC
level of the diclofenac sodium [288].
The in-vivo data correlates with the in-vitro release data. The in-vivo evaluation suggest
the in-situ thermorevesible solution to gel formulation of Pluronic, MC and their
combination could increase the diclofenac sodium content absorbed into the eyes and
prolong the elimination time, hence resulting in better bioavailability as compared to the
conventional eye drops.
177
4.2.6.4 In-vivo Evaluation of Timolol Maleate In-situ Gels
The drug release and pharmacokinetics studies were conducted following the instillation
of the formulations TP20, TP18, TPM 15/3, TMPG3/2 and TMPG1.5/10 onto rabbits’
cornea. The pharmacokinetic parameters determined include; Peak aqueous humour
concentration (Cmax),Time (Tmax) to attain Cmax, area under aqueous humour
concentration curve (AUC0-t), area under curve (AUC0-∞), area under the movement
curve (AUMC0-∞), mean residence time (MRT), volume of distribution (Vd), clearance
and Half life (t1/2).
The concentration of TM determined in the samples of rabbit’s aqueous humour was
analyzed by PK-summit® software to calculate the above mentioned pharmacokinetic
parameters.
After the administration of 50 µl (25µg) dose of TM commercial conventional (0.5%) eye
drops, used a s a reference formulation, and the prepared in-situ thermorevesible gel
formulations (TP20, TP18, TPM15/3, TMPG3/2 and TMPG1.5/10). The pharmacokinetic
parameters of TM in aqueous humour of these preparations are depicted formulations in
Table 4.17. Various pharmacokinetics parameters were statistically compared with the
reference formulation using t-test 95% confidence interval.
4.2.6.4.1 TP18 and TP20 TM In-situ Gel
Timolol (TM) aqueous humour concentrations was determined at different time intervals
following the instillation of TP 18 and TP 20 in-situ gel formulations (50 µl ≈25µg) and
178
conventional eye drops, used as reference formulation, onto the rabbits’ cornea. The TM
concentration in the aqueous humour was detected for 6 hours following drug instillation.
Aqueous humour drug concentration-Time profile of the TP 18 and TP 20 following
instillation of eye drops is shown in Figure 4.36. The pharmacokinetic parameters of TM
in aqueous humour of the preparation are given in Table 4.17. The data was statistically
evaluated using Minitab 14 and the difference between in-situ gel preparations and
reference formulation was conducted using student t-test at 95% confidence interval.
Fig 4.35: Representative chromatogram of in-vivo TM release in AH from TP 20 in-situ

gel at different time interval, A=0.5 hr, B=1 hrs, C=2 hrs, D=3 hrs, E=6 hrs.
The Cmax obtained was 3.637 ± 0.56, 6.126 ± 0.45 and 1.601 ± 0.12 for TP18, TP20 and
conventional eye drops respectively. The Cmax values for both in-situ formulations were
higher with significant P-Values compared with the reference conventional eye drops.
The Cmax was 2.7 and 3.83 fold higher than the conventional eye drops for TP18 and
179
TP20 respectively. The Tmax was one hour for both TP18 and TP20 in-situ gels as
compared to conventional eye drop which was half (0.5) hour.
The values of AUC0-t, AUC0-∞, AUMC0-∞, MRT and t1/2 were high for the formulation,
while the clearance rate was low for the in-situ gel formulations. The AUC, CL, MRT
and biological half-life values clearly suggest that the in-situ gels has higher
bioavailability as compared to conventional eye drop.
The AUC0-t values of TP18 and TP20 suggested that the bioavailability of TM was 4.0
and 6.8 fold respectively, higher than the conventional eye drops. As compared to the
reference formulation the relative bioavailability was 44% and 73%higher for DP 18 and
DDP 20 respectively. The MRT were 2.856 ± 0.84 and 2.872 ± 0.33 for DP 18 and DP 20
respectively, while the MRT value of the reference formulation was only 1.305. The
higher value of MRT suggests higher residence time of the in-situ gel formulation in the
ocular tissue in comparison to the conventional TM eye drops. The elimination rate of the
drug from the in-situ gel was significantly low indicating low drainage and high
residence of the gel in the ocular tissue. The elimination half-life was 1.266 ± 0.68, 1.44
± 0.15 and 0.668 ± 0.45 for TP 18, TP 20 and reference formulation respectively. The
half-life of the in-situ gel formulation was significantly high.
The pharmacokinetic parameters revealed that the TM was well absorbed from the TP18
and TP20 formulations, it might be due to the bio-adhesiveness and high consistency of
the gel that increase the residence and contact time [289] of the TM in the eye as
180
compared to the conventional eye drop that may be rapidly drained off and eliminated
from the eye tissue.
Aqueous humour drug concentration-time profile is depicted in Figure 4.36. The rapid
decline of the TM concentration in AH signify rapid erosion of the gel and diffusion of
the water soluble drug in aqueous humour[290].
The TM concentration in aqueous humour for TP18 and TP20 was 0.540 and
0.710µg/ml, respectively, after 6 hours indicating that the gels are able to maintain the
steady state concentration within the aqueous humour for the studied duration.
8.0
TPM 15/3
7.0
TP 20
6.0
Aq.Humour Drug Conc. (µg/ml)
TP 18
5.0 TMPG1.5/10
TMPG3/2
4.0
Reference
3.0
2.0
1.0
0.0
-1.0
0 1 2 3 4 5 6 7
-2.0
Time (h)
Fig. 4.36: Concentration (µg/ml, mean ± SD) of Timolol maleate in rabbits’ aqueous
humour at various times after instillation of a commercial diclofenac sodium eye drops
and TM in-situ gel formulations
The AUC0-t value of the TP20 was higher (p=0.008) than TP18. This significance
indicates that with increase in the pluronic concentration the bioavailability of TM
181
increases. The results obtained in current study are consistent with the earlier studies [91]
and also correlates with the in-vitro release profiles.
4.2.6.4.2 TPM 15/3 TM In-situ Gel
The formulation consists of 15% Pluronic F-127 and 3% methyl cellulose. The in-vitro
release profile indicated that the formulation sustained the drug release for 24 hours that
is higher as compared with TP20 that extended the release of TM for 12 hours. The mean
AH drug concentration–time profiles after administration of the conventional eye drops
and TPM15/3 in-situ gel of TM are illustrated in Figure 4.36. From the profile it is clear
that higher AH drug levels were achieved in case of the TPM15/3 gel compared with the
conventional eye drops. The pharmacokinetic and bioavailability data for the different In-
situ gels and conventional eye drops is shown in Table 4.17.
The Cmax values were 3.10 8 ± 0.70 µg/ml for the TPM15/3 in-situ gel while that of
conventional reference formulation was 1.601 ± 0.12 µg/ml. Statistical analysis revealed
that the Cmax was significantly (p<0.02) higher in case of the in-situ gel.
Moreover, the values for AUC0-t, AUC 0-∞, AUMC0-∞ and MRT were significantly high
for the in-situ gel compared with the conventional eye drops (Table 4.17); suggesting
high bioavailability of the drug from the thermorevesible gel. This might be due to the
bio-adhesive character of the gel imparted by MC[291]. The relative bioavailability of the
formulation compared with reference was 39% greater. The increase in bioavailability
might be due to highly viscose gel formed by the addition of the thickening agent MC to
182
the poloxamer solution. The results obtained in current study is consistent to the in the
early studies [146].
The MRT obtained for the formulation (2.988 ± 0.40) was higher than the reference
formulation (1.395 ± 0.25) the higher MRT value is due to the long residence and contact
of the formed gel as compared to the reference conventional eye drop that drains off
rapidly. The elimination rate of the drug from the in-situ gel was significantly low
indicating low drainage and high residence of the gel in the ocular tissue. The increased
residence and contact time in the eye is due to the adhesiveness of gel augmented by the
addition of MC.
4.2.6.4.3 TMPG 3/2 and TMPG 1.5/10 TM In-situ Gel
Aqueous humour drug concentration-Time profile of the TMPG 3/2 and TMPG 1.5/10
following instillation of these formulations is shown in Figure 4.36.The pharmacokinetic
parameters of TM in aqueous humour of the preparations are given in Table 4.17. The
data was statistically evaluated using Minitab 14 and the difference between in-situ gel
The pharmacokinetic data obtained for the in-situ formulations is shown in Table
4.17.The peak aqueous humour drug concentration for both the formulation was obtained
at one hour.
183
The Cmax value was in order of 3.145 ± 0.45µg/ml and 2.879 ± 0.41µg/ml and P-Values
were 0.018 and 0.035 for TMPG3/2 and TMPG 1.5/10, respectively. The Cmax obtained
for the in-situ gel formulations were significantly high compared with the commercial
ophthalmic solution.
The values of AUC0-t, AUC0-∞, AUMC0-∞, MRT and t1/2 were higher and statistically
significant. The higher values of these Parameters indicate that the bioavailability of the
drug from the in-situ gels was higher in comparison to the commercial eye drops. The
relative bioavailability of the formulations TMPG 3/2 and TMPG 1.5/10 was 33% and
26% more than that of the reference conventional eye drops.
The MRT values obtained using PK-Summit software was higher for the formulation
TMPG 3/2 and TMPG 1.5/10, suggesting high residence and slow drainage from the eye,
which is the prime objective of enhancing the ocular drug availability. The elimination
rates for TMPG3/2, TMPG 1.5/10 and conventional eye drops was 25.995 ml/hr/kg,
33.053 ml/hr/kg and 86.508 ml/hr/kg, respectively. The low elimination rate suggests that
the clearance of the TM was low after the instillation of the in-situ thermoreversible gel
compared with the conventional eye drops. The high Cmax, bioavailability and lower
elimination rate is due high residence and contact of the in-situ gels because of the muco-
adhesive character of the MC.
184
Table 4.17: Pharmacokinetic parameters of Timolol maleate commercial formulation and in-situ gel formulation after ocular
administration
P P P P P Conv. Eye
Pk Parameter Unit TP20 TP18 TPM15/3 TMPG 1.5/10 TMPG3/2
value value value value value Drops
Cmax µg/ml 6.126±0.45 0.009 3.637±0.56 0.019 3.108±0.70 0.028 2.879±0.41 0.035 3.145±0.45 0.018 1.601±0.12
Tmax Hr 1.0±0.0 1.0±0.0 1.0±0.0 1.0±0.0 1.0±0.0 0.50±0.0
AUC(0-t) µg-hr/ml 14.984±1.29 0.004 8.870±0.50 0.002 8.049±0.21 0.001 5.327±0.28 0.004 6.352±0.28 0.001 2.202±0.18
AUC (0-∞) µg-hr/ml 16.458±1.86 0.007 9.856±3.50 0.005 8.815±0.28 0.001 5.900±0.33 0.001 7.502±0.47 0.006 2.254±0.22
AUMC (0-∞) µg-hr*hr/ml 47.263±11.01 0.023 28.143±10.50 0.06 26.339±3.88 0.009 14.991±1.51 0.014 24.010±1.21 0.001 2.943±0.83
MRT Hr 2.872±0.33 0.003 2.856±0.84 0.175 2.988±0.40 0.017 2.541±0.27 0.089 3.201±0.42 0.011 1.305±0.25
Vd ml/kg 24.617±5.37 0.063 36.135±16.00 0.111 35.439±2.29 0.062 101.810±13.53 0.288 93.108±7.46 0.188 83.429±39.86
CL kg ml/hr/kg 11.848±1.26 0.003 19.786±3.02 0.007 22.121±0.66 0.006 33.053±2.80 0.02 25.995±2.81 0.01 86.508±6.90
Half-life from
Hr 1.440±0.15 0.623 1.266±0.68 0.754 1.110±0.13 0.729 2.135±0.35 0.033 2.482±0.20 0.033 0.668±0.45
Vd and CL
(n= 3, mean ± SD)
185
It can be concluded from the data that higher the concentration of MC higher the
bioavailability, also it can be inferred from the data that gels (TMPG1.5/10) with low
concentration of MC and high concentration of PEG can reasonably increase the
bioavailability and also extend the drug release for the studied period of time.
The bioavailability of all the in-situ gels was significantly high as compared to the
reference conventional ophthalmic solution. The elimination rate of the drug was
significantly low than that of conventional eye drops.
The TM content after the instillation of conventional commercial eye drop could not be
detected in the aqueous humour after four hours indicating that it was below the limit of
detection or cleared from the aqueous humour. While the TM content for the gels was
detected after six hours after the instillation. The concentration of the TM determined
with HPLC method for TP20, TP18, TPM15/3, TMPG3/2 and TMPG1.5/10 after 6 hours
was 0.710, 0.540, 0.478, 0.186 and 0.321 (µg/ml) respectively.
These values suggested that the in-situ thermorevesible gels prepared with Pluronic, MC
and their combination could increase the TM content absorbed into the eyes and prolong
the elimination time, hence resulting in better bioavailability as compared to the
conventional eye drops.
186
4.2.6.5 In-vivo Evaluation of Insulin In-situ Gels
The drug release and pharmacokinetics studies were conducted following the
subcutaneous administration of the formulation IPM 15/3 and commercial insulin
preparation to rabbits. The pharmacokinetic parameters determined include; peak plasma
concentration (Cmax), Time (Tmax) to attain Cmax, area under plasma concentration curve
(AUC0-t), area under curve (AUC0-∞), area under the movement curve (AUMC0-∞), mean
residence time (MRT), volume of distribution (Vd) and clearance and Half-life (t1/2).
The concentration of insulin determined in the rabbits’ plasma samples (Figure 4.37 and
4.38) after the administration of 2ml (2U) dose of Insulin commercial conventional
preparation, used as a reference formulation, and the IPM15/3 in-situ thermorevesible gel
formulations was analyzed by PK-summit® software.
The pharmacokinetic parameters of Insulin in plasma of these preparations are depicted
in Table 4.18. Various pharmacokinetics parameters were statistically compared with the
reference formulation using student’s t-test 95% confidence interval.
4.2.6.5.1 IPM 15/3 Insulin In-situ Gel
Insulin plasma concentration was determined at different time intervals following the
subcutaneous administration of IPM15/3 and conventional insulin preparation, used as
reference formulation. The plasma Insulin concentration (Figure 4.37) was detected for
240 hours (10 days) following administration of in-situ gel formulation in comparison
with the conventional insulin preparation for which the insulin content in plasma was
detected for only 12 hours (Figure 4.38).
187
45.0
Plasma Insulin Level (µU/ml)

40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
0 24 48 72 96 120 144 168 192 216 240 264
Time (hrs)
Fig 4.37: Plasma concentration (µU/ml) of Insulin in rabbits at various times intervals
after subcutaneous administration of Insulin in-situ gel formulations
30.0
Plasma Insulin level (µU/ml)
25.0
20.0
15.0
10.0
5.0
0.0
0 2 4 6 8 10 12 14
Time (hrs)
Fig 4.38: Plasma concentration (µU/ml) of Insulin in rabbits at various times intervals
after subcutaneous administration of a commercial insulin solution
The Cmax values attained after administration of the In-situ gel formulation and reference
formulation were 37.7 ± 5.1 and 26.0 ± 0.8, respectively. The Cmax of IPM15/3 was 1.45
times higher than the reference formulation. The Cmax for the in-situ gel was achieved
188
after 144 hours in comparison to one hour of the reference insulin formulation. A burst
release at 144 hours from the IPM15/3 was observed (Figure 4.37) indicating the
degradation of the polymer followed by decline plasma insulin level.
Higher AUC0-t, AUC0-∞, AUMC0-∞ and MRT values were obtained for the in-situ gel
compared to reference parenteral solution (Table 4.18). The P-Values for all the
pharmacokinetic parameters were significant at 95% confidence interval.
189
Table 4.18: Pharmacokinetic parameters of Human Insulin commercial formulation and

in-situ gel formulation after subcutaneous administration
IPM 15/3 Reference

PK Parameter Unit P Value
Mean ± SD Mean ± SD
E Half-life Hr 28.6±5.9 7.2±0.3 0.001
Cmax µg/ml 37.7±5.1 26.0±0.8 0.003
Tmax Hr 144.0±0.00 1.0±0.0 ----
AUC(0-t) µg-hr/ml 5794.0±940 123.8±4.3 0.001
AUC (0-∞) µg-hr/ml 6228.6±954 181.6±9.8 0.001
AUMC (0-∞) µg-hr*hr/ml 805888±117910 1881.8±178 0.001
MRT Hr 129.5±2.1 10.3±0.4 0.001
Vd ml/kg 1.0±0.4 8.6±0.3 0.001
CL ml/hr/kg 0.025±0.004 0.83±0.04 0.001
Half-life from
Hr 28.6±5.9 7.2±0.3 0.001
Vd and CL
(n=3, Mean ± SD)
190
The higher values of these parameters suggest higher bioavailability of the drug from the
thermorevesible gels. The relative bioavailability compared with reference formulation
was 360% higher for the in-situ gel formulation.
The volume of distribution (Vd) of the in-situ gel formulation was significantly low
compared with the reference formulation. The Vd was small in case of IPM 15/3 in-situ
gel formulations as the drug concentration was maintained at steady state this may be due
to the equilibrium between the drug absorption and elimination. Insulin was slowly
released from the gel formulation compared with the conventional reference formulation
that provides 100% of the drug immediately at the site of absorption.
The elimination rate of the drug from the in-situ gel was significantly low compared to
the reference conventional solution. The MRT was statistically significant; the MRT
value was 129±2.1 that was 12.5 times greater than the conventional reference
preparation.
The formulation IPM 15/3 maintains the steady state plasma insulin level between 10-40
µU/ml (Figure 4.37) throughout the study period which is the normal required basal
insulin level [292].
191
CHAPTER 5 CONCLUSION
CHAPTER 5
CONCLUSION
192
5. Conclusion
The objective of the current study was to develop sustained release in-situ solution to gel
polymeric delivery system for the drugs to be administered through subcutaneous or
ocular route. Four polymers (Pluronic F-127, Methyl cellulose, hydroxyl
propylmethylcellulose and polyethylene glycol 6000) were used in different
concentrations alone and in combination with each other in different ratios. Total of thirty
four formulations were prepared and evaluated for Tsol-gel.
Depending upon the gelling temperature and rheological behavior only five formulations
(P20, P18, PM15/3, MPG 3/2 and MPG 1.5/10) among the prepared thirty four
formulations were selected as drug delivery system. The selected formulations were clear
free flowing solution at room temperature (25oC) and converts to viscous gel below the
body temperature (37oC). All of the formulations showed perfect thermorevesible
behavior i.e. the solutions gels with increase in temperature and reverts to solution with
decrease in the temperature. The formed gels of the solutions (P18 and P20) containing
the pluronic as thermorevesible polymer were transparent, while the solutions prepared
by combination of pluronic and MC (PM15/3) form semitransparent gel. The gels of the
formulation (MPG 3/2 and MPG 1.5/10) containing MC and PG were turbid.
The studied preparations were Newtonian at room temperature and the formed gels were
thixotropic as indicated by the flow index and consistency index. It was also concluded in
the current study that sterilization by autoclaving have no significant effect on the Tsol-
gel and rheological properties.
193
Addition of DS and insulin to the hydrogel solutions showed a positive effect on Tsol-gel
i.e. these drugs increase the transition temperature while TM decreases the Tsol-gel. The
polymer solutions of the drug were clear indicating complete solubility of the drugs.
For the determination of the drug content in the in-vitro and in-vivo samples of the DS
and TM HPLC-UV method was developed and validated as per ICH guidelines. The
analytes were separated within 7 minutes with total run time of 10 minutes. The LOD
determined for DS and timolol were 2.0 ng/ml and 1.0 ng/ml respectively, while the LOQ
for the drugs were 0.80 ng/ml and 0.25 ng/ml respectively.
Autoclaving have no significant effect on the Tsol-gel, viscosity, and clarity of
thermoreversible formulation of DS and TM, though it decreases the drug content of DS
formulations significantly. The filtration process carried out for sterilization of insulin
formulations showed no significant effect on the physical and chemical properties of
these formulations.
The in-vitro drug release data of the DS preparation reveals that the formulation DP 20
extended the drug release for 144 hours that was fully supported by the in-vivo data. The
drug release from the DP18 and DP 20 followed Higuchi model while DMPG 3/2,
DMPG 1.5/10 and DPM 15/3 followed Hixson-Crowell Model.
TPM 15/3 extended the drug release for 24 hours, while TP18 and TMPG 1.5/10 could
only retard the drug release for 8 hours. All formulations followed first order release
194
kinetics. The linearity for Hixson –Crowell suggests the drug release through diffusion of
drug and erosion of polymer.
Among the formulations of insulin the formulation IPM 15/3 showed promising in-vitro
dissolution profile by extended the drug release for 264 hours (11 days). The drug release
form the formulation was governed by diffusion. The in-vivo studies indicate that the
formulation IPM15/3 is capable of maintaining the plasma insulin level for 10 days.
The study reveals that HPMC polymer didn’t gel below the body temperature (37oC) at
the studied concentration range (1-10%w/v).
The in-vivo data obtained correlates to the in-vitro dissolution profile. The
thermoreversible in-situ gel formulations of all three drugs showed greater and prolong
bioavailability compared with reference conventional formulations of these drugs. The
formulations of all the drugs studied were able to maintain the plasma and AH steady
state concentration over a longer period of time.
195
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222
ANNEXURE
PUBLICATION FROM THESIS

ANNEXURE
1. Nasir F, Iqbal Z, Khan A, Ahmad L, Shah Y, Khan AZ, Khan JA, Khan S.
Simultaneous determination of timolol maleate, rosuvastatin calcium and
diclofenac sodium in pharmaceuticals and physiological fluids using HPLC-UV.
Journal of Chromatography B. 2011 Nov 15;879(30):3434-43.

Thermo-Reversible Subcutaneous PH.D Thesis

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DEVELOPMENT AND EVALUATION OF

THERMO-REVERSIBLE SUBCUTANEOUS AND

THESIS SUBMITTED TO UNIVERSITY OF PESHAWAR IN

2. BACK GROUND ................................................................................................................... 9

2.1 Drug Delivery System ............................................................................................................... 9

2.2 Delayed Release ......................................................................................................................10

2.3 Extended Release ....................................................................................................................11

2.4 Site Specific Targeting..............................................................................................................11

2.5 Receptor Targeting ..................................................................................................................11

2.6 Fast Dissolve Drug Delivery System (Flash)...............................................................................12

2.7 Drug Modification ...................................................................................................................13

2.8 Dosage Form Modification.......................................................................................................13

2.9 Ophthalmic Drug Delivery:.......................................................................................................15

2.10 Ocular Routes ..........................................................................................................................19

2.12 Subcutaneous Drug Delivery .................................................................................................... 26

2.13 Parenteral Depot System .........................................................................................................29

2.14 Hydrogels ................................................................................................................................41

3. MATERIALS AND METHODS ........................................................................................ 68

3.1 Materials .................................................................................................................................68

3.2 Instrumentation ......................................................................................................................68

3.3 Study Design ...........................................................................................................................69

4. RESULTS AND DISCUSSION ......................................................................................... 97

4.2 Physical Evaluation of In-situ Solution to Gel Formulations .................................................... 108

5. CONCLUSION ................................................................................................................. 193

6. REFERENCES .................................................................................................................. 197

I would like to dedicate this thesis to my beloved mother who was

spring of spiritual, religious and moral support for me throughout

I also dedicate this thesis to my wife, my daughters Salwa, Meerab,

Aroosh and my son Muhammad for their sufferings and support.

patience and health to complete my Ph. D research work.

without their support and help.

value of continued excellence, education, and personal development throughout my

to continue our relationship for many years.

Pharmacy University of Peshawar.

of Pharmacy University of Peshawar for their co-operation.

converts to gel with increase in temperature.

below 37oC at appropriate concentrations (P= 18% w/v, 20%w/v, MC=4%w/v,

PM=15/3%w/v, MPG3/2w/v and MPG1.5/10% w/v).

To evaluate the efficacy of the in-situ thermoreversible formulations in controlling the

after autoclaving the solutions indicates that autoclaving results in degradation of DS

while it has no significant effect on TM.

Summit software was used.

insulin in steady state for longer duration of time.

sensitive and rapid method for the determination of DS and TM in pharmaceutical

possessing a unique thermoreversible property.

studied drugs through subcutaneous and ophthalmic routes.

common approaches are Drug modification and Dosage form modification[2].

The applications of the polymers in pharmaceutical sciences particularly in drug delivery

been reported for gelatin and agarose [3-6].

cellulose derivatives have an optimum balance of hydrophilic and hydrophobic moieties,

on the substitution of cellulose at the hydroxy group[7], Methyl cellulose and

Hydroxypropyl cellulose are typical examples[7]. With rise in temperature of solution of

become dominant resulting in a gel[8].

techniques such as spectroscopy [10], differential scanning calorimetry (DSC) and

polymeric solutions [10].

Many polymers show a decrease in solubility as their hydrophobicity increases upon

phenomenon is called hydrophobic effect [10-13]. As a result of the increase in

therefore return to solution after the temperature stimulus causing gelation is

great compatibility, degradability within the biological system and temperature

poly (propylene glycol)-poly(ethylene glycol) copolymer (PEG-PPG-PEG), known as

Poloxamer or Pluronic, has extensively been studied as a potential thermo-reversible drug

delivery system [9, 18].

spironolactone release was maintained over 2 months [19].